TITLE PAGE
STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID
ISOMERASE OF THE VISUAL CYCLE
by
PHILIP DAVID KISER
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Dr. Krzysztof Palczewski
Department of Pharmacology
CASE WESTERN RESERVE UNIVERSITY
May, 2010
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CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
______candidate for the ______degree *.
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DEDICATION
I dedicate this work to:
My family for their love and support throughout the years
and
My teachers for inspiration
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TABLE OF CONTENTS
TITLE PAGE...... i
SIGNATURE PAGE...... ii
DEDICATION ...... iii
TABLE OF CONTENTS ...... iv
LIST OF TABLES ...... viii
LIST OF FIGURES ...... ix
ACKNOWLEDGEMENTS...... xii
LIST OF ABBREVIATIONS ...... xiv
ABSTRACT ...... 1
CHAPTER 1: BACKGROUND ON RPE65 AND RETINOID ISOMERIZATION ... 3
1.1. Introduction to retinal structure and function ...... 4
1.1.1. Structure and function of the photoreceptor and pigment epithelium
layers of the retina...... 4
1.1.2. The retinoid (visual) cycle...... 5
1.2. Prior studies on RPE65 function and membrane binding...... 6
1.2.1. Functional characterization of RPE65 ...... 7
1.2.2. Biochemical studies on RPE65 membrane binding...... 10
1.3. Chemistry of Retinoid Isomerization ...... 14
1.3.1. Substrates and products...... 14
1.3.2. Thermodynamic and kinetic considerations...... 16
1.3.3. Mechanisms of retinoid isomerization...... 17
1.3.4. Substrate specificity...... 21
iv
1.3.5. The requirement of an iron cofactor...... 22
1.4. Experimental outline, goals and rationale ...... 24
Table...... 27
Figures...... 28
CHAPTER 2: EXPRESSION, PURIFICATION AND BIOCHEMICAL
CHARACTERIZATION OF RPE65...... 37
2.1. Expression and purification of Zebrafish RPE65...... 38
2.1.1. Rationale and Methods...... 38
2.1.2. Results and Discussion ...... 40
2.2. Expression and Purification of Human RPE65...... 41
2.2.1. Rationale and Methods...... 41
2.2.2. Results and Discussion ...... 43
2.3. Generation of an RPE65 monoclonal antibody and mapping of its epitope
...... 44
2.3.1. Methods...... 44
2.3.2. Results and discussion...... 47
2.4. Extraction and phase separation studies on native RPE65 ...... 48
2.4.1. Methods...... 48
2.4.2. Results and discussion...... 50
2.5. Purification of Native RPE65 from Bovine RPE ...... 52
2.5.1. Rationale and Methods...... 52
2.5.2. Results and discussion...... 55
Table...... 58
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Figures...... 59
CHAPTER 3: CRYSTALLIZATION AND STRUCTURE DETERMINATION OF
RPE65 ...... 77
3.1. Construction of an RPE65 homology model ...... 78
3.2. Attempted crystallization of recombinant RPE65 ...... 79
3.3. Crystallization of native bovine RPE65 ...... 79
3.4. Diffraction data collection and phasing ...... 80
3.5. Model building, refinement and analysis of the model ...... 86
Table...... 89
Figures...... 90
CHAPTER 4: DESCRIPTION AND INTERPRETATION OF THE RPE65
CRYSTAL STRUCTURE...... 98
4.1. A general description of the structure ...... 99
4.2. The membrane-binding surface ...... 100
4.3. The active site cavity and iron-binding site ...... 101
4.4. Dimeric structure found in the asymmetric unit and analysis of crystal
packing ...... 106
4.5. RPE65 amino acid substitutions associated with Leber congenital
amaurosis ...... 109
4.6. Comparison of the RPE65 and apocarotenoid oxygenase structures.... 110
Figures...... 114
CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS ...... 142
APPENDIX I: CLASSIFICATION OF MEMBRANE PROTEINS ...... 152
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BIBLIOGRAPHY...... 155
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LIST OF TABLES
Table 1: RPE65 substrate specificity ...... 27
Table 2: Detergents tested for extraction, purification and crystallization of bovine
RPE65 ...... 58
Table 3: Data collection, phasing and refinement statistics...... 89
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LIST OF FIGURES
Figure 1: Role of the RPE in maintaining photoreceptor health and function. .... 28
Figure 2: Schematic representation of the retinoid (visual) cycle...... 30
Figure 3: Potential mechanisms of RPE65 membrane association...... 32
Figure 4: Proposed catalytic mechanisms of retinoid isomerization...... 34
Figure 5: Expression of an MBP-His10-zRPE65a fusion protein in E. coli and its purification...... 59
Figure 6: Purification of recombinant human RPE65 from insect cells...... 61
Figure 7: Immunostaining of a mouse retinal section using the monoclonal
RPE65 antibody...... 63
Figure 8: Identification of the epitope-containing RPE65 segment recognized by a
RPE65 monoclonal antibody...... 65
Figure 9: SDS-PAGE analysis of bovine RPE microsomal proteins after
incubation with reagents known to solubilize certain classes of membrane
proteins...... 67
Figure 10: Behavior of RPE microsomal alkaline extracts after pH neutralization
or dialysis...... 69
Figure 11: Partitioning of RPE65 in Anapoe X-114 phase separation
experiments...... 71
Figure 12: Purification of native RPE65 from bovine RPE...... 73
Figure 13: Gel filtration analysis of purified RPE65...... 75
Figure 14: Photographs of typical RPE65 crystals...... 90
Figure 15: SDS-PAGE analysis of washed crystals...... 92
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Figure 16: A diffraction pattern recorded from an RPE65 crystal at the NSLS X29 beamline...... 94
Figure 17: An initial electron density map calculated with observed amplitudes and density-modified SAD phases from SHELXE...... 96
Figure 18: RPE65 structure and topology...... 114
Figure 19: The two tunnels that lead to the active site iron of RPE65...... 116
Figure 20: Predicted membrane-binding regions of RPE65...... 118
Figure 21: Locations of and electron density maps surrounding the cysteine residues previously proposed to participate in a palmitoylation switch mechanism...... 120
Figure 22: Stereoview of residues lining the RPE65 interior cavity...... 122
Figure 23: Stereoview of a possible water entry route to the RPE65 active site.
...... 124
Figure 24: Stereoviews of the RPE65 iron cofactor, surrounding residues, and residual electron density in the active site...... 126
Figure 25: Ligands found in the fifth and/or sixth coordination sites of all known 4-
His iron-binding proteins...... 128
Figure 26: The RPE65 dimer found in the asymmetric unit...... 130
Figure 27: Packing of RPE65 in the P65 unit cell...... 132
Figure 28: LCA- or RP-associated RPE65 amino acid substitutions...... 134
Figure 29: Locations and local interactions of residues commonly substituted in
RPE65-associated LCA or RP...... 136
Figure 30: Comparison of the RPE65 and ACO crystal structures...... 138
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Figure 31: Stereoview of the iron cofactor and its ligands and the putative substrates/products in the active sites of RPE65 and ACO...... 140
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ACKNOWLEDGEMENTS
First, I would like to thank my thesis advisor, Dr. Krzysztof Palczewski, for
giving me an interesting project to work on, for taking the time to frequently meet
with me to make sure I wasn't too far off the correct path and for being patient
with me during my growth as a scientist. He has helped make my experience in
graduate school everything I wanted it to be.
I would especially like to acknowledge two colleagues in the Palczewski
laboratory, Dr. David Lodowski and Dr. Marcin Golczak. When I joined the
Palczewski lab, David took me under his wing and taught me the basics of
protein crystallization and x-ray crystallography. It would have been much more
difficult to get started on my project without his help. Marcin helped me
understand the biochemistry of the retinoid cycle and helped me learn several biochemical techniques that proved to be essential for my project. I thank both of you for your time.
I would like to thank my colleagues in the Palczewski laboratory for all the
help they provided me during my time in the laboratory. Especially in the
beginning stages of my graduate studies, I greatly benefited from being in a laboratory with so many experienced postdoctoral scientists. I especially want to
thank Akiko Maeda for help producing monoclonal antibodies. Also, I would like
to thank our laboratory technicians, David Peck, Satsumi Roos, Susan Farr
Melissa Matosky for providing me with expertly dissected eyecups and other
excellent technical support.
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I owe special thanks to the members of my Ph.D. thesis committee, Drs.
Anthony Berdis, Michael Maguire, and Focco van den Akker for taking the time to meet with me and for providing valuable advice.
During my graduate studies I was supported by both the Molecular
Therapeutics Training Program (MTTP) and Visual Sciences Training Program
(VSTP) grants. I would like to thank the committees and the principle investigators of these training grants, Dr. Paul MacDonald and Dr. Susann
Brady-Kalnay, respectively, for continuous financial support.
I owe special thanks to Dr. Wuxian Shi at Brookhaven National Laboratory for assistance with data collection at the X29 beamline at the National
Synchrotron Light Source.
I want to thank my Pop and Mom, David and Gail Kiser, and my brother
Eric for your unconditional love and support throughout the years.
Finally, I want to thank my wife Jianying for her constant unconditional love and support, and for inspiring me to do better every day.
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LIST OF ABBREVIATIONS
Å - angstrom
ACO - apocarotenoid oxygenase
APS - Advanced Photon Source
ATP - adenosine triphosphate
BMH - bis-maleimidohexane
BTP - bis-tris propane or 1,3-bis(tris(hydroxymethyl)methylamino)propane
CAPS - N-cyclohexyl-3-aminopropanesulfonate
CCD - charge-coupled device
CCO - carotenoid cleavage oxygenase cDNA - complementary DNA
CHAPS - 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate
C8E4 - octyltetraoxyethylene
DAPI - 4',6-diamidino-2-phenylindole
DEAE - diethylaminoethyl
DNase I - deoxyribonuclease I
DTT - dithiothreitol
EDTA - ethylenediaminetetraacetic acid
ELISA - enzyme-linked immunosorbent assay
EMTS - ethyl mercuric thiosalicylate
ER - endoplasmic reticulum
FOM - figure of merit
GPI - glycosyl phosphatidylinositol
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HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonate
IPTG - isopropyl-β-D-thiogalactopyranoside keV - kiloelectron volt
LB - Luria-Bertani
LCA - Leber congenital amaurosis
LRAT - lecithin:retinol acyltransferase
MAD - multi-wavelength anomalous dispersion
MALDI - matrix-assisted laser desorption/ionization
MBP - maltose-binding protein
MES - 2-(N-morpholino)ethanesulfonate
MOPS - 3-(N-morpholino)propanesulfonate mS - millisiemens
NCS - non-crystallographic symmetry
Ni2+-NTA - nickel-nitrilotriacetic acid
NSLS - National Synchrotron Light Source
OD - optical density
PBS - phosphate-buffered saline
PCR - polymerase chain reaction
PDB - Protein Data Bank
PEG - polyethylene glycol
PLA2 - phospholipase A2
PMSF - phenylmethylsulfonyl fluoride
RBP - retinol-binding protein
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RDH - retinol dehydrogenase
RDH5 - 11-cis-retinol dehydrogenase
Rho - rhodopsin
RMSD - root mean square deviation
RP - retinitis pigmentosa
RPE - retinal pigment epithelium
RPE65 - retinal pigment epithelium-specific 65 kDa protein
SAD - single-wavelength anomalous dispersion
SDS-PAGE - sodium dodecylsulfate-polyacrylamide gel electrophoresis sER - smooth endoplasmic reticulum
SN1 - unimolecular nucleophilic substitution
SN2 - bimolecular nucleophilic substitution
TEV - tobacco etch virus
TLS - translation libration screw
TPCK - tosyl phenylalanyl chloromethyl ketone
Tris - tris(hydroxymethyl)aminomethane
VP14 - viviparous 14
XAS - x-ray absorption spectroscopy zRPE65a - zebrafish RPE65 isoform a
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Structural and Biochemical Studies of RPE65, the Retinoid Isomerase of the
Visual Cycle
ABSTRACT
by
PHILIP DAVID KISER
Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans- retinyl esters to 11-cis-retinol in the retinal pigment epithelium. Mutations in
RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis. Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Additionally, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To obtain structural insights into these questions the crystal structure of RPE65 from bovine retinal pigment epithelium was determined. The structure reveals the architecture of the enzymatic active site and a hydrophobic patch that likely mediates membrane binding. Extraction and phase separation experiments indicate a direct interaction between hydrophobic side chains of RPE65 and the acyl core of the lipid bilayer consistent with crystal structure. The structure also provides insights
1 into the mechanisms by which Leber congenital amaurosis-associated RPE65 amino acid substitutions lead to loss of function. To facilitate characterization of
RPE65 a monoclonal antibody was generated and its epitope was mapped within the RPE65 sequence. The antibody specifically stains retinal pigment epithelium cells in retinal sections and is useful for immunoaffinity purification of RPE65 and
RPE65-containing complexes. The studies described here provide key insights into the biology and biochemistry of this important visual cycle enzyme.
2
CHAPTER 1: BACKGROUND ON RPE65 AND RETINOID ISOMERIZATION
Portions of this Chapter were previously published in:
Kiser, P.D., Palczewski, K. (2010) Membrane-binding and enzymatic properties of RPE65, Prog Retin Eye Res Mar 18. [Epub ahead of print]
3
1.1. Introduction to retinal structure and function
1.1.1. Structure and function of the photoreceptor and pigment epithelium layers of the retina
Vision is a physiological process involved in nearly every aspect of human life. The light-sensing tissue in humans is the retina, which is located in the posterior portion of the eye. The main light-sensitive cells of the retina, called photoreceptor cells, consist of two different types; rod cells for vision under low- illumination conditions and cone cells for color vision in well-illuminated environments (1). Visual pigments are the light-sensitive molecules found in photoreceptor cells. Visual pigments consist of a protein moiety called opsin and a vitamin A-derived chromophore called 11-cis-retinal that is covalently bound to a Lys side chain amino group of the opsin via a protonated Schiff base linkage
(2). The covalently bound retinoid is called 11-cis-retinylidene. Visual pigments are seven-pass transmembrane proteins that are tightly packed together in pancake-like membrane structures known as disks, which are located in the outer segments of photoreceptor cells (2). Adjacent to the photoreceptor outer segments is a monolayer of epithelial cells known as the retinal pigment epithelium (RPE). The RPE transports nutrients to and removes waste products from the photoreceptors, absorbs excess light through melanin granules, phagocytoses fragments of shed outer segments and recycles chromophore for visual pigments (Fig. 1). In addition, all-trans-retinol is bi-directionally transferred between the systemic circulation and the RPE via a membrane transporter found
4 on the basolateral plasma membrane of RPE cells called STRA6 (3-4).
Therefore, this cell layer is vital for the health of the retina.
1.1.2. The retinoid (visual) cycle
Light sensation by photoreceptor cells in the retina begins with a photochemical event in which absorption of a single photon causes the geometrical photoisomerization of the pigment 11-cis-retinylidene chromophore to an all-trans configuration (2). This conversion activates the visual pigment allowing it to trigger a cascade of downstream signaling events that lead to the transmission of electrical signals to the visual cortex and the perception of light by the brain. After a brief period of time, signaling by the pigment is terminated by phosphorylation and subsequent binding of a silencing protein known as arrestin
(5). Following a light stimulus, the pigment is no longer capable of being photoactivated. Thus, a mechanism for the regeneration of light-sensitive pigments, that is the isomerization of all-trans-retinylidene back to the 11-cis configuration, is essential for continuity of vision (6) (Fig. 2). At least in vertebrates, the chromophore is not directly reisomerized while it is bound to opsin. Instead, the labile Schiff base linkage between the chromophore and opsin is hydrolyzed and free all-trans-retinal is released in a process known as bleaching (7). all-trans-Retinal is subsequently reduced in the photoreceptor cells by NAD(P)H-dependent retinol dehydrogenases (RDHs) to yield all-trans-retinol
(vitamin A). The vitamin A is transferred to the RPE where it is esterified by lecithin:retinol acyltransferase (LRAT) (8)(summarized in (9)). It is these retinyl
5
esters that serve as substrates for retinoid isomerization, which occurs in a
complex enzymatic reaction involving simultaneous hydrolysis of the ester
moiety. The retinoid product of this reaction, 11-cis-retinol, is then oxidized to 11-
cis-retinal and transported back to the photoreceptors where it condenses with
opsin to reform a light-sensitive pigment (summarized in (9)). This multistep
process of converting all-trans-retinal to 11-cis-retinal is known as the retinoid or
visual cycle. A 61 kDa RPE-specific protein called RPE65, because its apparent
molecular mass based on SDS-PAGE analysis is 65 kDa, is the enzyme
responsible for the light-independent conversion of all-trans-retinyl esters,
primarily palmitoyl esters, into 11-cis-retinol. Although the need for light-
independent isomerization activity in the regenerative visual cycle was
recognized early, the identification of RPE65 as the responsible enzyme was not straightforward and occurred only recently (10-12). RPE65 is involved not only
for regeneration of rhodopsin but also plays an important role in regeneration of
cone opsins and is critical for the health of cone photoreceptors (13). In contrast
to the RPE65-dependent chromophore regeneration pathway for rhodopsin,
there is substantial evidence that chromophore for cone opsins is at least
partially regenerated through an alternative metabolic pathway involving
enzymes located in cone photoreceptor and Müller cells (14-17).
1.2. Prior studies on RPE65 function and membrane binding
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1.2.1. Functional characterization of RPE65
The first description of RPE65 was dated in 1991 when a monoclonal
antibody obtained from mice exposed to chicken RPE cell antigens was shown to
recognize an RPE-specific protein with an apparent molecular mass of 63 kDa
(18). Immunocytochemical analysis revealed that the antigen was localized almost exclusively to the smooth endoplasmic reticulum (sER). The protein was not only found in chicken RPE but also in the RPE of a variety of different animals as well suggesting an important and highly conserved function. Although the amino acid sequence of the protein was not determined, its near-65 kDa apparent molecular mass and localization to the sER strongly suggest RPE65 was the protein identified in this study as these are now well-recognized features of this protein. RPE65 is most highly expressed in the RPE but its mRNA and
protein have also been reported by one group in cone photoreceptors of some amphibians and mammals (19-20).
RPE65 was initially thought to be a receptor for serum retinol-binding
protein (RBP), and in these studies it was referred to as p63 (21-23). However,
the localization of RPE65 to the sER rather than the basolateral plasma membrane, where the RBP receptors were known to reside (24-25), argued
against a physiological role for RPE65 as an RBP receptor. Furthermore, the
deduced amino acid sequence of RPE65, revealed by the cloning and
sequencing of RPE65 complementary DNA (cDNA), did not show potential
alpha-helical transmembrane segments, which would likely be present in a small-
7
molecule transporter (23, 26). Therefore, the RBP receptor hypothesis of RPE65 function fell out of favor.
The importance of RPE65 for proper human visual function was recognized in 1997 when patients afflicted with a severe retinopathy termed
Leber congenital amaurosis (LCA) were discovered by two groups, both utilizing a candidate gene approach, to have mutations in the RPE65 gene (27-28). LCA is a heterogeneous group of autosomal recessive severe retinopathies that often lead to blindness within the first year of life. The disease is relatively rare with an estimated population frequency of 1 in 81,000 (29). Mutations in the RPE65 gene have been estimated to account for ~ 6% of LCA cases (29). The involvement of
RPE65 in this extremely severe human disease is what led to a major interest in its structure and function.
In 1998, RPE65-null mice were shown to develop early-onset blindness and displayed a metabolic dysfunction characterized by near complete absence of ocular 11-cis retinoids together with substantial accumulation of all-trans- retinoids in the RPE (30). Together with the human disease association, these observations strongly suggested that RPE65 may play an important role in the metabolic processing of retinoids. In 1997, an ortholog of RPE65 found in plants called viviparous 14 (VP14) was shown to oxidatively cleave carotenoids (31).
The overall sequence homology and absolute conservation of four putative iron- binding histidine residues between the two proteins strongly suggested that
RPE65 too could possess enzymatic activity (32).
8
However, some observations and properties of RPE65 were thought not to
be consistent with this hypothesis. It was reported in 1998 that near-complete
removal of RPE65 from bovine RPE microsomes by high salt extraction did not
substantially impair retinoid isomerization activity of the microsomal fraction (33).
Attempts to demonstrate retinoid isomerization activity of purified RPE65 were
unsuccessful (34). Indeed, solubilization of the RPE microsomal proteins with
most detergents resulted in nearly complete loss of retinoid isomerization activity
(35-36). Furthermore, based on the sheer abundance of RPE65 in the RPE cell it
was suggested that the protein might act stoichiometrically, for example as
retinoid-binding protein, rather than enzymatically (37). Up until 2005, it was
generally thought that RPE65 was a retinyl ester-binding protein or a retinoid
chaperone that extracted and presented insoluble retinyl esters to a hypothetical
enzyme called isomerohydrolase (34, 37). The perceived action of RPE65 was
made more elaborate by the presentation of the "palmitoylation switch
mechanism" hypothesis which proposed a role for light-dependent, reversible
palmitoylation of specific Cys residues in the control of RPE65 membrane
binding and retinoid-binding specificity (38).
Strong evidence against a pure retinoid-binding function for RPE65 was
provided in 2005 when three groups independently demonstrated that
coexpression of RPE65 and LRAT in various mammalian cell lines as well as
insect cells conferred retinoid isomerization activity (10-12). RPE65 was
proposed to be the actual isomerase whereas LRAT was necessary to provide
retinyl ester substrates in situ. Despite the major progress made by these
9
studies, there still existed some lingering doubt regarding the identity of RPE65
as the isomerase in the assays since whole cells or cell lysates were used rather
than purified proteins (39). This doubt was, to a large extent, removed by the demonstration that purified RPE65, when reconstituted with retinyl ester- containing phospholipid vesicles, possesses retinoid isomerase activity (40), although the observed activity of the reconstituted protein was far below what is physiologically required (41). The study suggested that RPE65 interactions with an intact phospholipid bilayer are critically important for its activity.
1.2.2. Biochemical studies on RPE65 membrane binding
The localization of RPE65 to the sER was recognized during its initial characterization (18, 22, 42). The sER is an unusually abundant organelle in
RPE cells where it occupies a large fraction of the cytoplasmic space (43). An abundance of sER is typically found in cells that are involved in metabolism of lipophilic compounds, consistent with the retinoid processing function of the RPE
(44). As expected from its subcellular location, RPE65 cosediments with microsomal membranes during centrifugation, and this fact can be exploited for its purification (42).
A fairly large number of studies have investigated the nature of RPE65- membrane interactions, and at least four different proposals have been made
(Fig. 3; see Appendix A for a brief review of the different classes of membrane proteins). Initial studies indicated that RPE65 has many characteristics that are consistent with its classification as an integral membrane protein. Detergents,
10
especially Triton X-100 and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane
sulfonate (CHAPS), were found to be the most effective agents for extracting
RPE65 from microsomal membranes (21, 42). Additionally, Triton X-114-
solubilized RPE65 partitioned almost exclusively into the detergent-rich fraction
after temperature-induced phase separation, the behavior usually observed for
integral membrane proteins (21, 42, 45). However, other experiments indicated a
peripheral membrane association. It was noted that treatment of RPE
microsomes with alkaline carbonate solutions resulted in extraction of a
significant amount of RPE65 (21, 46). The alkaline pH may disrupt membrane-
protein or protein-protein salt bridges by deprotonation of titratable amino groups.
It has also been reported that exposure of RPE microsomes to neutral pH
solutions containing 0.75 M to 1 M KCl results in the release of significant
amounts of RPE65 (26, 33), although we have been unable to observe this effect
in our laboratory and routinely wash RPE microsomal membranes with 1 M KCl
prior to detergent extraction of RPE65 without appreciable loss of membrane-
bound RPE65 or retinoid isomerization activity (47). These observations together
with the lack of an N-terminal signal peptide and putative transmembrane
segments were taken as evidence of a peripheral membrane association for
RPE65 (26, 42, 48). However, there are some important points to be made here
that argue against this interpretation. First, it has never been shown that the
RPE65 released under the aforementioned conditions can be purified while preserving the native structure of the protein. Second, it has never been demonstrated that RPE65 remains soluble after removal of these extracting
11
agents as would be expected for a peripheral membrane protein. Additionally, in
the initial study reporting the KCl extraction phenomenon it was demonstrated
that the salt-extracted RPE65 partitions into the detergent-rich phase during
Triton X-114 phase separation just as the detergent-extracted protein does indicating that the salt-extracted protein is not a distinct "soluble" form (42).
Fatty acid acylation, particularly palmitoylation was, for several years, thought to confer membrane affinity to RPE65 (38). Palmitoylation-dependent membrane association is an attractive hypothesis for a protein like RPE65, which apparently lacks an intrinsic endoplasmic reticulum (ER) localization signal
sequence, as palmitoylation is known to influence membrane localization of
intracellular proteins (49). Initial data supporting this hypothesis came from
matrix-assisted laser desorption/ionization (MALDI) mass spectrometric
measurements of RPE65 (48). These measurements indicated the mass of native bovine RPE65 purified from membranes was significantly higher than the
calculated value for the unmodified polypeptide chain whereas cytosolic RPE65
had a mass only slightly above the theoretical value. It was also estimated that
the ratio of cytosolic to microsomal RPE65 in native bovine RPE is approximately
1:2, indicating the presence of a potentially distinct soluble form of the protein. In
2004, it was reported that RPE65 is reversibly palmitoylated by LRAT on three
specific residues, Cys 231, Cys 329 and Cys 330 (38). This palmitoylation was proposed to control RPE65 membrane affinity as well as alter retinoid-binding specificity. As intriguing as this hypothesis was, subsequent studies indicated that it was probably incorrect. The membrane localization of RPE65 in LRAT-/-
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mice compared to wild type mice was not substantially different (50). Mutation of all three Cys residues to Ala or Ser did not substantially affect RPE65 membrane binding (50-51). Mass spectrometric analyses of native, membrane-bound
RPE65 that were of higher quality than those originally performed revealed no palmitoylation of Cys residues 231, 329 and 330 (46, 50). A recently described palmitoylation site at Cys 112 was reported to be critical for membrane targeting based on the observation that Ala substitutions at that position led to the protein being expressed in inclusion bodies (52). A subsequent study has called into question the presence of palmitoylation on Cys 112 and suggests that RPE65 does not undergo any significant post-translational modifications (46). Thus, despite a number of biochemical studies, no clear consensus has emerged regarding RPE65 palmitoylation. However, these studies indicate that RPE65 is not palmitoylated on Cys 231, 329 and 330.
Another potential mechanism for RPE65 binding to membranes is through interactions with other membrane-bound proteins. Initial studies indicated that
RPE65 is part of a large molecular weight complex that possibly includes other
RPE microsomal proteins (21). Copurification of RDH5, a known integral membrane protein, with RPE65 or its fragments has been observed in several
studies (53-56) and the reported crosslinking between RPE65 and RDH5 provides evidence that the RDH5 catalytic domain is oriented towards the cytosolic space rather than the ER lumen (56). Protein-protein interactions may help direct RPE65 to the ER and assist in its membrane anchoring. However, the observations that purified RPE65 can bind directly to liposomes (40, 57) and that
13
it can extract retinyl esters directly from membranes (34) indicate that it has inherent membrane affinity. Furthermore, enzymatic activity of purified RPE65 can be restored upon reconstitution with retinyl ester-containing liposomes demonstrating that RPE65 is not dependent on another protein for membrane association and substrate extraction (40). In accord with this notion, PLA2 treatment of native RPE microsomal membranes, which loosens the membrane structure through removal of a fatty acid from the sn2 position of phospholipids, significantly impairs retinoid isomerization activity and disrupts the association of
RPE65 with other RPE microsomal proteins but does not result in dissociation of
RPE65 from the membrane (56). This observation suggests that RPE65 interacts directly with the membrane in a highly specific manner and that this interaction is critical for its enzymatic activity. Disruption of the membrane structure could affect the orientation of the retinoid substrate in the membrane such that it cannot be effectively extracted by RPE65. Substrate recognition elements of RPE65 may also be destabilized when the membrane structure is altered.
1.3. Chemistry of Retinoid Isomerization
1.3.1. Substrates and products
Originally, an enzymatic activity that produces 11-cis-retinol directly from all-trans-retinol or 11-cis-retinyl esters from all-trans-retinyl esters was thought to be responsible for light-independent chromophore regeneration in the eye (58).
The bulk of current data now suggest that it is all-trans-retinyl esters that are
14 directly converted to 11-cis-retinol by RPE65. Identification of the substrate was not trivial though owing to the presence of several other retinoid-modifying reactions, such as those catalyzed by acyl transferases and hydrolases, which occur simultaneously with retinoid isomerization in RPE microsomes and can complicate data analysis.
The proposal that retinyl esters rather than retinol were the substrates for the isomerization reaction was made primary on theoretical grounds. It was hypothesized that the energy released by ester hydrolysis could drive the thermodynamically unfavorable trans to cis isomerization (discussed below) (59).
Some experimental support for this hypothesis came from the observation that
RPE65-/- mice accumulated large stores of retinyl esters in the RPE, suggesting the metabolic blockade in these mice is at the retinyl ester-processing step (30).
Also, experiments that employed specific LRAT inhibitors, all-trans-retinyl bromoacetate or dodecyl chloromethyl ketone, demonstrated that 11-cis-retinol generation from all-trans-retinol only occurred when retinyl esters were allowed to form (60-61). More recently it was demonstrated in an chemically defined system that retinyl esters are converted to 11-cis-retinol in the presence of
RPE65 (40).
However, pulse-chase experiments performed by Stecher and colleagues using radiolabeled all-trans-retinol demonstrated that at a given time most of the retinyl esters in the RPE were not able to be enzymatically isomerized (62). It was speculated that only a small pool of the retinyl esters in the ER membranes were accessible to the isomerase. The observations in this study were nicely
15
explained by the discovery of dynamic retinyl ester-containing lipid bodies,
termed retinosomes, in the RPE through the use of two-photon microscopy (63).
1.3.2. Thermodynamic and kinetic considerations
As mentioned above, the reaction catalyzed by RPE65 is complex,
consisting of a geometrical isomerization of a trans double bond coupled with an
atypical ester hydrolysis. Trans-to-cis isomerization is energetically unfavorable
both thermodynamically and kinetically. It is well-known that cis double bonds are
generally higher in free energy than their trans counterparts except in certain
compounds such as some cycloalkenes. In the retinol molecule the difference in
free energy between the all-trans and 11-cis isomers is ~ 4 kcal/mol (64). It was
proposed that the energy released by ester hydrolysis (~5 kcal/mol) was
sufficient to drive the endergonic isomerization (59). However, the fact that 11-
cis-retinoid binding proteins are required for robust production of 11-cis-retinol in vitro indicates that product release may be the slow step of the isomerization reaction and that the reaction is actually driven by mass action (62, 65).
The retinyl moiety of an all-trans-retinyl ester is rigid owing to its conjugated π bond system. Therefore, geometrical isomerization of the ground- state molecule is kinetically highly unfavorable, requiring an activation energy of
~36 kcal/mol (65). For the isomerization reaction to occur at physiological temperatures, the activation energy for the process must be reduced, and this is accomplished by temporarily reducing the carbon-carbon bond order of the
16
system. The bond order, defined by equation (1), is approximately equal to 1.5
for the carbon-carbon bonds in the polyene chain of ground state retinol (66).
equation (1): bond order = (number of electrons in bonding orbitals - number of
electrons in antibonding orbitals)/2
Thus, this equation suggests two mechanisms by which double bond
isomerization chemistry can occur: 1) temporary electron removal from bonding p
orbitals, or 2) generation of transient antibonding orbitals, e.g. via photochemical
processes. As the visual cycle isomerization reaction is known to occur in the
absence of light it probably involves the first mechanism.
1.3.3. Mechanisms of retinoid isomerization
Interestingly, many important characteristics of the isomerase enzymatic
reaction mechanism were discovered long before RPE65 was identified as the
visual cycle isomerase and before the discovery that all-trans-retinyl esters (40,
60-61) rather than all-trans-retinol were its substrates. Identification of RPE65 as the visual cycle isomerase combined with recent structural data gathered on it
(47) and a related protein, apocarotenoid oxygenase (ACO) (67) have revived interest in the retinoid isomerization mechanism. Initial evidence for a coupled isomerase/hydrolase reaction was derived from experiments showing that both activities were always found together during fractionation (35). Isotope labeling studies established that a retinoid in the alcohol oxidation state (retinol or a
17
retinyl ester) is the species that is enzymatically isomerized rather than retinal
(68). An intriguing early observation was that the stereochemical configuration of
retinoid carbon 15 inverts during the isomerization reaction, indicating breakage of the carbon 15-oxygen single bond at some point during the isomerization reaction (69) (see Fig. 4A for standard retinoid numbering). Further isotope labeling studies demonstrated that the oxygen present in the all-trans-retinoid substrate is replaced by a different oxygen atom in the 11-cis product (65), an
oxygen atom later found to be derived from water (47).
Two different mechanisms of retinoid isomerization have been proposed.
The first is a dual bimolecular nucleophilic substitution (SN2) mechanism (Fig.
4B). In this reaction an enzyme active site nucleophile, presumably a cysteine sulfur atom, attacks the retinyl C11 atom with simultaneous dissociation of a leaving group from C15 (69). This results in an intermediate that can undergo low-energy rotation about C11-C12. In the second part of this reaction, a nucleophile (presumably hydroxide) attacks C15 resulting in the expulsion of the
C11-bound nucleophile and restoration of the conjugated π bond system, now in the 11-cis configuration. Based on this mechanism, the isomerase predictably would be highly specific with respect to isomerization of the 11-12 double bond.
However, this high specificity is not experimentally observed as discussed below.
There are also some chemical considerations that make this mechanism improbable as discussed previously (65). The major difficulty is that the first step involves a nucleophile attacking an already electron-rich atom, i.e. C11. SN2-type
nucleophilic substitutions involving allylic carbons are known to be chemically
18
unfavorable (70). Furthermore, this reaction would lead to formation of a highly
stable carbon-heteroatom bond, most likely a thioether or ether group, which
would be difficult to break in the subsequent step.
The second mechanism involves a unimolecular nucleophilic substitution
(SN1) (65) (Fig. 4C). Here, the leaving group dissociates first via alkyl-oxygen
cleavage to generate a resonance-stabilized carbocation intermediate. Formation
of a carbocation intermediate is known to be the slow step of an SN1 reaction and
a good leaving group is required. Although an ester can leave as a relatively
stable, resonance-stabilized acyloxy anion, this reaction could be promoted by
the interaction of the ester acyl oxygen with a Lewis acid (a proton or metal ion)
(65). The retinyl carbocation intermediate has a reduced polyene carbon-carbon
bond order which results in a substantial decrease in the activation energy
needed for trans to cis isomerization from ~ 36 to ~18 kcal/mol (65). The latter
value is consistent with the experimentally determined isomerization activation
energy of 17 kcal/mol derived from Arrhenius plots (65). Because the carbocation
is delocalized, it could be predicted that an isomerase which operates via this mechanism may not be 11-cis specific. Indeed, both 11-cis and 13-cis-retinol are produced by RPE65 and the isomer that accumulates depends on which retinoid- binding proteins are available for product sequestration (65, 71). After isomerization, attack by a nucleophile on C15 would quench the carbocation and lock the molecule in the cis configuration. In an SN1 mechanism, the strength of
an attacking nucleophile is less important compared to ease of leaving group
dissociation (70). Thus, a water molecule, which would subsequently be
19
deprotonated, rather than hydroxide could very well be the nucleophile in this
SN1 reaction. On the basis of this proposed mechanism, a putative carbocation transition state analog, all-trans-retinylamine, was developed. This compound was found to be a highly effective blocker of 11-cis-retinol production both in vitro
and in vivo (72-73) and was later shown to bind directly to RPE65 (74). The first
step of this mechanism, the alkyl-oxygen cleavage, deserves a bit more
discussion. In virtually all biochemical ester hydrolysis reactions, it is thought that
the reaction proceeds through a tetrahedral intermediate with the oxygen atom of
water (or hydroxide) attacking the carbonyl carbon (75). But it is also known from
non-biological organic chemistry that cleavage is favored at the alkyl rather than
the acyl carbon in SN1 reactions when the carbocation intermediate can be stabilized by hyperconjugation or by resonance (70). The retinyl ester molecule
presents such a situation wherein a generated carbocation is stabilized by
extensive resonance delocalization. For general SN1 reactions performed in
solution, it is often the case that the chirality of the electrophilic center is
racemized; thus this consideration could argue against an SN1 mechanism for
retinoid isomerization because it has been observed that a high percentage of
the C15 atoms in the retinoid products undergo inversion of configuration (69,
76). However, because the reaction occurs in the active site of RPE65, it is easy
to imagine that the enzyme only allows water to approach C15 from a specific
direction, which would result in the formation of only one enantiomer.
20
1.3.4. Substrate specificity
A number to studies have examined the structural features of the retinyl
ester molecule that are required for enzymatic isomerization (Table 1). As the
currently proposed mechanisms of RPE65-mediated retinoid isomerization both
minimally require the presence of the 13-14 double bond, it was of interest to
determine if compounds that were saturated at this position as well as the other
positions along the polyene chain could be enzymatically isomerized.
Interestingly, saturation of any double bond of the polyene chain partially blocks
RPE65-dependent isomerization indicating a possible role of the entire
conjugated double bond system in the reaction (77). This is a surprising
requirement for the SN2 mechanism because the concerted reaction appears to involve only carbon atoms 11 through 15. However, the SN1 mechanism relies on
extensive resonance stabilization of a carbocation; thus, the requirement of a
fully conjugated double bond system would be expected. Interestingly, the more
extensive conjugated double bond system in vitamin A2 (all-trans-3,4-
dehydroretinol) does not inhibit or promote its isomerization.
The requirements for the C18, C19 and C20 methyl groups have also been investigated. An initial report indicated that the presence of C18 and C20 methyl groups were not required for isomerization, whereas all-trans-9-des- methylretinol could not be isomerized (78). A subsequent study on 9 and 13- desmethyl-all-trans-retinol showed that these compounds could be isomerized albeit less effectively than all-trans-retinol (79). These studies demonstrated that
21
a mechanism of retinoid isomerization involving proton abstraction from the C18,
C19 and C20 methyl groups was highly unlikely.
Fluorinated retinoids have also been prepared and tested for the ability to
be isomerized. All-trans-19,19,19-trifluororetinol was found not to be
enzymatically isomerized (78). Similarly, all-trans-11-fluororetinol did not undergo enzymatic isomerization (65). Based on these results, it was hypothesized that the electron-withdrawing character of the fluoro group could make the formation of a carbocation intermediate more difficult. Thus, these experiments are consistent with the SN1 nucleophilic substitution mechanism.
Although it is often stated that retinyl esters are the substrates for RPE65,
not all retinyl esters are capable of being isomerized. In vivo the most abundant
retinyl ester present is retinyl palmitate because a palmitoyl group is most often
found in the sn1 position of lecithin, which is used by LRAT as an acyl donor. It
has been observed that short esters such as retinyl acetate and retinyl
heptanoate are unable to be processed by RPE65. The specificity for long chain
fatty acid esters has not yet been explained. However, shorter esters may orient incorrectly in the membrane for uptake into the active site of RPE65 or they may not bind properly in the active site since a long saturated hydrophobic chain could greatly contribute to the binding affinity but is missing in these compounds.
It is highly unlikely that the fatty acid hydrocarbon chain directly participates in
the isomerization reaction though.
1.3.5. The requirement of an iron cofactor
22
Another interesting property of RPE65-dependent isomerization is its
requirement for iron, specifically divalent iron (80). The iron requirement of this
enzyme was anticipated based on its evolutionary relationship to lignostilbene
and carotenoid cleavage oxygenases (CCO) (12, 31-32). All members of this
family, including RPE65, possess a set of four absolutely conserved histidine
residues that directly bind iron. The requirement of iron for RPE65 activity has
been well documented by mutating key iron-binding residues (12, 81) and by chelation experiments (47, 80). Additionally, metal add-back experiments demonstrated that Fe3+, Cu2+, Mg2+ and Zn2+ were unable to restore RPE65
isomerase activity whereas addition of Fe2+ resulted in partial restoration (80).
For members of the CCO family other than RPE65, iron is thought to activate
oxygen for cleavage of carotenoids or lignostilbenes (82-83). However, RPE65 is
not known to possess carotenoid-cleavage or other oxygenase activities, so the
iron cofactor in this enzyme could be used for some other purpose. One major
unresolved mystery of RPE65 enzymology is the requirement of the reduced
form of a redox-active metal cofactor for the non-redox isomerization reaction. As
mentioned above, the rate-limiting, alkyl-oxygen cleavage step of the SN1-type mechanism of retinoid isomerization is facilitated by interaction of the carbonyl oxygen of a retinyl ester with an acid (70). It is possible that the iron atom either
directly interacts with the carbonyl oxygen (47) or binds a water molecule that
can subsequently donate a proton to the carbonyl oxygen (71-72).
23
1.4. Experimental outline, goals and rationale
From the discussions above, it is clear that while great progress has been made in understanding RPE65 function, there is still controversy regarding its precise enzymatic mechanism and mode of membrane binding. Lack of structural information is one major reason why these issues have been difficult to resolve.
We hypothesized that by determining the three-dimensional structure of RPE65, critical insights into these unresolved issues could be obtained.
The 61 kDa molecular mass of RPE65 makes x-ray crystallography the most appropriate technique for obtaining high-resolution structural information.
The study of protein structure by x-ray crystallography is a multi-step process. A protein sample that is as homogeneous (i.e. pure and monodisperse) as possible must first be obtained. It should be noted that absolute homogeneity is not always required for successful crystal growth. The sample is then used for crystallization trials. In these trials, the protein sample is mixed with a buffered solution that contains, among other things, a compound that will reduce the solubility of the protein such that precipitation or crystal growth is favored. Protein crystal growth is far from being a science and one cannot predict a priori the conditions under which the protein of interest will crystallize. Furthermore, unless a crystal is observed in a trial, the results of each crystallization experiment provide little guidance on which set of conditions should be tested next.
Therefore, one may have to screen many conditions before crystals are obtained and this may require relatively large amounts of the protein of interest, generally on the milligram scale. Owing to the stringent requirements of high quality protein
24 for crystallization, a sizeable portion of my effort was spent expressing and purifying RPE65 from various sources. Indeed, obtaining high quality protein that would yield diffraction-quality crystals was rate-limiting step for this particular project. To gain detailed information about the three-dimensional structure of
RPE65, we required crystals that diffract x-rays to less than 3 angstrom (Å) resolution so that amino acid side chains would be clearly visible in the electron density maps. One cannot calculate the correct electron density map of the protein with only the measured diffraction intensities because the phase value for each reflection is not directly measured in the diffraction experiment. This missing phase information must be obtained by various methods such as molecular replacement, isomorphous replacement and/or anomalous scattering.
The phasing methods that were utilized in this study are described in more detail later.
Additionally, the nature of RPE65-membrane interactions has been critically reevaluated using biochemical methods in order to explain the crystallographic data. As mentioned above, there is considerable controversy surrounding the mechanism by which RPE65 binds to microsomal membranes.
RPE65-membrane binding is likely to be important for the function of this enzyme so a clear understanding of these interactions is important. To gain insights into the nature of RPE65-membrane interactions, we have performed a variety of extraction and phase separation experiments that can help differentiate between various modes of membrane association.
25
To facilitate the characterization of RPE65, a significant amount of time
was devoted towards the development of an RPE65 monoclonal antibody. While
there are RPE65 antibodies that are commercially-available, these antibodies are ineffective for use in immunohistochemistry and probably other applications that require recognition of native protein. Additionally, some studies required large amounts of an RPE65 antibody for purification of RPE65 or RPE65-containing
complexes. Thus, a novel monoclonal antibody was developed that is useful for
all of these purposes, and its epitope was mapped within the RPE65 sequence.
26
Table
Table 1: RPE65 substrate specificity
27
Figures
Figure 1: Role of the RPE in maintaining photoreceptor health and function.
The photoreceptor outer segments and RPE cell layers are adjacent to one another in the retina. Each RPE has microvillar extensions that surround several outer segments the number of which depends on the exact location in the retina
(84). A number of proteins that are critical for regeneration of 11-cis-retinal and storage of retinyl esters in retinosomes (red circle) are located in these cells. The
RPE supports the photoreceptor by providing nutrients (glucose, vitamin A) and oxygen and removing waste products such as chloride and carbon dioxide. The
RPE also phagocytoses spent photoreceptor outer segments. The cytoplasm of the RPE contains large numbers of melanin granules (brown oval) that absorb excess light.
28
Figure 1
29
Figure 2: Schematic representation of the retinoid (visual) cycle.
Vision begins when light (hv) causes photoisomerization of the 11-cis- retinylidene chromophore of ground-state rhodopsin (Rho). Subsequently, the
Schiff base linkage loses a proton enabling Rho to activate G proteins (i). After remaining active for a short period of time, the isomerized chromophore is released via hydrolysis, generating free all-trans-retinal and opsin (ii). The all- trans-retinal is enzymatically reduced (iii) and the resultant all-trans-retinol is exported from the rod outer segment to the RPE. Here all-trans-retinol is metabolized by LRAT to produce all-trans-retinyl esters (iv), which can be either stored in retinosomes or further processed. RPE65 is the key enzyme that catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol (v). 11-cis-
Retinol is enzymatically reduced to 11-cis-retinal (vi), which is then transported
back to the photoreceptor outer segment where it recombines with opsin to form ground-state rhodopsin (vii). Continuous operation of this cycle is what sustains vision under conditions where rods are primarily active.
30
Figure 2
31
Figure 3: Potential mechanisms of RPE65 membrane association.
Four potential modes of RPE65-membrane interactions have been proposed
based on extraction experiments and other biochemical and biophysical studies.
A) Anchoring via electrostatic interactions between RPE65 side chains and the charged headgroups of phospholipids. Blue circles indicate positively charged residues and red circles indicate negatively charged phospholipid moieties. This mode of interaction was proposed based on the results of high-salt and carbonate extraction experiments. B) Anchoring via covalently attached Cys- palmitoyl groups. Green circles and lines indicate the Cys and palmitate moieties, respectively. This mode of interaction was proposed on the basis of mass spectrometry experiments. C) Anchoring via direct interactions between
hydrophobic side chains (colored brown) and the lipid matrix. The mode of
interaction is supported by detergent extraction and phase separation experiments as well as structural and enzymological observations. D) Attachment
via interactions with other membrane-bound proteins. The observation that
RPE65 appears to form complexes with other RPE microsomal proteins might
suggest this mode of interaction. A hypothetical transmembrane protein is shown
colored orange.
32
Figure 3
33
Figure 4: Proposed catalytic mechanisms of retinoid isomerization.
Two mechanisms for retinoid isomerization have been proposed that account for the experimentally-observed loss of the C15-bound oxygen from the all-trans- retinyl ester during enzymatic processing. A) A retinoid molecule showing the standard numbering scheme. R indicates a saturated hydrocarbon chain B) In this dual SN2-type nucleophilic substitution mechanism a protein associated nucleophile (X) (e.g. a Cys side chain sulfur atom) attacks C11 (i) resulting in the loss of the ester moiety via alkyl-oxygen cleavage and the formation of an intermediate (ii) with a freely rotatable C11-C12 single bond. In the second step
(iii) another nucleophile, presumably a hydroxide ion, attacks C15 when the intermediate is in a cis-like conformation, resulting in loss of the C11-bound protein group and restoration of the conjugated double bond system. C) In this
SN1-type nucleophilic substitution mechanism, the rate-limiting step, i.e. loss of the ester via alkyl-oxygen cleavage, is catalyzed by a Lewis acid (X+) such as a metal ion or a proton (i). Dissociation of the ester (ii) and the generation of a resonance-stabilized carbocation (iii) lowers the polyene chain carbon-carbon bond order and the activation energy needed for trans to cis isomerization. After rotation to a cis-like conformation, a water molecule, which is either concomitantly or subsequently deprotonated by a base (B), attacks C15, restoring the conjugated double bond system in the 11-cis configuration (iv).
Dissociation of the ester leaving group regenerates the Lewis acid catalyst (v). In both mechanisms the thermodynamic energy needed for the endergonic isomerization reaction may derive from the ester hydrolysis or from a
34 downstream process such as interaction of the 11-cis products with retinoid- binding proteins.
35
Figure 4
36
CHAPTER 2: EXPRESSION, PURIFICATION AND BIOCHEMICAL
CHARACTERIZATION OF RPE65
Portions of this Chapter were previously published in:
Golczak, M., Kiser, P.D., Lodowski, D.T., Maeda, A., Palczewski, K. (2010) Importance of membrane structural integrity for RPE65 retinoid isomerization activity, J Biol Chem Jan 25. [Epub ahead of print].
Kiser, P. D., Golczak, M., Lodowski, D. T., Chance, M. R., and Palczewski, K. (2009) Crystal structure of native RPE65, the retinoid isomerase of the visual cycle, Proc Natl Acad Sci U S A 106, 17325-17330.
Golczak, M., Maeda, A., Bereta, G., Maeda, T., Kiser, P. D., Hunzelmann, S., von Lintig, J., Blaner, W. S., and Palczewski, K. (2008) Metabolic basis of visual cycle inhibition by retinoid and nonretinoid compounds in the vertebrate retina, J Biol Chem 283, 9543-9554.
Kiser, P. D., Lodowski, D. T., and Palczewski, K. (2007) Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions, Acta Crystallogr Sect F Struct Biol Cryst Commun 63, 457-461.
37
2.1. Expression and purification of Zebrafish RPE65
2.1.1. Rationale and Methods
Based on unpublished data from a collaborator that indicated zebrafish
RPE65 isoform a (zRPE65a) behaved a soluble protein when heterologously expressed in E. coli, we first attempted expression and purification of this orthologous protein. To potentially increase solubility and yield, we made a fusion protein between RPE65 and maltose-binding protein (MBP). zRPE65a
(gi:55775677) was cloned into a modified pMAL expression plasmid (New
England Biolabs, Ipswich, MA, USA) containing a tobacco etch virus (TEV) protease site and a His10 tag between the regions encoding MBP and RPE65
(85). Six L of (Luria-Bertani) LB media supplemented with 0.3% glucose were each inoculated with 10 mL of an overnight culture and grown at 37 °C until an optical density (OD) at 600 nm of 0.4-0.6 was achieved, at which point the temperature was lowered to 25 °C and protein expression was induced with
100 µM isopropyl-β-D-thiogalactopyranoside (IPTG). Four h after induction, E. coli cells were harvested by centrifugation, flash-frozen in liquid nitrogen and stored at -80 °C until needed. The bacterial pellet was triturated under liquid nitrogen using a mortar and pestle and lysed at 4 °C in a buffer consisting of
1 mg/ml lysozyme in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonate
(HEPES), pH 8.0, containing 300 mM NaCl, 25 mM imidazole, 10 mM β- mercaptoethanol, 0.1 mM ethylenediaminetetraacetic acid (EDTA) and the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride (PMSF),
0.1 mM tosyl phenylalanyl chloromethyl ketone (TPCK), 7.6 µM leupeptin and
38
0.15 µM soybean trypsin inhibitor (buffer A). After ~30 min, the viscocity of the
lysate was elevated indicating that lysis was complete. Deoxyribonuclease I
(DNase I) and MgCl2 were then added to 20 µg/ml and 4 mM, respectively, and
the mixture was stirred at room temperature until the viscosity of the lysate
decreased (~10 min). The lysate was centrifuged at 150,000g for 40 min and the
resulting supernatant was collected. The supernatant was diluted twofold with
buffer A lacking EDTA and protease inhibitors (buffer B) and was applied onto a
5 mL nickel-nitrilotriacetic acid (Ni2+-NTA) superflow column (Qiagen, Valencia,
CA, USA) that was pre-equilibrated with buffer B. The column was washed with
ten column volumes (50 ml) of buffer B and the protein was eluted with buffer C
(buffer B containing 250 mM imidazole pH 8.0). One-half mL fractions were collected and those containing protein were identified using a modified version of
the Bio-Rad protein assay and SDS-PAGE (Bio-Rad, Hercules, CA, USA) (Fig.
5A). Protein-containing fractions were pooled, TEV protease (86) was added to a
concentration of 3% w/w (based on protein concentration) and the mixture was
dialyzed against 4 L of buffer B overnight. The dialyzed protein solution was
again applied onto a pre-equilibrated Ni2+-NTA column in order to remove the cleaved MBP-His10 tag, uncleaved fusion protein and TEV protease, all of which
contain His6 or His10 tags, and the flowthrough containing cut fusion protein was
collected in 0.5 mL fractions. Protein-containing fractions were again identified using the modified Bio-Rad assay and SDS-PAGE analysis (Fig. 5B). After the second Ni2+-NTA affinity purification a prominent ~60 kDa band, which is
approximately the correct molecular mass of zRPE65a, was observed on a
39
Coomassie-stained SDS-PAGE gel. Fractions containing this ~60 kDa band were pooled and the protein was concentrated to ~8 mg/ml using a 50 kDa molecular weight cutoff (MWCO) Centricon (Millipore, Billerica, MA, USA) (Fig. 5C). The buffer containing the concentrated protein consisted of 10 mM HEPES, pH 8.0, containing 300 mM NaCl, 25 mM imidazole and 10 mM β-mercaptoethanol.
2.1.2. Results and Discussion
Using the expression and purification method described above we obtained a nearly pure preparation of a ~ 61 kDa protein. Although the molecular mass and chromatographic behavior of this protein strongly suggested that in was the desired protein, recombinant zRPE65a, we later discovered, through mass spectrometric analysis, that the purified protein was actually the well-known bacterial chaperonin GroEL. It is likely that zRPE65a was expressed in E. coli in a non-native state that led to its recognition and sequestration by GroEL. The 6-
His tag of the fusion protein was apparently not hidden inside GroEL because the fusion protein-GroEL complex could be purified by metal-affinity chromatography.
Importantly, GroEL alone has not been observed to bind Ni2+-NTA resin. Heavy
GroEL contamination was also observed when amylose resin, an affinity resin for
MBP, was used in place of Ni2+-NTA resin for purification of the fusion protein.
The inclusion of various compounds that could promote the dissociation of GroEL from the fusion protein such as adenosine triphosphate (ATP), magnesium and potassium in the column wash buffer were ineffective. A variety of other zRPE65a constructs were tested for expression in E. coli, including non-MBP
40
fusions and 6-His C-terminal tags, but in each case we observed significant
copurification of GroEL with the expressed protein (data not shown). Changing
the E. coli strain or the plasmid promoter used for expression did not remedy the situation. Because the recalcitrant nature of the GroEL contamination problem, attempts at RPE65 expression in E. coli were abandon.
2.2. Expression and Purification of Human RPE65
2.2.1. Rationale and Methods
Because RPE65 did not fold properly when expressed in E. coli we decided to switch to the baculovirus/insect cell expression system, which may express folding machinery that more closely resembles that naturally found in the
RPE cell. Additionally, because there was no reason to believe that zRPE65a would be more easily expressed and purified than mammalian RPE65, and also because our interest was in the human rather than the zebrafish protein, expression of human RPE65 was attempted. Full-length human RPE65 containing a C-terminal 1D4 tag (the last nine residues of bovine rhodopsin) was cloned into the pFastBac HT A plasmid, which introduced an in-frame, TEV protease-cleavable, 6-His tag to the N-terminus of RPE65, and the resulting construct was used to produce baculovirus according to the manufacturer's instructions (Invitrogen Bac-to-Bac handbook). For protein expression, 50 mL of high titer P3 baculovirus was added to 600 mL of cultured Sf9 cells at a density of 2 × 106 cells/ml, and the cells were shaken at 27 °C. After 36–48 h, cells were harvested by centrifugation and resuspended in 10 mL of phosphate-buffered
41
saline (PBS), pH 7.4, containing one dissolved tablet of EDTA-free complete
protease inhibitors (Roche Applied Science). Cell suspensions were flash-frozen in liquid nitrogen and stored at –80 °C. Suspensions were thawed in 40 mL of 20
mM bis-tris propane (BTP), pH 7.0, containing 150 mM NaCl, 10 mM 2-
mercaptoethanol, 5% v/v glycerol, 2 mM CHAPS, and one dissolved tablet of
EDTA-free protease inhibitors (Roche Applied Science). Cells were disrupted by
Dounce homogenization, and lysates were centrifuged at 145,000g for 30 min.
The supernatant was collected, diluted 2-fold with lysis buffer, and loaded onto
either a 3 mL Ni2+-NTA resin (Qiagen) column or a 3 mL Talon resin (Clontech)
column equilibrated with lysis buffer. The column was washed with 30 column
volumes of 20 mM BTP, pH 7.0, 500 mM NaCl, 10 mM 2-mercaptoethanol, 5%
v/v glycerol, 2 mM CHAPS, and 5 mM imidazole, pH 7.0. Protein was eluted with
a buffer consisting of 20 mM BTP, pH 7.0, 150 mM NaCl, 10 mM 2-
mercaptoethanol, 5% v/v glycerol, 2 mM CHAPS, and 150 mM imidazole, pH 7.0.
The fractions were analyzed by SDS-PAGE (Fig. 6A). The protein was purified
further on a Superdex 200 gel filtration column and fractions containing RPE65
were identified by SDS-PAGE and immunoblot analysis (Fig. 6A,B). RPE65
preparations were >99% pure after gel filtration chromatography based on silver-
stained SDS-PAGE (Fig. 6C). Immunoaffinity purification of the recombinant
RPE65 protein using the ID4 tag fused the C-terminus of the protein was also attempted. We found that the protein was able to bind to a 1D4 column, but elution with a 1D4 peptide was problematic. The protein eluted in a large number of fractions resulting in significant dilution. Thus, we did not employ 1D4 affinity
42
chromatography for the purification of recombinant RPE65 for use in
crystallization trials.
2.2.2. Results and Discussion
Using the expression and purification methodology described above we
were able to obtain 1-2 mg of purified RPE65 per eight liters of insect cell
suspension culture, which is a modest expression level of for an insect cell
expression system. We observed a large amount of contaminating proteins when
RPE65 was purified using Ni2+-NTA resin. The level of contaminating proteins was greatly diminished when a cobalt-based resin was used instead of Ni2+-NTA resin. The remaining contaminants were removed by gel filtration chromatography resulting in chromatographically pure RPE65. RPE65 was predominantly monomeric based on the gel filtration elution profile although some dimeric aggregates were present as well. Immunoblotting indicated that the
N and C termini were intact in the purified protein. The purified protein could be stably concentrated to ~5 mg/ml. It was noted that if CHAPS was omitted from the purification buffers, RPE65 aggregated immediately after elution from the metal affinity column. This purified protein was utilized for ligand-binding studies, generation of RPE65 antibodies and crystallization trials.
43
2.3. Generation of an RPE65 monoclonal antibody and mapping of its
epitope
2.3.1. Methods
An anti-RPE65 monoclonal antibody was raised against full-length,
recombinant human RPE65 protein expressed in Sf9 cells and purified as
described in Golczak et al. (74). The purified protein was used to immunize mice
as described previously (87). Purified RPE65 was emulsified with the Sigma
Adjuvant System (Sigma-Aldrich) according to the manufacturer’s protocol, and
the antigen (50 µg/body) was injected intraperitoneally into 4 week old BALB/c
mice. The same immunization procedure was repeated three times every 10
days. After immunization, mouse serum titers were checked by immunoblotting
with purified RPE65 and bovine RPE microsomes. Purified native bovine RPE65
(30 µg/body) in PBS was used for the final immunization performed 48 h before
the fusion. Hybridoma cell lines were prepared by fusion of SP2 mouse myeloma
cells (American Type Culture Collection, Manassas, VA) (7.7 x 106) with
splenocytes (2.3 x 107) from an immunized mouse by using polyethylene glycol
(PEG) 1500 (Roche, Branford, CT). Culture supernatants from resulting
hybridomas were screened with purified RPE65 and bovine RPE microsomes by
enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Positive
hybridomas were subcloned three times by the method of limiting dilution in
microtiter plates. IgG isotypes were examined with a mouse monoclonal
isotyping test kit (Roche).
44
To map the epitope recognized by the RPE65 monoclonal antibody,
fragments of RPE65 were expressed in E. coli and tested for their ability to be
recognized by the antibody. Five overlapping portions of a human RPE65 cDNA
(GI:67188783), together encompassing the entire coding region, were amplified
by polymerase chain reaction (PCR) using PfuUltra high-fidelity DNA polymerase
(Stratagene, La Jolla, CA) and the following primers:
A) 5'-caccatgtctatccaggttgagcatcc-3' and
5'-ctaggaaaatatattcttgcaggg-3',
B) 5'-caccatggctttcccagatccctgc-3' and
5'-ctaagtcagaccaaaactatgaacg-3',
C) 5'-caccatgaagccatcttacgttcatag-3' and
5'-ctacccattgtcttcatagg-3',
D) 5'-caccatgttccatcacatcaacacc-3' and
5'-ctatttcccacaatacttctgg-3',
E) 5'-caccatgtttgagtttcctcaaatcaattacc-3' and
5'-ctaagattttttgaacagtcc-3'.
The resulting blunt-end PCR products were agarose gel-purified and cloned into
the pET100/D-TOPO vector (Invitrogen, Carlsbad, CA) for protein expression in
E. coli. A second round of epitope mapping, using information from the first
round, was performed to more precisely locate the epitope-containing region.
The following PCR primers were used to amplify segments of the RPE65 cDNA
that encode various portions of the C-terminal 115 amino acid residues where
the epitope was located in the first round of mapping:
45
1) 5'-cacccctcaaatcaattaccagaagtattgtggg-3' and
5'-agattttttgaacagtccatgaaaggtgacaggg-3',
2) 5'-caccggggtttggcttcatattgc-3' and
5'-gccaagtccatacgcatatgtgtaagg-3',
3) 5'-caccgggtttctgattgtggatctctgc-3' and
5'-ccaagtttctttagttttgacattcagc-3',
4) 5'-caccgggtttctgattgtggatctctgc-3' and
5'-gctcaccaccacactcagaactacacc-3',
5) 5'-caccgggtttctgattgtggatctctgc-3' and
5'-ggcaacttcacttaagtccttggc-3',
6) 5'-caccgggtttctgattgtggatctctgc-3' and
5'-ggcaacttcacttaagtccttggc-3'.
The resulting blunt-end PCR products were agarose gel-purified and cloned into
the pET102/D-TOPO vector for expression as thioredoxin fusion proteins
(Invitrogen). Construct sequences were all confirmed by DNA sequencing. The resulting plasmids were used to transform Rosetta 2 (DE3)pLysS cells (Novagen,
San Diego, CA). Bacterial cultures grown in the presence of 50 µg/mL
carbenicillin and 34 µg/mL chloramphenicol in LB media to an OD600 nm of 0.5
were induced with 1 mM IPTG for 4 h to allow expression of the fusion proteins.
After 4 h the cells were harvested by centrifugation and lysed with SDS-PAGE
loading buffer. Proteins were separated by SDS-PAGE and analyzed by
Coomassie staining and immunoblotting by using the anti-RPE65 monoclonal
antibody described above.
46
2.3.2. Results and discussion
In this study we developed a monoclonal antibody for use as a tool in the
biochemical characterization of RPE65 and mapped the epitope of this antibody
within the sequence of RPE65. The antibody obtained was an IgG1κ that
specifically recognized RPE65 amongst all other RPE proteins.
Immunohistochemical analysis demonstrated that the antibody specifically
recognizes the RPE cell layer of the retina (Fig. 7). This finding indicated the
antibody was capable of recognizing RPE65 in its native conformation and in its
natural environment in the RPE. Indeed, we observed that detergent-solubilized
RPE65 and RPE65-containing complexes could be immunopurified using this
antibody. The high purity of the immunoaffinity-purified, RPE65-containing bis-
maleimidohexane (BMH)-crosslinked complex greatly facilitated the identification
of other members of the complex as well as the specific linkage between RPE
and RDH5 (56).
We mapped the epitope recognized by this monoclonal antibody within the
RPE65 sequence into order to better understand the favorable properties of the
antibody and so that a peptide useful for dissociating RPE65 from the antibody
could be generated. First, we subdivided the sequence of RPE65 into 5
fragments which were expressed as N-terminal 6-His fusion proteins in E. coli.
This initial screen localized the epitope to a C-terminal region of the protein
consisting of residues 419-533. To more precisely locate the epitope, constructs were created that code for progressively longer portions of the RPE65 C-
47
terminus (Fig. 8A). The fragments were expressed as thioredoxin fusion proteins
at high levels in E. coli (Fig. 8B, left). Immunoblot analysis revealed that the
antibody specifically recognizes fragments 5 and 6 indicating that the epitope is located within or immediately surrounding residues 492-514 (Fig. 8B, right).
Based on the RPE65 crystal structure, these residues form surface-exposed loops and a β-strand that could easily be recognized by the antibody in their
native state (Fig. 8C). Thus, the biochemical and structural properties of the
epitope are in agreement.
2.4. Extraction and phase separation studies on native RPE65
2.4.1. Methods
Suspensions of bovine RPE microsomes with protein concentrations of 20
mg/ml were diluted 1:10 into buffered solutions containing a variety of reagents
described in the figures. With the exception of the pH titration experiments,
solutions were buffered with 10 mM tris(hydroxymethyl)aminomethane (Tris)-HCl,
pH 7.0, and all contained 1 mM dithiothreitol (DTT). Mixtures were incubated on
ice for 1 h prior to centrifugation except for those undergoing hydroxylamine, pH
7.0, treatment which was performed at 22 °C. Mixtures were centrifuged at
150,000g for 1 h to sediment insoluble material. Resulting pellets were
resuspended in a volume of buffer equal to that of the recovered supernatant.
Equal volumes of each fraction were analyzed by SDS-PAGE. The separated
proteins were examined by Coomassie-staining and immunoblotting.
48
To analyze RPE microsome alkaline extracts, RPE microsomes were
suspended in a solution consisting of either 100 mM Na2CO3, pH 11.5, or 100
mM N-cyclohexyl-3-aminopropanesulfonate (CAPS), pH 11.0, and 1 mM DTT,
incubated on ice for 1 h and then centrifuged at 150,000g for 1 h to sediment
insoluble material. Supernatant fractions were collected and used for subsequent
experiments. In one set of experiments the pH of the extracts was adjusted to pH
6.0 by addition of 2-(N-morpholino)propanesulfonate (MES), pH 6.0, to a final concentration of 50 mM followed by slow addition of a predetermined amount of
12.1 N HCl. After 1 h incubation on ice, the samples were centrifuged at
150,000g for 1 h to sediment insoluble material. The supernatant was collected and the pellet was resuspended in a volume of alkaline buffer equal to that of the supernatant. In the second set of experiments 100 µL of alkaline extract was injected into a 20,000 kDa MWCO slide-a-lyzer dialysis cassette and dialyzed overnight against 1 L of PBS containing 1 mM DTT at 4°C. The retentate was removed from the cassette and centrifuged at 150,000g for 1 h to sediment
insoluble material. The supernatant was collected and the pellet was resuspended in a volume of PBS equal to that of the supernatant. In both experiments, the pH of the samples was tested to ensure that the desired value had been achieved. Equal volumes of each sample were analyzed by SDS-
PAGE followed by Coomassie staining and immunoblotting.
For Anapoe X-114 phase separation experiments, suspended RPE microsomes or the 20,000g supernatants obtained after centrifuging RPE
homogenate at protein concentrations of ~ 1 mg/ml were incubated in a solution
49
containing 10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM DTT and 1% w/v
Anapoe X-114 (Anatrace) for 1 h on ice to allow solubilization of membrane
proteins. Mixtures then were centrifuged at 150,000g for 1 h to sediment
insoluble material. The resulting supernatant fractions were used for phase
separation experiments. The phase separation procedure was performed
essentially as described by Bordier (45) except that no pre-condensation of the
detergent was performed because the detergent used in this study was purified
by the manufacturer, and the last detergent rinse of the aqueous phase was
omitted. Final aqueous and detergent-rich phases were equalized in volume by adding 10% w/v Anapoe X-114 and the solubilization buffer, respectively. Equal
volumes of each fraction were analyzed by SDS-PAGE followed by Coomassie
staining and immunoblotting.
2.4.2. Results and discussion
To determine the predominant type of interaction responsible for binding of RPE65 to native RPE microsomal membranes, we exposed microsomes to a
variety of treatments known to solubilize peripheral or integral membrane
proteins. As shown in Fig. 9A, virtually no RPE65 was extracted when
microsomes were exposed to solutions containing low or high salt
concentrations, the chaotropic agent KSCN, or the divalent cation chelating
agent EDTA. Furthermore, treatment with a high concentration of hydroxylamine,
which cleaves thioester bonds, did not extract significant amounts of RPE65.
These data suggest that binding of RPE65 to native membranes is not
50
dominated by electrostatic interactions or by palmitoyl anchoring. However, as
reported in the literature (88), exposure of microsomes to strong alkaline
conditions results in the extraction of significant amounts of RPE65 (Fig. 9B,C). It
was hypothesized that exposing basic residues on the surface of RPE65 to
increasing pH would liberate the protein by disrupting its electrostatic interactions
with membrane phospholipid head groups. If this hypothesis is correct and
RPE65 is a true peripheral membrane protein, it should remain soluble after lowering the pH back to a more physiological value or removing the solubilizing agent (89). Alternatively, the apparently soluble RPE65 could be generated by denaturation of the protein in strong alkaline conditions or by formation of non- sedimentable lipoprotein particles, which is well known to occur in alkaline conditions (90). To test these hypotheses, we adjusted the pH of Na2CO3 or
CAPS alkaline microsomal extracts to pH 6.0 and after 1 h incubation on ice centrifuged the sample at 150,000g for 1 h to sediment particulate matter. In a separate experiment, we dialyzed these alkaline extracts overnight against PBS containing 1 mM DTT to remove the solubilizing agent and return the pH to 7.4.
As shown in Fig. 10, most of the RPE65 became insoluble after either treatment suggesting that alkaline-extracted RPE65 was either denatured or not truly in solution. The behavior of RPE65 in this experiment further argues against a peripheral association of the protein with the native membrane. Detergent, especially the non-ionic detergent octyltetraoxyethylene (C8E4) at concentrations
above its CMC, is the most effective agent for extracting RPE65 (Fig. 9A) and the
resulting solubilized protein is highly stable. The structural integrity of this
51
detergent-solubilized protein is supported by its successful and robust
crystallization (47). The last finding suggests that the interaction of RPE65 with
native membranes is predominantly hydrophobic. To further demonstrate that
hydrophobic rather than electrostatic forces dominate the RPE65-membrane
interaction, we performed Anapoe X-114 phase separation experiments using a well-established procedure (45). Here water-soluble and peripheral membrane proteins can be separated from integral membrane proteins based on the selective partitioning of integral proteins into a detergent-rich phase that develops after raising the temperature of the solution. We performed this experiment using the Anapoe X-114-solubilized RPE microsomes, where RPE65 is the single most abundant protein, as well as the 20,000g supernatant fraction obtained after centrifugation of homogenized bovine RPE, where RPE65 makes up a small fraction of total protein. Consistent with previous reports (21, 42), we found in both experiments that RPE65 resided predominantly in the detergent-rich phase thus demonstrating the integral membrane behavior of the protein in this assay
(Fig. 11A,B). Because Anapoe X-114 is a non-ionic detergent, the interaction of
RPE65 with the detergent micelle must be attributed to hydrophobic rather than electrostatic forces.
2.5. Purification of Native RPE65 from Bovine RPE
2.5.1. Rationale and Methods
The failure of recombinant RPE65 to crystallize (discussed below) prompted us to seek an alternative source of the protein. Since the laboratory
52
was already obtaining bovine eyes for other projects, we decided to collect RPE
and purify RPE65 from this tissue. Fresh bovine eyes, obtained from a local
slaughter house (Mahan Packing, Bristolville, OH, USA), were hemisected and
retinas were removed. Approximately 1 mL of 48 mM sodium 3-[N-
morpholino]propanesulfonate (MOPS), pH 7.0, containing 0.25 M sucrose and 1
mM DTT was added to each eyecup, the RPE cell layer was detached by
brushing, and the resultant cell suspension filtered through cotton gauze to
remove large particulate matter and stored at -80 °C until needed. RPE
microsomes were prepared as previously described with some modifications
(62). Briefly, the RPE cell suspension was thawed in a 33 °C water bath, and
cells were disrupted by Dounce homogenization. The lysate was centrifuged at
20,000g for 20 min to pellet melanin granules, choroidal material, unbroken cells
and large organelles. The supernatant was removed and centrifuged at 100,000g for 75 min in order to pellet microsomal membranes. The supernatant was removed and the microsomes rinsed with de-ionized water and then resuspended and incubated in ~12 mL of 10 mM Tris acetate, pH 7.0, containing
1 mM DTT and 1 M KCl on ice for 1 h in order to remove peripherally bound membrane proteins. The microsomes were then harvested by centrifugation at
100,000g for 1 h and again rinsed with de-ionized water. All centrifugations were performed at 4 °C.
A variety of detergents were tested for the ability to extract RPE65 from microsomal membranes (Table 2). C8E4 was selected as the detergent for
RPE65 purification owing to its ability to extract and keep RPE65 stable in
53
solution as well as the favorable physical properties it exhibits such as the ability
to be removed by dialysis. Washed RPE microsomes were resuspended in ~12
mL of 10 mM Tris acetate, pH 7.0, containing 1 mM DTT and 24 mM C8E4
(Anatrace, Maumee, OH, USA) and allowed to incubate on ice for 1 h. The mixture was centrifuged at 100,000g for 1 h in order to pellet insoluble material.
RPE65 was purified from the supernatant by anion-exchange chromatography on a 1 mL diethylaminoethyl (DEAE)-Macroprep column (Bio-Rad, Hercules, CA,
USA) pre-equilibrated with 10 mM Tris acetate, pH 7.0, containing 16 mM C8E4 and 1 mM DTT. The column was washed with 5 mL of the same buffer, and the protein was eluted with a 0-500 mM linear NaCl gradient. RPE65 eluted from the column at an approximate conductivity of 15 millisiemens (mS)/cm. Fractions containing RPE65 were pooled and concentrated to 10-15 mg protein/mL in a 50 kDa MWCO Amicon centrifugal filter (Millipore, Billerica, MA, USA). The concentrated protein solution (typically 100-200 µl total volume), which exhibited a pronounced reddish brown hue, was then dialyzed overnight against 50 mL of
10 mM Tris acetate, pH 7.0, containing 1 mM DTT and 19.2 mM C8E4 to remove excess detergent and salt. The resulting protein preparation was used directly for crystallization trials and biochemical experiments. It was noted that a significant amount of retinoids, primarily retinyl esters, copurified with RPE65. Exogenous iron was not added at any time during purification or crystallization trials. Column chromatography was performed at 4 °C.
In order to assess the oligomeric state of native RPE65, purified RPE65 at
an approximate concentration of 5 mg/mL was loaded onto a Superdex 200
54
10/300 gel filtration column (Amersham Biosciences, Piscataway, WI, USA) equilibrated with 10 mM Tris acetate, pH 7.0, containing 150 mM NaCl, 1 mM
DTT and 19.2 mM C8E4. The column was developed at a flow rate of 0.5 mL/min in a buffer identical to the equilibration buffer. Bio-Rad gel filtration standards
(Bio-Rad, Hercules, CA, USA), which were separated under conditions identical to those of RPE65, were used to calibrate the column so that the apparent molecular mass of RPE65 could be quantified.
2.5.2. Results and discussion
Owing to the fact that, despite numerous trials, the heterologously- produced, purified human RPE65 protein failed to crystallize (see below) we decided to attempt purification of native RPE65 from bovine eyes. There are a number of factors that potentially could complicate purification of RPE65 from this tissue and these factors were what deterred us from initially taking this approach. First, when purification experiments were initiated we did not yet have a suitable monoclonal antibody that could be used for immunoaffinity chromatography. Therefore, traditional biochemical methods were used for purification of native RPE65. Second, although RPE65 is a fairly abundant bovine RPE protein, it makes up only a small fraction of the total protein that is collected during the isolation of RPE cell from bovine eye because of unavoidable contamination from blood cells, neuroretina, choroidal material and other extracellular proteins. Thus, a large purification factor is required to obtain homogeneous RPE65. Third, it has been estimated that an adult bovine eye
55
contains a total of only ~ 11 µg of RPE65 (48). Thus, to obtain an amount of
protein suitable for crystallization trials large numbers of bovine eyes would be
required for each purification attempt and the purification protocol would have to
be designed to minimize RPE65 loss. Lastly, previous reports suggested that
native RPE65 may be heterogeneously palmitoylated. This heterogeneity may
complicate both purification and crystallization of the protein. Despite these
potential problems we were able to develop a high-yield purification protocol that
resulted in an approximately 95% pure RPE65 preparation. The first key step that
resulted in high enrichment and no appreciable loss of RPE65 was the careful
isolation of RPE microsomes (Fig. 12A). A variety of detergents could effectively
extract RPE65 from microsomal membranes (Table 2). Selective membrane
extraction with C8E4 separated RPE65 from several other microsomal membrane
proteins (Fig. 9B). Further purification on a weak ion exchange column removed additional contaminants and concentrated the protein 3-4 fold (Fig. 12B). The
purified RPE65 could be concentrated to at least 20 mg/mL without visibly
aggregating. Detergent was undoubtedly concentrated with the protein and
excess detergent is thought to be detrimental for crystal growth. Therefore, the
purified, concentrated RPE65 sample was dialyzed overnight to remove the excess detergent as well as salt. The detergent concentration before and after dialysis was never precisely measured but it was clear that the viscosity of the sample after dialysis was reduced indicating that detergent concentration had been lowered. The behavior of the native RPE65 protein during purification did not indicate any obvious heterogeneity. From 300 adult bovine, we typically
56
obtained a 150 µL sample at a protein concentration of 15 mg/ml and a purity of
95% based on Coomassie-stained gels (Fig. 12C).
To determine the oligomeric state of purified RPE65 and the
monodispersity of the sample, we performed gel filtration chromatography on the final purified and concentrated sample. RPE65 eluted in a small number of fractions indicating that the sample was monodisperse. Based on the elution pattern of standard proteins, RPE65 had an apparent molecular mass of ~83 kDa which was interpreted as an RPE65 monomer (61 kDa) complexed with a C8E4 micelle (~20 kDa) being the prodominant species in the detergent-containing solution (Fig. 13).
57
Table
Table 2: Detergents tested for extraction, purification and crystallization of bovine RPE65
58
Figures
Figure 5: Expression of an MBP-His10-zRPE65a fusion protein in E. coli
and its purification.
In panel A, a Coomassie-stained SDS-PAGE gel shows the results of a typical
expression and purification using Ni2+-NTA resin. After induction, the fusion
protein (position on the gel is indicated by a red arrowhead) is found in both the
soluble (SN) and pellet fractions. Lanes 3 through 9 show the eluates from the
metal affinity column. B) After TEV protease cleavage (left gel, right lane) the
~100 kDa band (blue arrowhead) was diminished in intensity while bands at ~60
kDa (presumed to be zRPE65a) and ~40 kDa (MBP) (red and yellow
arrowheads, respectively) appeared to intensify. After passing the TEV protease-
cleaved material back over the re-equilibrated Ni2+-NTA column to remove His-
tagged proteins (i.e. MBP, TEV protease, and uncut fusion protein) the 60 kDa protein became the predominant band on the gel, as expected. Lanes 1 through
25 show the flow-through fractions from the Ni2+-NTA column. Fractions
containing the 60 kDa protein were pooled and concentrated to ~8 mg/ml. C) A
Coomassie-stained gel of the concentrated material shows a highly purified
sample.
59
Figure 5
60
Figure 6: Purification of recombinant human RPE65 from insect cells.
A) Coomassie-stained SDS-PAGE analysis of the eluates from sequential cobalt- affinity and gel filtration chromatography. Numbers above the lanes indicate fraction numbers from the respective columns. FT - flow through, L - molecular weight standards, W - wash. The red arrowhead indicates the position of the
RPE65 band. The majority of RPE65 eluted at a volume consistent with a monomer (Fraction 82). B) Immunoblot analysis of the protein confirmed its identity as tagged RPE65. C) RPE65-containing fractions around fraction 82 from the gel filtration column were pooled, concentrated to 5 mg/ml and analyzed by
SDS-PAGE followed by silver staining. The silver-stained gel confirms the purity of the concentrated sample.
61
Figure 6
62
Figure 7: Immunostaining of a mouse retinal section using the monoclonal
RPE65 antibody.
The green staining, which indicates RPE65-immunoreactivity, is confined to the
RPE cell layer demonstrating the high specificity of the antibody and its ability to recognize RPE65 in its native setting. 4',6-Diamidino-2-phenylindole (DAPI) staining (red) shows the nuclear layers of the retina. Image courtesy of Dr. Akiko
Maeda.
63
Figure 7
64
Figure 8: Identification of the epitope-containing RPE65 segment
recognized by a RPE65 monoclonal antibody.
A) Outline of the strategy used to identify the epitope-containing segment. The black arrow represents the RPE65 coding region and the horizontal lines indicate the region of the protein covered by each fragment. Numbers to the left of each horizontal line indicate the fragment number. B) Expression and immunoblotting
of the RPE65 fragments. Each of the RPE65 fragments was expressed as a
thioredoxin fusion protein in E. coli. SDS-PAGE analysis of bacterial cell lysates
with Coomassie stain (left) shows that after a 4 h induction with 1 mM IPTG each
fusion protein was expressed at a high level. Immunoblotting (right) of the
bacterial lysates with RPE65 monoclonal antibody showed reactivity only with the
fusion proteins containing RPE65 fragments 419-533, 322-514 and 322-533,
consistent with the epitope residing within or immediately adjacent to residues
492-514. C) Location of the epitope-containing region within the structure of
RPE65. The amino acid sequence of the epitope-containing region is shown at
the top. Location of the full or partial epitope-containing segment (colored red)
within the tertiary structure of RPE65 is shown on bottom. This structure is
rotated 120 degrees around the horizontal axis in the right panel relative to the
left panel. The epitope is found in a region of the protein predicted to face the
cytosol and is located far from both the predicted membrane-binding face of the
protein and the Cys 231 residue that cross-links with RDH5. This finding explains
why the antibody is effective for both immunohistochemical studies and for
purification of the RPE65-RDH5 cross-linked complex.
65
Figure 8
66
Figure 9: SDS-PAGE analysis of bovine RPE microsomal proteins after incubation with reagents known to solubilize certain classes of membrane proteins.
A) Supernatant (S) and pellet (P) fractions obtained after incubating microsomes with the indicated compounds for 1 h on ice followed by centrifugation at
150,000g for 1 h were separated by SDS-PAGE and the resulting gels were either stained with Coomassie Brilliant Blue (upper panels) or used for immunoblotting with an anti-RPE65 antibody (lower panels). The results clearly show that of these treatments only C8E4 treatment at concentrations above the
CMC resulted in significant extraction of RPE65. The arrowhead indicates the position of RPE65. B) and C) Supernatant (S) and pellet (P) fractions obtained after incubating microsomes with the indicated compounds for 1 h on ice followed by centrifugation at 150,000g for 1 h were separated by SDS-PAGE and the resulting gels were either stained with Coomassie Brilliant Blue (upper panels) or used for immunoblotting with anti-RPE65 and anti-LRAT antibodies (lower panels). Significant extraction of RPE65 occurred when microsomes were incubated with 50 mM CAPS, pH 11.0, but not at lower pH values. The final concentration of buffers in this experiment was 50 mM. RPE65 extraction increased after incubation with higher concentrations of Na2CO3. After incubation in 100 mM Na2CO3, pH 11.5, approximately 50% of the RPE65 was extracted from the microsomes. The arrowhead indicates the position of RPE65.
WB - western blot
67
Figure 9
68
Figure 10: Behavior of RPE microsomal alkaline extracts after pH
neutralization or dialysis.
RPE microsomes were treated with either 100 mM Na2CO3, pH 11.5, or 100 mM
CAPS, pH 11.0, for 1 h on ice and then centrifuged for 1 h at 150,000g. The
supernatant fractions after Na2CO3 and CAPS treatment are shown in lanes 1
and 6, respectively. After adjusting the pH of these extracts to six and incubating
them on ice for 1 h, samples were centrifuged at 150,000g for 1 h to determine if
RPE65 remained in the supernatant. The supernatant and pellet fractions from
this experiment for the pH-adjusted Na2CO3 and CAPS extracts are shown in
lanes 2 and 3 and lanes 7 and 8, respectively. Alternatively, we dialyzed the
alkaline extracts overnight at 4 °C against PBS containing 1 mM DTT.
Afterwards, the retentates were collected and centrifuged at 150,000g for 1 h to
assess the solubility of RPE65 after removal of the extraction agent and
adjustment of pH to 7.4. The supernatant and pellet fractions from this
experiment with Na2CO3 and CAPS extracts are shown in lanes 4 and 5 and
lanes 9 and 10, respectively. In both experiments RPE65 became insoluble upon return to more physiological pH values. The arrowhead indicates the position of
RPE65. WB - western blot
69
Figure 10
70
Figure 11: Partitioning of RPE65 in Anapoe X-114 phase separation experiments.
RPE microsomes A) or the 20,000g supernatant B) obtained after centrifugation of homogenized bovine RPE were solubilized in 1% w/v Anapoe X-114
(Anatrace) and mixtures were centrifuged at 150,000g for 1 h to sediment insoluble material. Supernatant fractions obtained after centrifugation were used for the phase separation studies performed as described in Materials and
Methods. "Input" indicates total proteins found in the supernatant fractions after high speed centrifugation, "aqueous phase" indicates proteins remaining in the detergent-poor top phase after phase separation and "detergent rich-phase" indicates the proteins found in the small oily droplet at the bottom of the sucrose cushion after phase separation and low-speed centrifugation. RPE65 strongly partitioned into the detergent-rich phase in both experiments, demonstrating its amphiphilic nature. The arrowhead indicates the position of RPE65. WB - western blot
71
Figure 11
72
Figure 12: Purification of native RPE65 from bovine RPE.
A) Distribution of RPE65 amongst the various fractions obtained during the isolation of bovine RPE microsomes. The upper Coomassie-stained gel demonstrates that RPE65 can be substantially enriched by isolating microsomes.
Note that the prominent ~65 kDa band in the supernatant fractions is albumin.
Immunoblot analysis demonstrates that nearly all RPE65 sediments during centrifugation with only a trace amount left in the 150,000g supernatant fraction.
Each fraction is normalized by volume. B) Purification of RPE65 by anion exchange chromatography. A chromatogram showing results of a typical purification is shown in the top panel. The green, purple, and grey traces sequentially represent absorbance at 280 nm, conductivity, and percentage of buffer B containing 500 mM NaCl (from 0% to 100%). RPE65 elutes from the column at a conductivity of approximately 15 mS/cm. Coomassie-stained SDS-
PAGE analysis of fractions from the anion exchange column is shown in the lower panel. RPE65 was concentrated in fractions 50-54. C) A Coomassie- stained SDS-PAGE analysis of the concentrated sample demonstrating its purity.
The arrowheads indicate the position of RPE65 in each gel.
73
Figure 12
74
Figure 13: Gel filtration analysis of purified RPE65.
The black dots represent the elution values obtained for the standards on a
Superdex 200 10/300 gel filtration column. RPE65 eluted at an apparent molecular mass of 83 kDa (red asterisk) as shown by SDS-PAGE analysis (inset) of the gel filtration eluates, which is consistent with an RPE65 monomer complexed with a detergent micelle. Ve/Vo stands for elution volume/void volume.
75
Figure 13
76
CHAPTER 3: CRYSTALLIZATION AND STRUCTURE DETERMINATION OF
RPE65
Portions of this Chapter were previously published in:
Golczak, M., Kiser, P.D., Lodowski, D.T., Maeda, A., Palczewski, K. (2010) Importance of membrane structural integrity for RPE65 retinoid isomerization activity, J Biol Chem Jan 25. [Epub ahead of print].
Kiser, P. D., Golczak, M., Lodowski, D. T., Chance, M. R., and Palczewski, K. (2009) Crystal structure of native RPE65, the retinoid isomerase of the visual cycle, Proc Natl Acad Sci U S A 106, 17325-17330.
Bereta, G., Kiser, P. D., Golczak, M., Sun, W., Heon, E., Saperstein, D. A., and Palczewski, K. (2008) Impact of retinal disease-associated RPE65 mutations on retinoid isomerization, Biochemistry 47, 9856-9865.
77
3.1. Construction of an RPE65 homology model
An RPE65 homology model was created for use in structural analysis of a
pathogenic RPE65 mutation identified in our laboratory (91) and also for use as a molecular replacement search model after obtaining well-diffracting RPE65 crystals. Initial RPE65 homology models were generated with the Fugue (92),
Phyre (93), and 3D-Jigsaw (94) servers. All three algorithms identified
Synechocystis ACO (Protein Data Bank (PDB) accession code 2BIW) as the
most appropriate template for construction of the RPE65 homology model. In
general, each of the models was similar in those regions containing His residues
180, 241, 313, and 527 which are highly conserved amongst all RPE65 family
members, including the apocarotenoid and β-carotenoid oxygenases (12). The
model produced by Fugue was selected as our starting model since it appeared
to portray the seven-bladed β-propeller fold most completely. Amino acid side
chains were inserted using Coot (95) and manually adjusted to optimize both the
stereochemistry and inter-residue contact distances. The model was then
energy-minimized by using CNS (96) with harmonic restraints placed on the
residues surrounding the active site iron. Because residues 380−415 were
predicted to constitute a long unstructured loop in all three homology models, this
region was omitted from our final model because it was likely to be portrayed
incorrectly.
78
3.2. Attempted crystallization of recombinant RPE65
A variety of sparse matrix screens and rational grid screens were used for
recombinant RPE65 crystallization trials. Purified protein at a concentration of 5
mg/mL was mixed with the various crystallization solutions in 1:1 ratios and the
resulting drops, typically 2 µL in volume, were incubated over the respective
crystallization solutions, typically 500 µL in volume, in the hanging drop format.
The drops were incubated either at room temperature or at 4 °C. Although
approximately 700 different conditions were screened, no diffraction quality
crystals were obtained.
3.3. Crystallization of native bovine RPE65
Unlike the situation observed for recombinant human RPE65, we found
that purified native RPE65 prepared using the method described above readily
crystallized. Indeed, small crystals were observed after only 96 crystallization
trials using the PEG/ion crystallization screen from Hampton Research. An
exploration of the crystallization space of native RPE65 revealed that alteration of
buffer type, pH value and type of PEG utilized as the precipitant did not prevent
crystal growth. By contrast, the type of detergent used during purification of
RPE65 was critical for growth of crystals. Although RPE65 could be stably
purified and concentrated in other detergents such as CYMAL-4 and
nonylmaltoside, such samples did not yield any crystals despite extensive
screening (Table 2). Of the detergents tested, only RPE65 purified in C8E4 crystallized. The best RPE65 crystals, which were used for diffraction analysis,
79
were grown by the hanging-drop vapor diffusion method by mixing 1 µl of protein
solution, at ~15 mg/ml, with either 1 µl of 0.3 M sodium acetate, pH 8.0,
containing 11% w/v PEG 3350 (condition A) or 1 µl of 100 mM sodium MES, pH
6.0, containing 30% v/v PEG 200 and 2 mM DTT (condition B) at room
temperature and incubating the drops over 0.5 mL of the same crystallization
solution at 8 °C. Crystals typically appeared within one day and continued to
grow over the course of several weeks (Fig. 14A,B). To confirm that the
constituent molecules of the crystals were indeed RPE65, single large crystals
were collected from the drop, washed three times in protein-free synthetic mother
liquor, dissolved in water, and analyzed by silver staining and immunoblotting. A
silver-stained gel showed only one band at ~ 65 kDa and this band was also
recognized by an anti-RPE65 monoclonal antibody (Fig. 15A,B). The largest
crystals analyzed had dimensions of approximately 100 x 100 x 300 µm with a
hexagonal shape when viewed down their long axis indicating trigonal or
hexagonal point group symmetry (Fig. 14B). Prior to flash cooling, crystals grown
under condition A were cryoprotected by soaking in crystallization solution A
containing 9.6 mM C8E4 and 15% glycerol by volume. Crystals grown under
condition B did not require additional cryoprotection. Crystals were flash cooled
in liquid nitrogen prior to x-ray exposure.
3.4. Diffraction data collection and phasing
The diffraction properties of the RPE65 crystals were first tested on the
Case Western Reserve University pharmacology home x-ray source. Closely
80
spaced diffraction spots, consistent with a macromolecular crystal, that extended
to ~3.2 Å resolution were clearly visible after a 15 min x-ray exposure. The
diffraction intensities were generally weak, and this problem could not be
overcome by longer exposures. Indexing based on one image indicated the
lattice symmetry of the crystal was primitive hexagonal in accord with the
observed hexagonal-shaped crystal morphology. The weakly diffracting nature of the crystals indicated that high-intensity synchrotron radiation would be required to obtain a high-quality dataset.
The crystals were next tested at the Advanced Photon Source (APS) 23-
ID and 24-ID beamlines as well as the National Synchrotron Light Source (NSLS)
X29 beamline. The greater beam intensities, compared to the home x-ray source, were clearly beneficial as evinced by the overall high signal-to-noise ratio and the improved resolution of the diffraction intensities with the best crystals diffracting to ~2 Å resolution (Fig. 16). Consistent with the results obtained from data collected at home, the data were indexed in a primitive hexagonal lattice with the following unit cell constants: a = b ≈ 176 Å, c ≈ 87 Å. Regardless of growth
conditions, all crystals analyzed had highly similar unit cell constants. Data
merged well in P3(1 or 2) and imposing hexagonal crystal symmetry did not
significantly raise the Rmerge value. Examination of the 00l reciprocal lattice plot
revealed significant intensity values only for every sixth reflection consistent with
the P6(1 or 5) or P6(1 or 5)22 space groups. Attempts to impose 6/mmm Laue class
symmetry on the data led to unacceptably high Rmerge values indicating 6/m Laue
symmetry was correct. Based on the Matthew's coefficient the most probable
81 number of RPE65 monomers per asymmetric unit was two, which would result in a solvent content of ~62%. Analysis of several datasets with phenix.xtriage revealed that about 80% of examined crystals were merohedrally twinned with the twin fraction being highly variable between crystals (97-98).
The high resolution datasets used for structure analysis were collected at the APS 23-ID-D beamline using a MAR300 charge-coupled device (CCD) detector. Two 90° datasets on crystals grown under condition B, one extending to
2.14 Å resolution (Native 1) and the other to 1.9 Å resolution (Native 2) were collected on a MAR300 CCD detector both with a crystal-to-detector distance of
250 mm, an x-ray wavelength of 1.0332 Å, an oscillation angle of 1°, and an exposure time of 1 second per frame. All data were reduced using HKL2000 (99) and TRUNCATE from the CCP4 suite (100). Data collection statistics are shown in Table 1. No twinning was evident in the 2.14 Å dataset, whereas the 1.9 Å dataset was estimated to have a twin fraction of 30-35% by the program phenix.xtraige.
Because the general fold of RPE65 was already known based on the crystal structure of ACO, we first attempted to solve the structure by molecular replacement using the ACO structure or the RPE65 homology model described above as search models. Despite an extensive number of trials using the program Phaser (101-102), we were unable to obtain a correct solution using this method. This result was not unexpected though because RPE65 and ACO are only about 25% identical and sequence identity values less than 30% generally indicate that the corresponding model will be ineffective for molecular
82 replacement trials. Another rule of thumb rule for successful molecular replacement is that the known and unknown structures should have a Cα root mean square deviation (RMSD) value of ~1.5 Å (103). As discussed later, the
RMSD between ACO and RPE65 was found to be ~2.5 Å, which explains the failure of the molecular replacement method.
So in order to obtain phase information we also collected diffraction data on derivatized crystals. The tuneability of the x-ray energy on the NSLS X29 beamline made it possible to collect anomalous scattering data for multi- or single-wavelength anomalous dispersion (MAD or SAD) phasing. Crystals grown under condition A and then soaked in mercury acetate or ethyl mercuric thiosalicylate (EMTS) at concentrations of 1 mM for 48 h exhibited no diffraction whereas crystals soaked in 1 mM iridium hexachloride for 48 h mostly maintained their diffraction properties with some loss in resolution. A 180° dataset was collected on an iridium-soaked crystal at X29 using a ADSC Q315 detector, a crystal to detector distance of 375 mm, an oscillation angle of 1° and an exposure time of 5 seconds per frame at an x-ray energy just above the theoretical iridium LIII white line at 11.3203 kiloelectron volts (keV). Data collection and refinement statistics are shown in Table 1. No twinning was evident in this dataset. Although the anomalous signal in the data was strong enough to allow location of the iridium substructure, we ultimately did not use this dataset for phasing.
X-ray absorption spectroscopy (XAS) data collected on RPE65 in solution at the NSLS X3B beamline indicated the presence of iron, a known RPE65
83 cofactor, in the sample. This finding prompted us consider iron MAD/SAD as a way to phase the diffraction data. Previous data indicated that iron bound to
RPE65 in a 1:1 stoichiometry (80). Iron is a weak to moderate anomalous scatterer with a maximum theoretical f" value (K edge) of 4 electrons. The low anomalous scatterer to protein ratio together with the relatively low anomalous signal emitted by iron atom indicated that iron-SAD phasing may be difficult.
However, the theoretical Bijvoet ratio, a measure of anomalous signal in diffraction data, for a 1:1 iron to RPE65 complex calculated using equation (2)
(104-105) is equal to 1.3%, with a value of 0.6% suggested to be the theoretical lower limit for successful SAD phasing (106). <|ΔFanom|> is the absolute value of the mean anomalous difference,
1/2 Equation (2): <|ΔFanom|>/
Therefore, it seemed possible that the iron atom could provide enough signal to phase the structure by the SAD method. We collected a 360° dataset on a crystal grown under condition A at an x-ray energy just above the iron K edge (7.12119 keV) with a crystal to detector distance of 182 mm, an oscillation angle of 1° and an exposure time of 5 seconds per frame. No inverse-beam or special crystal
84 alignment strategies were used. Data collection statistics are shown in Table 1.
No twinning was detected in this dataset.
After data reduction, the scalepack file containing unmerged Bijvoet pairs was input into the SHELX C/D/E macromolecular phasing suite for iron substructure location, phasing of reflections and solvent flattening (107). Data within the resolution range of 50-3 Å resolution were used to locate the iron substructure. A group of correct solutions evidenced by high correlation coefficients and Patterson figures of merit were readily found using SHELXD.
Initial phases were calculated for all reflections, out to 2.5 Å resolution, using
SHELXE. Fifty cycles of density modification using SHELXE revealed a large difference in the map contrast and connectivity between the original and inverted hands of the iron substructure, which confirmed that the phasing procedure was working and that the correct space group was P65 rather than P61. The SHELXE electron density maps had flat solvent regions and good connectivity in the protein region (Fig. 17A,B) with highly-plausible iron sites. However, the side chain density was too weak to allow model building based on the SHELXE maps.
The histogram matching algorithm implemented in RESOLVE was used to further improve the phases (108-109). Following this density modification procedure, large side chains could readily be identified and model building was initiated.
There were several factors that were responsible for the success of the iron-SAD phasing experiment. The major positive attributes of the crystals were their strong diffraction properties combined with their resistance to radiation damage at the long x-ray wavelength required to maximize the Bijvoet
85
differences. The high symmetry and low mosaicity of the crystals both facilitated
the accurate measurement of Bijvoet differences. Additionally, the relatively high
solvent content of the crystals made solvent flattening a highly effective method
for phase improvement (Table 1).
3.5. Model building, refinement and analysis of the model
The quality and resolution of the density-modified experimental electron density maps allowed automated model building using the program ARP/wARP
(110). The program was able to build approximately 90% without manual intervention. Refinement of this initial model against the 2.5 Å resolution dataset using the program REFMAC resulted in an Rfree value of ~ 30% (111-112). The
missing segments, with the exception of residues 1, 2 and 109-126 were built by
hand into the electron density maps calculated with refined phases using the
program Coot (113). Electron density for residues 1, 2 and 109-126 was virtually
non-existent. The model was then refined against the 2.14 Å resolution dataset
using REFMAC (Table 1). Because the datasets were isomorphous, Rfree reflections in the 2.5 Å dataset were maintained in the 2.14 Å dataset and in the
1.9 Å dataset mentioned below. Non-crystallographic symmetry (NCS) restraints were applied to residues 196-202, which exhibited somewhat weak electron density and high B-factors, in order to make the refinement of these residues more stable. Translation libration screw (TLS) refinement of the B-factors, with one TLS group per RPE65 monomer, resulted in a 1-2 % drop in Rfree (112). A
Babinet bulk solvent model was used to model scattering from solvent regions.
86
After several cycles of refinement followed by manual rebuilding of the model led
to final Rwork and Rfree values of 18% and 21.6%, respectively with RMSDs for bond length and bond angles of 0.013 Å and 1.4°. The model was validated using the MolProbity server (114). No Ramachandran plot outliers were present in the model, and the MolProbity and clash scores were in the 96th and 97th percentiles for structures with resolutions of 2.14 ± 0.25 Å, respectively indicating excellent model geometry. The model and structure factor amplitudes have been deposited in the PDB under accession code 3FSN.
The model was also refined against the twinned 1.9 Å dataset using
REFMAC (Table 1). The amplitude-based twin refinement algorithm in REFMAC was employed to account for the significant merohedral twinning in the 1.9 Å dataset. The twin fraction was refined to a value of 33.5% using the following twin
operator: h+k, -k, -l. The refinement procedure was similar to that explained
above except that NCS restraints and TLS refinement were not employed.
Despite the overall higher resolution of the dataset, we observed that electron density for residues 1, 2, 108-126, 198-200 and 261-271 was so weak that modeling these segments was not justified. Therefore, these regions were omitted from the final model. After several cycles of refinement followed by manual rebuilding of the model led to final Rwork and Rfree values of 14.6% and
17.1%, respectively with RMSDs for bond length and bond angles of 0.016 Å and
1.5°. It should be noted that the somewhat lower than expected R values are a
consequence of the altered statistical properties associated with twinned crystals.
The model was validated using the MolProbity server. No Ramachandran plot
87 outliers were present in the model, and the MolProbity and clash scores were in the 98th and 94th percentiles for structures with resolutions of 1.9 ± 0.25 Å, respectively indicating excellent model geometry. The model and structure factor amplitudes have been deposited in the PDB under accession code 3KVC.
Sequence-based alignments were produced with ClustalW (115).
Structure-based alignments and three-dimensional superpositions were produced with the DALI server (116). Contact surface area calculations were performed with the PISA server (117). MOLE was used to visualize tunnels leading to the active site iron atom (118). All structure figures were made with
PyMOL v1.0 (119).
88
Table
Table 3: Data collection, phasing and refinement statistics.
Data Collection* Crystal Native 1 Native 2 Native 3 Iridium (iron-SAD)† SAD† X-ray source APS APS NSLS NSLS 23-ID-D 23-ID-D X29 X29 Temperature, K 100 100 100 100 Wavelength, Å 1.03324 1.03324 1.74100 1.09520 Space group P65 P65 P65 P65 Unit cell constants, a=b, c, Å 176.53, 176.36, 176.94, 178.63, 86.87 86.72 86.96 86.47 Resolution, Å 50-2.14 100-1.9 50-2.5 50-2.76 (2.22-2.14) (1.97-1.9) (2.59-2.5) (2.86-2.76) Unique reflections observed 83509 119836 53648 40674 Completeness, % 99.5 (98.3) 99 (92) 99.6 (99) 100 (100) Multiplicity 6.3 (5.7) 4.7 (3.2) 18.2 (9.3) 10.5 (8.9) I/σI 14.2 (2.16) 17.5 (2.26) 31 (3.29) 23.7 (3) Rsym(I), % ‡ 11.6 (73.4) 10.4 (56.5) 11.5 (65.6) 10.3 (68.1) Average mosaicity, ° 0.42 0.41 0.38 0.51 Wilson B factor, Å2 32 23.2 54 70 Chains per asymmetric unit 2 2 2 2 Solvent content, % 62 62 62 62 SAD phasing Sites per asymmetric unit 2 1
89
Figures
Figure 14: Photographs of typical RPE65 crystals.
A) Crystals were typically 50 to 300 microns in their longest dimension and 20 to
100 microns in their smallest dimension. B) The hexagonal symmetry of the crystals was evident when they were viewed down their long axis.
90
Figure 14
91
Figure 15: SDS-PAGE analysis of washed crystals.
A) A single large crystal was harvested from its mother liquor, washed extensively in well solution, dissolved in water and analyzed by SDS-PAGE followed by silver staining. Only one band is observed at ~65 kDa. B) A similarly
prepared sample was also subjected to immunoblot analysis with an RPE65
monoclonal antibody. The immunoreactivity of the band confirmed its identity as
RPE65.
92
Figure 15
93
Figure 16: A diffraction pattern recorded from an RPE65 crystal at the NSLS
X29 beamline.
The yellow arrowhead points to a reflection with a d-spacing of ~2 Å.
94
Figure 16
95
Figure 17: An initial electron density map calculated with observed
amplitudes and density-modified SAD phases from SHELXE.
A) A zoomed-out view of the map showing flat solvent regions. B) Close-up view showing electron density for an α helix.
96
Figure 17
97
CHAPTER 4: DESCRIPTION AND INTERPRETATION OF THE RPE65
CRYSTAL STRUCTURE
Portions of this Chapter were previously published in:
Kiser, P.D., Palczewski, K. (2010) Membrane-binding and enzymatic properties of RPE65, Prog Retin Eye Res Mar 18. [Epub ahead of print]
Golczak, M., Kiser, P.D., Lodowski, D.T., Maeda, A., Palczewski, K. (2010) Importance of membrane structural integrity for RPE65 retinoid isomerization activity, J Biol Chem Jan 25. [Epub ahead of print].
Kiser, P. D., Golczak, M., Lodowski, D. T., Chance, M. R., and Palczewski, K. (2009) Crystal structure of native RPE65, the retinoid isomerase of the visual cycle, Proc Natl Acad Sci U S A 106, 17325-17330.
98
4.1. A general description of the structure
The basic RPE65 structural motif is a single-domain, 7-bladed β-propeller
with single strand extensions on blades VI and VII and a two-strand extension on
blade III (Fig. 18A,B). Blades I, II and V adopt the canonical four consecutive β
strand arrangements seen in prototypical β propeller proteins (120). Blades VI
and VII both have one strand extensions that are formed by the N-terminus of the
protein. Blade III has an interesting two blade extension that is formed from a
long insertion between the two outer β strands (24 and 27) of sheet IV. This
insertion contains several residues that mediate a large dimer contact observed
in the asymmetric unit of the crystal (discussed in detail below). The first and last
β strands of the core propeller structure (strands 1 and 2 are not a part of the
propeller core) "seal" the structure within blade VII. There are a total of 36 beta
strands and 10 helical segments (Fig. 18B). The first helix, composed of residues
6-10, is in a 310 conformation. The top face of the β-propeller, defined by the
positions of the segments connecting the outer β-strand of one sheet with the
inner β-strand of the next sheet, is covered by a helical cap that houses the
active site. The segments connecting the β strands on the bottom face are generally much shorter. The iron cofactor is located roughly on the propeller axis near the top face of the propeller and is coordinated by four His residues and three outer shell Glu residues. Notably, each blade of the propeller, including the associated connecting segments, contributes a single residue to the iron
coordination system. A hydrophobic tunnel enters the structure between the helical cap and top propeller face and leads to the active site defined by the
99 bound iron atom (Fig. 19). The active site is also accessible from a second, much narrower, tunnel that enters the protein from another portion of the helical cap.
Also, there is a water-filled cavity coincident with the propeller axis that is accessible from the bottom face of the propeller and extends deeply into but does not transverse the protein and does not provide access to the iron atom.
4.2. The membrane-binding surface
The structure exhibits a single prominent hydrophobic surface that likely anchors the protein to the lipid bilayer (Fig. 20A). This surface is formed by residues 196-202, 234-236 and 261-271 (Fig. 20B). The first two groups of residues are in loop conformations whereas the last group adopts an alpha helical conformation. These segments are enriched in residues known to be preferentially interact with headgroups (Ser, Trp, Tyr, Lys and Arg) and the hydrophobic core (Phe, Leu and Ile) of the phospholipid bilayer (121). One potentially alpha helical, amphipathic segment composed of residues 109-126 that may contribute to membrane affinity is disordered in the crystal (Fig. 20C).
The hydrophobic face surrounds the opening of the large channel that leads to the catalytic site of RPE65. Given that RPE65 can directly extract retinyl esters from the lipid bilayer for metabolic processing (34, 40) and also considering that these extremely hydrophobic retinyl esters are dissolved in the lipid matrix of the bilayer, it seems reasonable to conclude that this hydrophobic surface of the protein is embedded in the bilayer allowing the protein to gain access to the retinyl ester substrate.
100
Consistent with biochemical studies on RPE65 palmitoylation (50-51), no
evidence of Cys 231, 329 and 330 palmitoylation was observed in the RPE65 crystal structure (Fig. 21A,B). In fact, none of these Cys side chains are surface exposed indicating that significant conformational changes would be required for
them to become palmitoylated. By contrast, Cys 112 is found on the N-terminal
side of a ~ 15 residue-long, potentially amphipathic sequence that surrounds the entrance to the RPE65 active site and may form part of the membrane docking surface (47) (Fig. 20A,C). Thus, palmitoylation of this residue appears quite feasible from a structural perspective, but the modification could not be confirmed in the crystal structure because Cys 112 is located in a highly disordered and unresolved portion of the protein. The predicted close proximity of this Cys to the membrane suggests it could be subject to non-enzymatic palmitoylation by palmitoyl-coenzyme A present in the sER lipid bilayer (122).
4.3. The active site cavity and iron-binding site
The active site of the RPE65, defined by the bound iron cofactor, is found in an interior cavity of the protein. As the hydrophobic retinyl ester substrates are dissolved in the lipid core of the bilayer, the protein must have a mechanism for physically transferring them from the membrane to the active site. The RPE65 structure reveals a single hydrophobic tunnel that could serve this purpose (Fig.
22). As mentioned above, the entrance of this tunnel is surrounded by several hydrophobic residues that are likely integrated into the hydrophobic core of the
membrane in order to provide an energetically favorable passageway for
101
retinoids. The retinyl ester could conceivably be orientated in the membrane such
that the retinyl or palmitate moiety enters the active site first. Furthermore, it
cannot be excluded that the retinyl ester enters the tunnel in a bent conformation
with the ester moiety entering first, although the tunnel may be too narrow for this
to occur. Owing to the presence of a single passageway of suitable width to the
active site, it is likely that products of the isomerization reaction exit the active
site through the same tunnel and reenter the membrane for further processing
rather than being transferred directly to a retinoid-binding protein.
The interior cavity of RPE65 is lined primarily by apolar residues as
expected based on the hydrophobicity of its retinoid substrates (Fig. 22).
Interestingly, a large majority of these lining residues are aromatic hydrophobes.
The aromatic side chains of Phe, Tyr and Trp are known to be good stabilizers of
carbocation intermediates because of their ability to form cation-π interactions,
and similarly lined active sites have been observed for other enzymes that
catalyze reactions that transition through carbocation intermediates (123-124).
Thus, RPE65 appears to have an active site that could support the SN1
mechanism mentioned above. A recent mutagenesis study supports this
hypothesis by demonstrating that the ratio of 11 to 13 cis retinoids produced by
RPE65 can be altered by substitutions of active site residues, including some
aromatics (71). Exposed Cys residues that could act as nucleophiles in an SN2- type nucleophilic substitution reaction are notably absent in the interior cavity.
But there are a few interestingly positioned Tyr residues that may well have a role in the RPE65 catalytic mechanism. There is a hydrogen bonding network
102 involving Tyr 338, Tyr 239, Glu 148 and His 241 that lines part of the putative substrate entry tunnel and the active site cavity near the iron atom. These residues may interact with or be involved in proton shuttling to and from the retinoid substrate during catalysis. Indeed, the requirement of Glu and Tyr residues in positions 148 and 239, respectively, for RPE65 catalytic activity suggests a functional role for this group of residues in catalytic isomerization (71,
125). The hydrophobic cavity of RPE65 potentially could be a favorable binding site for non-physiological lipophilic or amphiphilic molecules such as detergents.
A detergent molecule was identified in the active site in one crystal form of the
RPE65 homolog ACO, suggesting that such binding may also occur with RPE65
(67). This possibility suggests that competitive binding to the RPE65 active could be a second mechanism, the first being direct disruption of membrane structure, by which detergents inhibit RPE65 isomerase activity (56).
As mentioned earlier, there also exists a narrow tunnel that enters the protein via a surface far removed from the predicted membrane-binding face.
Although, the functional significance of this tunnel is not yet clear, it appears to be large enough to allow passage of small molecules such as water. The 1.9 Å resolution structure of RPE65 revealed well-ordered water molecules in this tunnel and in the interior cavity of the protein suggesting that the waters might have entered via the small passageway (56) (Fig. 23). Thus, water molecules used in the retinoid isomerization reaction may gain access to the substrate by this route (47). Notably, there is a thin tube of poorly defined electron density leading from the narrow tunnel to a portion of the active site near the iron atom
103 that may originate from several mobile water molecules (Fig. 23). A Phe side chain (residue 418) appears to cause bending of this electron density and may restrict the approach of water to the substrate in order to promote its nucleophilic attack on the correct carbon atom, i.e. C15 rather than the carbonyl carbon.
Thus, it would be interesting to determine the effects of Phe418Trp or Tyr substitutions on retinoid isomerization activity as these mutants, by potentially restricting water access to the substrate, might result in longer lived reaction intermediates and potentially alternative products.
The iron cofactor of RPE65 is directly coordinated by a set of four His residues (residues 180, 241, 313 and 527) arranged in a distorted, partially-filled, octahedral geometry (Fig. 24A). Although the His-Fe-His bond angles indicate that the best description of this geometry is octahedral, it could also be described as trigonal bipyramidal (D3h point group symmetry) depending on ligand interactions in the open coordination sites (e.g. mono vs. bidentate binding). A
Val residue (residue 134) side chain located 4.9 Å away from the iron may block access of ligands to one of the open iron coordination sites. Of the four His residues, three (residues 241, 313 and 527) individually form hydrogen bonding interactions with one member of a set of three highly conserved Glu residues
(residues 148, 417 and 469), which form the second shell of the iron coordination sphere, whereas the His 180 side chain forms a hydrogen bond interaction with a water molecule (Fig. 24A). This general mode of iron coordination is virtually identical to that of ACO, another CCO family member (67). Aside from ACO, the four-His iron coordination motif has been observed in only three other proteins,
104 photosynthetic reaction center (PDB code 5PRC), photosystem II (PDB code
1S5L) and 15-lipoxygenase (PDB code 1LOX); however, these three proteins lack second shell Glu residues. Mutagenesis experiments combined with mutations observed in LCA patients have established that the second shell Glu residues are essential for RPE65 isomerase activity and even highly similar residues such as Asp and Gln are unable to functionally substitute for them (12,
81, 125-126). It might be expected that the negatively charged second shell could help strengthen the iron-His coordinate bonds by partially deprotonating the δ N atoms of the three His residues. However, the four Fe-His bond lengths are identical, within the resolution of the diffraction data (2.2-2.3 Å), suggesting that this is not the case. Thus, the function of the second shell is probably to help position the inner shell His ligands correctly and fine-tune the electronic properties of the iron atom for catalysis. Interestingly, there are hydrogen bonding interactions between the second shell Glu residues and residues even more distal to the iron atom, namely Tyr 239, Trp 331 and Arg 44. Substitutions at Trp 331 and Tyr 239 were detrimental for RPE65 retinoid isomerization activity
(71), whereas Arg44Gln substitutions have been found in patients with LCA
(127).
Despite reports showing that iron chelating agents such as 1,10- phenanthroline can inhibit RPE65 retinoid isomerization activity presumably by removal of the metal cofactor from the active site (12, 47, 80), our efforts to completely remove iron from the protein either before or after crystallization using such agents (40-100 mM EDTA or 10 mM 1,10-phenanthroline) have been
105 unsuccessful as demonstrated crystallographically (data not shown). A satisfactory explanation for this discrepancy remains elusive.
We observed that native RPE65, as purified, contained electron density, not accounted for by protein atoms, in its active site (47). This electron density occupies a portion of the putative substrate entry/product exit tunnel and appears to interact directly with the iron cofactor, possibly filling one or both of the vacancies in its octahedral coordination sphere (Fig. 24B). Based on its appearance, we hypothesized that the density represented a bound fatty acid molecule, possibly derived from an all-trans-retinyl ester. If this hypothesis is true, it could mean that iron acts directly to bind and polarize the ester moiety thus promoting its dissociation. Interestingly, for three of the proteins mentioned above that contain four-His iron coordination motifs, a carboxylate or bicarbonate ligand occupies a position similar to the carboxylate moiety of the putative fatty acid molecule found in the active site of RPE65 (Fig. 25). Confirmation of the identity of this electron density in RPE65 will require additional structures, for example containing a bound all-trans-retinyl ester molecule.
4.4. Dimeric structure found in the asymmetric unit and analysis of crystal packing
An RPE65 dimer with approximate two-fold rotational symmetry is contained in the crystallographic asymmetric unit (Fig. 26A). The dimer has a parallel arrangement such that entrances to the active sites of each RPE65 protomer face the same side of the dimer. This extensive dimer interface buries
106
~1550 Å2 of surface area which is about three times greater than any other protein-protein contact surface in the crystal. Many of the interacting residues between monomers are part of an extension to blade III of the β-propeller.
Interestingly, this extension is absent in Synechocystis ACO, and as a possible consequence, a symmetric dimer similar to that seen in the RPE65 structure is not observed in either of the two reported crystal forms (67). The dimer is held together by a number of hydrogen bonding and ion-ion interactions as well as a number of longer range van der Waals interactions (Fig. 26B). Analysis of the dimer with the PISA server (117) shows that the P-value of the observed solvation free energy gain upon dimer formation is 0.33, where values can range from 0 to 1 with values <0.5 indicating a high probability of the interaction surface being specific. For comparison, this statistic was calculated for known dimeric monotopic membrane proteins prostaglandin H2 synthase-1 (PDB ID: 1PRH),
squalene-hopene cyclase (PDB ID: 1SQC), fatty acid amide hydrolase (PDB ID:
1MT5) and glycerol-3-phosphate dehydrogenase (PDB ID: 2QCU) which yielded
values of 0.17, 0.16, 0.30, and 0.75, respectively. Both the absolute value of this
statistic for RPE65 and its similarity to a known dimeric monotopic membrane
protein, fatty acid amide hydrolase, indicate that the interface is sufficiently
specific that it could represent a physiological interaction. In order to determine
whether an RPE65 dimer pre-forms in detergent-containing solution prior to
crystallization, we performed gel filtration chromatography on purified RPE65 in
the presence 150 mM NaCl. RPE65 eluted at a molecular mass of 83 kDa
relative to standards consistent with an RPE65 monomer complexed with a
107
single C8E4 micelle (Fig. 13). The large buried surface area and symmetric
arrangement of the RPE65 dimer observed in the crystal suggest it may be
functionally relevant, and although RPE65 is monomeric in solution, it may form
dimers in native RPE membranes. Alternatively, the dimer interaction surface
could possibly be a binding surface for another microsomal protein such as
RDH5, which RPE65 has been shown to interact with (53-56).
A molecular packing analysis of hexagonal RPE65 crystals provided
additional insight into the surface of this protein that interacts with membranes.
All RPE65 molecules in the P65 unit cell are oriented such that the hydrophobic,
putative membrane-binding surface of RPE65 faces a large solvent channel in
the crystal that runs parallel to the crystallographic 65 screw axis (Fig. 27). Given
the strong interaction of RPE65 with non-ionic detergent, it is quite likely that a
protein-detergent mixed micelle was the building block of the crystal. Thus, we
speculate that formation of the large solvent channel observed in the crystal was
necessary in order to accommodate the associated detergent molecules. This
packing arrangement is consistent with a type II membrane protein crystal (128-
129). It is notable that prototypical monotopic membrane proteins, such as
prostaglandin H2-synthase-1 and squalene-hopene cyclase, form parallel symmetric dimers like RPE65 does and are also packed in the crystal with their
hydrophobic surfaces facing solvent channels (130-131).
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4.5. RPE65 amino acid substitutions associated with Leber congenital amaurosis
A number of RPE65 mutations have been shown to cause LCA or a milder retinal dystropy retinitis pigmentosa (RP) (for recent summary see (91)). Because human and bovine RPE65 amino acid sequences are 99% identical, it can be assumed that they adopt virtually identical folds. This close relationship allowed us to map human LCA and RP-associated RPE65 amino acid substitutions onto the RPE65 structure to evaluate possible mechanisms by which these substitutions induce changes in function (Fig. 28). It is apparent that a great majority of the amino acid substitutions found in LCA and RP patients are located within or adjacent to the blades of the β propeller rather than in the connecting loops and helices. Blade VII, where the main β-propeller closure occurs, is frequently affected. Mutations in this region could result in disruption of the β- propeller fold owing to improper "sealing" of the propeller structure. Because each blade of the propeller structure contributes a residue to the iron ion coordination system, substitutions in any sheet could displace the respective critical residue leading to disruption of iron binding. Missense mutations that result in substitutions of either the first or second shell iron ligands have been found in LCA patients (125, 127, 132) consistent with biochemical studies that demonstrated both first and second shell iron ion ligands are essential for RPE65 isomerase activity (12, 81).
Arg 91, Tyr 368 and His 182 are among the most frequently affected positions in patients with RPE65-associated LCA or RP (91, 125, 133-135). An
109
analysis of these positions in the RPE65 structure reveals that each amino acid
side chain makes specific interactions with surrounding residues that cannot be
mimicked by other side chains (Fig. 29). In many cases, such as His182Arg and
Tyr368His substitutions, significant structural distortions would be required to
accommodate the volume of the substituted side chain. Arg 91 is especially interesting because it is located outside of the β-propeller core in an α-helix. In the crystal structure this helix is adjacent to the proposed RPE65 membrane- binding motif. Arg 91 forms a salt bridge with Glu 127 which is located on the C- terminal side of the amphipathic stretch of amino acids that are disordered in the crystal structure. This salt bridge could be critical for the proper positioning of
RPE65 membrane-binding elements. Indeed, Arg 91 substitutions have been reported to affect RPE65 subcellular localization (136).
4.6. Comparison of the RPE65 and apocarotenoid oxygenase structures
The availability of two crystal structures of CCO family members, a cyanobacterial protein with oxidative cleavage activity (67) and a mammalian protein with isomerase activity, allows us to compare and contrast these distantly related enzymes. As predicted, both proteins exhibit a seven-bladed β-propeller chain fold. A structural alignment performed with the DALI server shows that
ACO and RPE65 superimpose with an RMSD of 2.5 Å (116) (Fig. 30A). The most notable differences occur in three regions as shown by structure-based alignment (Fig. 30B). In RPE65 residues 337-357 form a group of three helices
(H7, H8, and H9) that sit on the top face of the β-propeller, the first two of which
110 help form the active site cavity. The corresponding residues 325-342 of ACO have less helical structure and are less tightly packed. In ACO this region forms a potential exit tunnel for the product(s) of the oxidative cleavage reaction. It was proposed that the hydrophilic dialdehyde product of the apocarotenoid cleavage reaction exits the protein through this opening into the cytosol whereas the more hydrophobic all-trans-retinal exits the same way it entered back into the lipid bilayer. In RPE65, a structurally analogous tunnel exists but it is much smaller in diameter than the tunnel in ACO. The small diameter strongly suggests that neither the retinoid nor fatty acid product of the isomerization reaction exit the active site through this route. Rather they both probably exit through the large, hydrophobic tunnel through which the retinyl ester substrate entered, which is consistent with their hydrophobic nature. However, the water molecule that is involved in the isomerization reaction may enter through the narrow tunnel as discussed above.
The second region in RPE65 consists of residues 376-402, which are totally absent in Synechocystis ACO. Residues 376-391 form a loop leading to a two-stranded anti-parallel β-sheet that forms an extension to sheet III of the β- propeller (Fig. 18B). Strands 21b and 26 of sheet III form a short parallel β-sheet interaction. Residues 376-402 provide most of the RPE65 dimerization surface. A symmetric dimer arrangement is not observed in the ACO crystal structure, consistent with the hypothesis that this region in RPE65 is critical for its quaternary structure. In contrast to ACO, human CCOs do contain a stretch of amino acids in this region that are reasonably well conserved with respect to the
111
RPE65 sequence. This may indicate that dimerization of these enzymes is
important for their structure and function.
The third region consists of residues 109-126 in RPE65 and residues 116-
129 in ACO. Most of this segment was disordered in the RPE65 crystal structure.
However, a comparison of the sequences in this region shows that the RPE65
sequence is longer with a greater number of hydrophobic, aromatic amino acid
residues. If this segment in RPE65 is modeled as an α-helix, as suggested by
secondary structure prediction programs, the resulting structure is amphipathic with several exposed hydrophobic aromatic residues (Fig. 20C). This hydrophobic segment probably forms a major surface responsible for anchoring
RPE65 to the lipid bilayer. The presence of the palmitoylated Cys 112 residue at
the beginning of this sequence strongly supports this conclusion (47, 52).
Interestingly, this segment was proposed to interact with the lipid bilayer in an early paper describing the cloning of bovine RPE65 (26). The somewhat shorter length of this segment in ACO is consistent with its solubility properties. Refolded
ACO was shown to be soluble in the absence of detergents and required detergent only for crystal formation (67).
Despite similarities in the iron location and coordination system between the two proteins (Fig. 31), it appears that the metal ions perform fundamentally different functions within in the respective catalytic mechanisms. The iron cofactor in ACO was proposed to directly bind molecular oxygen resulting in its activation and correct positioning for oxidative cleavage of the apocarotenoid substrate. In contrast, there is no role for molecular oxygen in any of the retinoid
112 isomerization reactions proposed to date. Although molecular oxygen activation is apparently not needed for RPE65 function, the iron center has been maintained in a nearly unaltered form. As discussed above, we propose that iron is necessary to help bind, orient, and polarize the ester functionality and make it a better leaving group.
Recently, a member of the CCO family called NinaB from the Galleria mellonella moth with isomerooxygenase activity was described suggesting that members of this family originally developed isomerase activity while maintaining carotenoid oxygenase activity. (137). This finding supports the hypothesis that the iron cofactor is not directly involved in the isomerase activity of NinaB or
RPE65.
113
Figures
Figure 18: RPE65 structure and topology.
A) The structure of an RPE65 monomer viewed from the bottom face of the 7- bladed β-propeller. Blades are numbered I through VII with the blade where
"velcro" closure of the core propeller fold occurs labelled as blade VII in accord with established convention. Absolutely conserved His 180, His 241, His 313, and His 527 residues are shown as sticks coordinating the natively bound iron ion, shown as an orange sphere. B) An RPE65 topology diagram showing the positions of residues involved in iron ion-coordination as well as the position of a palmitoylated Cys residue. Numbers beside these residues indicate their position in the RPE65 amino acid sequence.
114
Figure 18
115
Figure 19: The two tunnels that lead to the active site iron of RPE65.
The blue mesh outlines the hydrophobic, substrate entry/product exit tunnel
(tunnel A) and the red mesh outlines the narrower tunnel (tunnel B). The axis of
the propeller is roughly vertical in this figure. The iron atom is shown as a brown sphere.
116
Figure 19
117
Figure 20: Predicted membrane-binding regions of RPE65.
A) RPE65 topology relative to a phosphatidylcholine bilayer. Predicted
membrane-binding residues are colored red. A palmitoyl group identified by mass
spectrometry is modeled on Cys 112. Residues 109-126, which could not be
experimentally modeled owing to weak electron density, are modeled as an α
helix in this figure as suggested by secondary-structure prediction programs. The
iron atom marking the RPE65 active site is shown as a brown sphere. B)
Stereoview of the putative membrane-binding surface of RPE65. Segments of
the chain that surround the entrance to the active site are enriched in residues that are favored in interfacial (Arg, Lys, Ser, Trp and Tyr) and lipid core regions
(Phe, Leu and Ile) of the membrane. Therefore, these regions probably interact extensively with the membrane including the lipid core, consistent with the need for this enzyme to assess membrane-dissolved retinyl esters. The view is approximately down the substrate entry/product exit tunnel with the iron cofactor shown as an orange sphere. The two green spheres indicate the positions of Phe
108 and Glu 127, which flank the unresolved region of the RPE65 chain. C) A helical wheel plot of the unresolved region showing the pronounced amphipathicity of this segment when it is modeled as an α helix.
118
Figure 20
119
Figure 21: Locations of and electron density maps surrounding the
cysteine residues previously proposed to participate in a palmitoylation
switch mechanism.
A) Locations of Cys residues 231, 329 and 330 in the RPE65 structure. None of
the sulfur atoms of these residues in surface exposed or located on the predicted
membrane-binding face of the protein. B) Crystallographic determination of the palmitoylation status of Cys residues 231, 329, and 330. The blue mesh represents the final σ A-weighted 2Fo-Fc electron density map contoured at 1 σ.
There are no signs of residual density that could represent a palmitoyl group. The
red mesh represents a 4 σ NCS-averaged anomalous difference electron density
map calculated using the anomalous differences from the Native 2 dataset and
the refined phases. The latter map confirms the correct positioning of the Cys
sulfur atoms. There is no evidence for palmitoylation of these residues in the
electron density maps.
120
Figure 21
121
Figure 22: Stereoview of residues lining the RPE65 interior cavity.
The internal cavity of RPE65 housing the iron cofactor is lined with several aromatic residues possibly involved in stabilizing a carbocation intermediate of the retinoid isomerization reaction. The blue mesh delineates the boundaries of this cavity. Carbon atoms of hydrophobic residues and polar or charged residues are colored orange and green respectively, and the iron cofactor is displayed as an orange sphere.
122
Figure 22
123
Figure 23: Stereoview of a possible water entry route to the RPE65 active
site.
In the 1.9 Å resolution RPE65 structure, water molecules (red spheres) can
clearly be identified in the initial portions of tunnel B as well as in the interior cavity. The bent tube of residual Fo-Fc electron density follows a path from the
interior cavity to a position near the iron cofactor. This density may result from a
string of several poorly localized water molecules. Phe 418 appears to be
responsible for the bent shape of the electron density. The arrows show the
possible routes water molecules could take from the protein exterior to the
interior cavity. The apparent orifice indicated by the arrowhead is an artifact
produced by this particular view. The Fo-Fc electron density map is contoured at 3
σ.
124
Figure 23
125
Figure 24: Stereoviews of the RPE65 iron cofactor, surrounding residues,
and residual electron density in the active site.
A) The iron inner coordination sphere consists of four histidine residues arranged in a distorted octahedral geometry. The Val 134 side chain may block access to one of the two open iron coordination sites. His residues 241, 313 and 527 hydrogen bond with Glu residues 148, 417 and 469, respectively. Additionally, there are hydrogen bonding interactions between these Glu residues and Tyr
239, Trp 331 and Arg 44, respectively. With the exception of Val 134, all of these residues are known to be critical for RPE65 activity based on data from either from in vitro isomerase activity assays or LCA patients with RPE65 mutations.
This arrangement suggests a highly tuned metal-binding site that is extremely susceptible to perturbation by amino acid substitutions. The iron cofactor and a water molecule are shown as brown and red spheres, respectively. Dashed lines indicate interactions, either hydrogen bonds or coordinate bonds, between atoms. B) Residual electron density found in the RPE65 active site. The green mesh represents a σ A-weighted Fo-Fc electron density map contoured at 3.5 σ
calculated prior to modelling the fatty acid. The appearance of the electron
density next to the iron atom is highly suggestive of a bound fatty acid molecule
which has been modelled. As discussed in Figure 23 above, the second tube of
electron density may result from the presence of mobile water molecules.
Numbers indicate bond distances in Å.
126
Figure 24
127
Figure 25: Ligands found in the fifth and/or sixth coordination sites of all
known 4-His iron-binding proteins.
Carbon atoms for bovine RPE65 (3FSN), rabbit 15-lipoxygenase (1LOX),
Rhodopseudomonas photosynthetic reaction center (5PRC),
Thermosynechococcus photosystem II (1S5L), and Synechocystis ACO (2BIW)
are colored green, light grey, orange, purple and black respectively. Negatively
charged carboxylic or bicarbonate ligands appear to be favored in the sites not
occupied by His ligands.
128
Figure 25
129
Figure 26: The RPE65 dimer found in the asymmetric unit.
A) The top panel illustrates the extensive and complementary nature of the dimer
interface. Approximately 1,550 Å2 of surface area is buried between protomers
(colored green and blue). The bottom panel shows the positions of the predicted
membrane-binding residues (colored orange) in each protomer. The yellow arrowheads indicate the mouths of the predicted substrate entry/product exit tunnels. The observed parallel orientation suggests the observed dimer
interaction could be physiological. The red curved arrow indicates a 180°
horizontal rotation. B) A two dimensional projection of the residues mediating the
dimer interaction. The residues are ordered as they appear in the projection of
the dimer in the top panel of A). Single-headed and double-headed arrows
indicate hydrogen bonding and ionic interactions, respectively. The inner and
outer columns indicate the atom identifiers and residues, respectively. The cut-off distances used for determining these interactions were < 3.4 Å for ionic interactions and < 3.2 Å for hydrogen bonding interactions. A number of longer range van der Waals interactions also contribute to the dimer interaction surface
(not shown).
130
Figure 26
131
Figure 27: Packing of RPE65 in the P65 unit cell.
The predicted membrane-binding face of RPE65 consisting of residues 196–202,
234–236, and 261–271 (colored orange) faces the largest solvent channel in the
crystal. Based on the locations of residues 108 and 127, it is clear that the disordered but potentially amphiphilic segment, consisting of residues 109-126,
also faces this channel. This packing arrangement was probably selected to
accommodate a protein-detergent mixed micelle in the crystal, as has been
noted for other monotopic membrane proteins. The grey box is an outline of a
unit cell, and the black symbols indicate crystallographic symmetry elements.
132
Figure 27
133
Figure 28: LCA- or RP-associated RPE65 amino acid substitutions.
An RPE65 topology diagram showing amino acid residues found to be altered in
LCA or RP patients (colored in red). The human RPE65 sequence is shown. The numbers in this panel indicate the position in the RPE65 amino acid sequence of
the C-terminal residue in each secondary structure element.
134
Figure 28
135
Figure 29: Locations and local interactions of residues commonly
substituted in RPE65-associated LCA or RP.
A) A view of RPE65 from the top face of the β propeller showing the locations of the commonly substituted residues in LCA or RP shown in more detail in panels
B, C and D. The maroon dashed line indicates the disordered segment (residues
109-126). B) Arg 91 and Glu 127 form a salt bridge interaction. Arg 91, a residue
frequently found substituted in RPE65-associated LCA, is located on the surface of the protein in the H2 α helix forming a salt bridge with Glu 127. Glu 127 is just upstream of one of the putative RPE65 membrane-binding regions. Substitutions in the nearby Glu 95 residue have also been found in LCA patients indicating that the structure of this region is critical for the proper function of RPE65. C)
Interaction of His 182 with surrounding residues of RPE65. His 182, a residue frequently involved in LCA-associated pathogenic substitution, forms hydrogen bond interactions with the hydroxyl moiety of Tyr 423 and the backbone carbonyl oxygen of Phe 243. Loss of these specific interactions may destabilize the protein and displace the critical His 180 and His 241 iron-binding residues. D)
Interaction of Tyr 368 with surrounding residues. Tyr 368 is another position
found substituted in RPE65-associated LCA. The Tyr side chain forms a polar
interaction with Arg 366 as well as several hydrophobic interactions with
surrounding side chains. Numbers indicate bond distances in Å.
136
Figure 29
137
Figure 30: Comparison of the RPE65 and ACO crystal structures.
A) Structural superposition of RPE65 (blue, PDB ID: 3FSN) and ACO (green,
PDB ID: 2BIW). The two structures superimpose with an RMSD of 2.5 Å over
443 matched Cα positions. The most notable differences are found in the helical cap (top) of the propeller and in blade III, where RPE65 contains a ~30 residues extension. The dashed maroon line indicates the approximate position the disordered loop in RPE65 consisting of residues 109-126. B) A structure-based alignment of bovine RPE65 and Synechocystis ACO amino acid sequences. Red cylinders and green arrows represent helices and strands, respectively. Yellow and green letter highlighting indicates sequence identity and conservation, respectively. The alignment was performed with the DALI server.
138
Figure 30
139
Figure 31: Stereoview of the iron cofactor and its ligands and the putative substrates/products in the active sites of RPE65 and ACO.
The conformations of the first and second shell iron ligands are quite similar between RPE65 A) and ACO B). The non-protein ligands occupy similar positions with respect to the iron centers except that the apocarotenol substrate does not directly interact with the iron atom in the ACO structure.
140
Figure 31
141
CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS
142
RPE65 catalyzes the key isomerization step in the visual cycle and thus is
essential for proper human vision. There has been great interest in the RPE65
molecule both because of its involvement in human disease and because of the
biochemically unique reaction it catalyzes. Although a great deal of progress has
been made in understanding the general function of RPE65, many critical details
regarding this protein have remained elusive. In particular, despite a number of
studies there was no consensus on the mode of RPE65 membrane binding.
Additionally, it has been difficult to distinguish between the two enzymatic
mechanisms proposed for this enzyme, which differ in their requirements for
specific active site properties. While it was recognized that three-dimensional
structure information would be required to help address these outstanding
issues, attempts to purify and crystallize RPE65 were apparently unsuccessful
and the structure remained elusive. The primary focus of this study was to determine the crystal structure of RPE65.
Three different approaches were employed to obtain a high-quality protein preparation required for successful crystallization. We first attempted to express both zebrafish and human RPE65 fused to MBP or His tags in E. coli. However, we were unable to successfully purify RPE65 produced using this method
because it was tightly bound to the bacterial chaperonin GroEL presumably in a
misfolded conformation. Interestingly, the RPE65-GroEL complex could be purified by metal affinity chromatography by virtue of polyhistidine tags fused to the RPE65 polypeptide and this method proved to be an effective way of isolating native GroEL from E. coli (138). Altering the position of the affinity tags did not
143 remedy the problem of GroEL contamination. Therefore, attempts at RPE65 expression in E. coli were abandoned.
Human RPE65 with an N-terminal 6-His tag and a C-terminal 1D4 tag was successfully expressed in Sf9 cells and could be purified to homogeneity by a combination of metal affinity and gel filtration chromatography. The purified protein could be stably concentrated to ~5 mg/ml. Despite a number of trials, no diffraction-quality crystals of this protein could be produced.
The final approach taken was to purify RPE65 from a natural source, bovine RPE. After examining a number of methods, a purification protocol was developed that consisted of differential centrifugation to isolate RPE microsomal membranes containing RPE65, washing the membranes in a high ionic strength buffer to remove peripherally bound proteins, selective extraction from the membranes using the non-ionic detergent C8E4, anion exchange chromatography and dialysis following concentration to remove excess detergent. This protocol resulted in a ~95% pure RPE65 preparation with high recovery. RPE65 prepared in this manner crystallized in a highly reproducible manner under a quite wide range of conditions.
The RPE65 crystals belonged to the space group P65, contained two
RPE65 monomers per asymmetric unit, and routinely diffracted to between 2 and
2.5 Å resolution. We were able to phase the structure by using the SAD method utilizing the anomalous signal from the single iron cofactor that copurified with the protein. Solvent flattening and histogram matching were employed to improve the experimental phases. The resulting electron density maps were readily
144 interpretable and a large fraction of the initial model was built automatically using the program ARP/wARP. The final models, refined against an untwinned 2.14 Å dataset and a 1.9 Å merohedrally twinned dataset, had highly acceptable crystallographic statistics.
As stated above, two important aspects of RPE65 biochemistry, namely its mode of membrane binding and catalytic mechanism are controversial. The
RPE65 crystal structure has provided the structural framework required for a clearer understanding of these biochemical properties.
The crystal structure and the extraction and phase separation experiments performed in this study strongly suggest that RPE65 should be classified as an integral monotopic membrane protein (see Appendix I). In other words, RPE65 strongly binds to membranes via a direct interaction between its hydrophobic side chains and the acyl core of the lipid bilayer. This classification is in contrast to previously proposed models that attributed RPE65 membrane affinity solely to palmitoylated cysteine residues or positively charged residues. However, this mode of membrane binding is quite reasonable from a physicochemical prospective because retinyl esters are likely localized exclusively to the hydrocarbon core of the lipid bilayer and the protein must recognize and extract these esters by inserting segments into this region. The implication of this classification is that RPE65 remains permanently bound to the ER membrane after insertion, rather than cycling between an active membrane bound form and an inactive soluble form as has been previously proposed. In this previous proposal, the cycling was proposed to be mediated by reversible palmitoylation of
145
specific Cys residues. The RPE65 crystal structure demonstrates that
palmitoylation of these Cys residues is highly improbable based on their locations
and solvent accessibility.
In the future, it would be useful to obtain a structure with the segment
consisting of residues 109-126 clearly resolved to confirm that this region is
oriented such that interaction with membrane would be favored and also to allow
crystallographic evaluation of the palmitoylation status of Cys 112. However, if
this segment requires interactions with a lipid bilayer for stability, such a structure
may be difficult to obtain. We have previously observed that disruption of the
RPE microsomal membrane structure via PLA2 or detergent treatment can
significantly reduce RPE65 isomerization activity (56). The relatively high
calculated B-factors and disorder for atoms comprising the membrane-binding surface of RPE65 may indicate that these regions are important for substrate recognition and uptake from the bilayer into the active site and may become more mobile and less structured upon detergent extraction or PLA2 treatment,
which leads to loss of isomerization activity. To test this hypothesis, I propose
that the structure of residues 109-126 as well as the other predicted membrane- binding residues be probed using hydrogen/deuterium exchange and/or synchrotron footprinting techniques. Washed RPE microsomal membranes containing RPE65 will be subjected to either PLA2 activity or detergent as well as the appropriate controls and the samples will be assayed for retinoid isomerization activity. The exchange rate of backbone amide protons, which is a measure of solvent accessibility and secondary structure stability, in the various
146
conditions will be determined by incubating the samples in deuterium oxide for
multiple periods of time, quenching exchange by acidification, and analyzing the polypeptide chains after pepsin digestion using mass spectrometry. It is possible
that mass spectrometric analysis of the samples may be difficult because RPE
microsomal membranes contain proteins other than RPE65. If this is the case,
the use of purified RPE65 reconstituted in liposomes in the proposed experiments may simply data analysis. A complementary approach that gives information about side chain solvent accessibility is synchrotron footprinting.
Samples will be treated as described above and then exposed to x-rays that generate hydroxyl radicals from water molecules. Such hydroxyl radicals can covalently modify amino acid side chains based on their solvent accessibility and proximity to stably-bound water molecules and the modifications can be detected by mass spectrometry. A potential advantage of this technique over hydrogen- deuterium exchange is that the modifications are stable. Thus, the protein can be modified and subsequently purified in order to facilitate mass spectrometric analysis without limitations placed on the amount of time required for purification.
The RPE65 monoclonal antibody described in Chapter 3 that recognizes an epitope distantly located from the region of interest could be useful for purifying modified RPE65. My prediction is that treatment of the RPE microsomal
membranes with PLA2 or a variety of detergents will result in an increase in
mobility and solvent accessibility of the presumed substrate recognition and
membrane-binding segments while the remainder of the protein will be relatively
unaffected by these treatments.
147
Based on the presence of several lining aromatic residues, the active site
of RPE65 appears to be most able to support a mechanism of retinoid
isomerization that involves a carbocation intermediate; that is the SN1
mechanism discussed above. No candidate groups capable of acting as strong
nucleophiles, which would be required for an SN2-type isomerization mechanism,
are located in the active site. The observed residual active site electron density
that appears to interact with the iron cofactor has characteristics that suggest it
could represent a fatty acid molecule. The direct interaction between the iron
atom and the carboxylate moiety of the putative fatty acid suggest that the iron
could promote catalysis by directly binding and polarizing the ester thus
facilitating its dissociation via alkyl-oxygen cleavage, which would generate the required retinyl carbocation intermediate.
The detailed structural information on the RPE65 active site has allowed identification of specific amino acid side chains that may be important for the isomerization reaction. Of particular interest are a pair of Tyr side chains that are located near the iron cofactor and line the RPE65 substrate-binding site. These residues may be important for the shuttling of protons or electrons during the retinoid isomerization reaction. Tyr 239 forms a hydrogen bonding interaction with Glu 148, one of the second shell iron ligands. A recent mutagenesis study demonstrated that a variety of substitutions at this position including a conservative Tyr to Phe substitution abolished RPE65 isomerization activity (71).
Tyr 338 forms a potentially important hydrogen bonding interaction with Tyr 239.
Mutation of this residue to Phe or Trp could allow determination of importance of
148
the hydroxyl moiety of Tyr 338 in the isomerization reaction. Another residue that
was suggested to be potentially important based on the crystal structure is Phe
418. This residue lines a portion of the interior cavity of RPE65 that may form the
passageway through which water travels to gain access to the active site.
Substitution of this residue with another containing a smaller hydrophobic side chain such as Val or one containing a larger side chain such as Trp could be useful in probing the importance of the channel diameter in retinoid isomerization. Alteration of the channel shape and diameter could change the specific site on the retinoid molecule where water reacts or, alternatively, it could restrict the access of water to the active such that longer-lived intermediates of the isomerization reaction are generated. To test these hypotheses, I would generate RPE65 point mutants that encode the RPE65 variants described above and coexpress the mutant proteins with LRAT to allow for the generation of retinyl esters in situ. The expression level of the mutant RPE65 proteins relative to wild-type protein will be quantified by western blotting. Cells expressing the various RPE65-constructs will be assayed for retinoid isomerization activity with a standard procedure used in the Palczewski laboratory (91). The products of the reaction will be analyzed by high-performance liquid chromatography and mass spectrometry.
Obtaining a co-crystal structure of RPE65 with a bound retinyl ester or a retinoid inhibitor such as all-trans-retinylamine would greatly clarify many of the uncertainties regarding substrate orientation and the exact moieties responsible for facilitating both the alkyl-oxygen cleavage step and the double bond
149 isomerization. A variety of attempts have been made to obtain such structures, but so far they have not been successful.
Further spectroscopic characterization of the RPE65 iron center is also desirable given its presumed role in the isomerization reaction. Our previous attempts to study the iron center using XAS have been partially successful primarily because it is difficult to obtain large enough amounts of RPE65 at the high concentrations required for these studies. Therefore, the establishment of an expression system that will allow the production of large quantities of enzymatically active RPE65 would greatly facilitate these studies. The expression studies described above indicate that heterologous expression of
RPE65 in E. coli may not be feasible owing to a lack of proper folding machinery in these bacteria. Expression in insect cells was somewhat successful but the yields obtained were too low to be useful. It may be worthwhile to attempt expression in yeast since these cells possess eukaryotic folding machinery and can often express foreign proteins at high levels.
The close similarity between the iron centers of RPE65 and ACO suggest that they could have similar or identical functions in the respective catalytic mechanisms of these enzymes. In ACO, the iron cofactor functions to bind and activate molecular oxygen for oxidative cleavage of apocarotenoids. There is currently no biochemical evidence to suggest that molecular oxygen is necessary for the retinoid isomerization activity of RPE65. However, no published studies have specifically tested this possibility and it is conceivable that molecular oxygen is somehow involved in the isomerization reaction. To test this possibility,
150
I propose to perform the isomerization reaction in both anaerobic, normoxic and
hyperoxic environments and evaluate the rate of isomerization under each
condition. As a control, the rate of carotenoid cleavage by a close RPE65 relative
with carotenoid oxygenase activity, such as BCMOI, could also be measured under the same conditions.
Finally, the functional role of the dimer observed in the RPE65 crystal structure still requires clarification. Its two-fold rotational symmetry and parallel arrangement together with the large buried surface area found at its interface strongly suggest this dimer may form on the ER membrane. Site directed mutagenesis of residues involved in the dimer interaction may provide some insights into the functional significance of the dimer. It would be interesting to see if the dimer can be trapped by crosslinking on the membrane. Observation of the dimer in another crystal form would also provide strong evidence in support of its physiological relevance.
151
APPENDIX I: CLASSIFICATION OF MEMBRANE PROTEINS*
The criteria that are used for classification of membrane proteins are not
always consistent between investigators. Therefore, it is important to describe
the different types of membrane proteins and how they have historically been
defined. Singer and Nicolson first defined two broad classes of membrane
proteins, the peripheral and the integral membrane proteins (89). Peripheral
membrane proteins typically associate with membranes through electrostatic
interactions with lipid head groups, by fatty acid acylation, prenylation, or glycosyl
phosphatidylinositol (GPI) anchors or by interactions with integral membrane
proteins (139-140). These proteins can usually be extracted from membranes
under relatively mild conditions such as high or low ionic strength, treatment with
divalent cation chelators (e.g. EDTA), or by incubation in alkaline carbonate
buffers (90, 140). Following extraction from membranes, peripheral membrane
proteins are usually stable after removal of the extraction agent and can be
handled like typical soluble proteins (90). By contrast, integral membrane
proteins bind to membranes in a much tighter, often irreversible fashion and
interact directly with the membrane hydrophobic core. Importantly, the interaction
between an integral membrane protein and the lipid matrix is usually of functional
significance (140-141). Quantitative extraction of integral membrane proteins
requires the presence of membrane-disrupting concentrations of detergents, and
after extraction detergent must remain in the purification buffers to keep the protein soluble (140). The integral membrane proteins can be further subdivided
based on their topology with respect to the membrane into monotopic, bitopic or
152 polytopic proteins as described by Blobel (142). Bitopic and polytopic membrane proteins possess one or multiple membrane spanning domains, respectively, and therefore have portions of their chains located in two topologically distinct spaces. There are numerous examples of both bitopic and polytopic membrane proteins. Monotopic membrane proteins, on the other hand, have hydrophobic segments that extensively interact with the lipophilic core of the membrane but these segments do not span the bilayer. This group can be further subdivided based on the presence or absence of a hydrophobic ER signal peptide sequence. The last distinction is important because different mechanisms of membrane insertion are required for each type (142). The former class can be cotranslationally inserted into the ER membrane by translocon machinery, whereas the latter must be translated on a free ribosome and then targeted to the correct membrane by a different mechanism such as interaction with another membrane-bound protein. Although monotopic membrane proteins are thought to be much rarer in nature than transmembrane proteins, structures of several of these proteins are now known. The first monotopic membrane protein to have its structure revealed was ovine prostaglandin H2 synthase-1 in the laboratory of
Garavito in 1994 (131). This microsomal protein is a prototypical example of a monotopic membrane protein targeted to the ER membrane by an N-terminal signal peptide. Subsequently, the structure of a squalene-hopene cyclase from
Alicyclobacillus acidocaldarius was determined in the Schulz laboratory (124).
The squalene-hopene cyclase is an example of a monotopic membrane protein that lacks an N-terminal signal peptide. A mammalian monotopic membrane
153
protein that is localized to the ER membrane but lacks a signal peptide is
lanosterol synthase (143-144). Thus, the absence of potential transmembrane
segments and/or an N-terminal signal peptide in the primary amino acid sequence of a protein does not necessarily exclude the possibility that it is an integral membrane protein. In reality, the distinction between peripheral and integral membrane proteins is sometimes difficult to make on the basis of extraction experiments (90, 139). For proteins that bind via direct interactions to the bilayer, the ratio of polar to non-polar interactions can take on a continuum of values; thus classification of proteins as peripheral or integral membrane can be ambiguous and somewhat arbitrary. Therefore, structural and functional information must also be considered when classifying protein-membrane interactions as peripheral or integral.
* Portions of this Appendix were previously published in:
Kiser, P.D., Palczewski, K. (2010) Membrane-binding and enzymatic properties of RPE65, Prog Retin Eye Res Mar 18. [Epub ahead of print]
154
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