Crystal structure of human mitochondrial trifunctional protein, a β-oxidation metabolon

Chuanwu Xiaa,1, Zhuji Fua,1, Kevin P. Battaileb, and Jung-Ja P. Kima,2

aDepartment of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226; and bIndustrial Macromolecular Crystallography Association- Collaborative Access Team, Hauptman-Woodward Medical Research Institute, Argonne, IL 60439

Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved February 11, 2019 (received for review September 20, 2018)

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes HELLP (hemolysis, elevated liver enzymes, and low platelets) β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA syndrome and AFLP (acute fatty liver pregnancy) syndrome (5), hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3- both of which are life-threatening obstetric conditions. ketothiolase (KT) activities consecutively. Inherited deficiency of TFP Structures of soluble multifunctional proteins have been de- is a recessive genetic disease, manifesting in hypoketotic hypoglyce- termined, including two bacterial TFPs from Pseudomonas fragi mia, cardiomyopathy, and sudden death. We have determined the (PfTFP, ref. 6) and from Micobacterium tuberculosis (MtTFP, ref. crystal structure of human TFP at 3.6-Å resolution. The biological unit 7) and rat peroxisomal multifunctional enzyme 1 (PMFE-1, ref.

of the protein is α2β2. The overall structure of the heterotetramer is 8), the latter of which is a bifunctional protein with ECH and the same as that observed by cryo-EM methods. The two β-subunits HAD activities, similar to the α-subunit of mitochondrial TFP. make a tightly bound homodimer at the center, and two α-subunits However, no membrane-bound mitochondrial TFP or peroxi-

are bound to each side of the β2 dimer, creating an arc, which binds somal multifunctional enzyme structures have been determined, on its concave side to the mitochondrial innermembrane. The catalytic despite intensive biochemical and structural studies. Only very residues in all three active sites are arranged similarly to those of the recently (while we were preparing this manuscript), the structure corresponding, soluble monofunctional enzymes. A structure-based, of human TFP determined by cryo-EM was published (9). Here, substrate channeling pathway from the ECH active site to the HAD we report the crystal structure of human TFP determined at 3.6- and KT sites is proposed. The passage from the ECH site to the HAD Å resolution and compare and contrast the two human TFP α β BIOCHEMISTRY site is similar to those found in the two bacterial TFPs. However, the structures. While the 2 2 structure and membrane orientation passage from the HAD site to the KT site is unique in that the acyl- are nearly identical, there are notable differences between the CoA intermediate can be transferred between the two sites by pass- models determined by cryo-EM and crystallography, presumably ing along the mitochondrial inner membrane using the hydrophobic due to the different methodology and resolution. One major nature of the acyl chain. The 3′-AMP-PPi moiety is guided by the difference is the structure of α6β6, a different oligomeric state of positively charged residues located along the “ceiling” of the channel, human TFP found in the crystal structure. Also included in this suggesting that membrane integrity is an essential part of the chan- report is a detailed description of putative substrate channeling nel and is required for the activity of the enzyme. pathways between the three active sites. Furthermore, the struc- ture allows us to propose a mechanism of remodeling fatty acid oxidation | mitochondrial trifunctional protein | activity (monolysoacyltransferase) of the TFP α-subunit. substrate channeling | metabolon Significance atty acid β-oxidation is the principal energy-yielding process Fin organisms ranging from bacteria to humans. It is carried Fatty acid β-oxidation is the major energy-producing process in out by a series of enzymes that successively cleave acetyl-CoA all tissues and is performed by four consecutive reactions that fragments from fatty acyl-CoA thioesters until the fatty acyl-CoA cleave fatty acids. Mitochondrial trifunctional protein (TFP) is completely degraded. The resulting acetyl-CoA is further ox- performs the last three of these four reactions. Herein, we re- idized in the tricarboxylic acid cycle. Each cycle of β-oxidation port the crystal structure of human TFP and compare and pathway is composed of four reactions: acyl-CoA de- contrast this with the recently reported cryo-EM structure. The hydrogenase (ACAD), 2-enoyl-CoA hydratase (ECH), 3- crystal structure reveals the channel through which the sub- hydroxyacyl-CoA dehydrogenase (HAD), and 3-ketothiolase strate/product can pass from the first reaction site to the sec- (KT). In mammalian mitochondria, four separate soluble en- ond and onto the third, thereby minimizing leakage of the zymes carry out each of the four reactions during the processing of intermediates/product to the outside of the protein. Our find- short and medium chain fatty acids (for review; ref. 1). However, ings provide a better understanding of how this enzyme degradation of very long chain fatty acids is carried out by two functions and reveal insight into the development of inhibitors proteins that are bound to the mitochondrial inner membrane; the or agonists for the regulation of fatty acid degradation. first reaction is carried out by very long chain acyl-CoA de- hydrogenase (VLCAD), and the next three reactions are carried out Author contributions: C.X., Z.F., and J.-J.P.K. designed research; C.X., Z.F., K.P.B., and by a single protein, trifunctional protein (TFP) (2). TFP is com- J.-J.P.K. performed research; C.X., Z.F., K.P.B., and J.-J.P.K. analyzed data; and C.X., Z.F., and J.-J.P.K. wrote the paper. posed of two subunits: the α-subunit contains the first two of the The authors declare no conflict of interest. remaining three activities (ECH and HAD), while the β-subunit bears the KT activity (Fig. 1). This article is a PNAS Direct Submission. Mutations in either subunit of TFP can result in reduced ac- Published under the PNAS license. tivity of all three TFP enzyme activities, leading to disease. In Data deposition: The atomic coordinates and structure factors have been deposited in the TFP-deficient patients, at least 32 mutations in the α-gene and Protein Data Bank, www.wwpdb.org (PDB ID code 6DV2). 30 mutations in the β-gene have been reported. Clinical 1C.X. and Z.F. contributed equally to this work. phenotypes include hypoketotic hypoglycemia associated with 2To whom correspondence should be addressed. Email: [email protected]. metabolic acidosis, cardiomyopathy, and retinopathy (3, 4). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Deficiency of the long chain HAD (LCHAD) activity in the 1073/pnas.1816317116/-/DCSupplemental. α-subunit has also been found in children of women who develop Published online March 8, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1816317116 PNAS | March 26, 2019 | vol. 116 | no. 13 | 6069–6074 Downloaded by guest on September 30, 2021 The Structure of the α2β2 Heterotetramer. Fig. 4 shows two different views of the α2β2 heterotetramer. The overall structure of the α2β2 heterotetramer is the same as that observed by the cryo-EM method (9). The two β-subunits make a tightly bound homo- dimer as found in other known thiolase structures. Two α-subunits are bound to each side of the β2 dimer. The overall folds of each α- and β-subunit alone are essentially the same as the corresponding subunits of two known bacterial TFP struc- tures, one from P. fragi (Pf TFP) (6) and the other from M. tuberculosis (MtTFP) (7). However, the domain/subunit arrangement in human TFP (hTFP) is very different from those of bacterial TFPs. Comparison of the three structures and se- quence alignments reveal that there are a few major insertions and some structural divergences in each subunit. These minor structural differences in the hTFP structure compared with the β Fig. 1. Schematic diagram of fatty acid -oxidation in mitochondria. TFP bacterial TFPs play important roles in defining the subunit in- and VLCAD are associated with the mitochondrial inner membrane. LCAD, ’ MCAD, and SCAD represent long-, medium-, and short-chain acyl-CoA de- terfaces, resulting in the protein s membrane-binding property, hydrogenase, respectively. which, in turn, leads to the formation of various quaternary structural assemblies of hTFP that have been observed pre- viously. It is likely that the biological assembly of hTFP on the α β α β α β Results and Discussion mitochondrial inner membrane is an 2 2, not 4 4 nor 6 6, Biochemical Characterization of the Escherichia coli-Expressed Human because in the latter two structures, the membrane-binding side TFP. Human TFP (hTFP) was expressed in an E. coli system and of the molecule is secluded and would not be exposed to bind to purified to homogeneity (SI Appendix, Fig. S1). The recombinant the mitochondrial membrane. enzyme has overall activity (0.7 unit/mg of purified protein; Fig. 2A) comparable to the native enzyme purified from pig heart Membrane-Binding Affinity. Comparison of the structures and se- mitochondria (1.0 unit/mg of purified protein; ref. 10). Although quences of three TFPs (i.e., membrane-bound hTFP and two α β soluble bacterial TFPs) clearly revealed a major insertion in the the soluble bacterial TFPs exist as 2 2 heterotetramers, the β oligomeric state of mitochondrial TFP has been generally ac- -subunit of the hTFP structure, corresponding to residues Met179-Leu207, which include the hydrophobic H4-H5 (In- cepted as α4β4. Native rat liver TFP was reported to exist as α4β4 as determined by size-exclusion chromatography (11), and sertion 1 in SI Appendix, Fig. S2A). A closer inspection of the recombinant human TFP was found by sedimentation velocity three TFP structures showed that, starting from Ser169, the main chain of the human TFP-β structure differs from the two bacterial analysis to be a mixture of α2β2, α4β4, and α6β6, with the α4β4 form in the majority. Our size-exclusion chromatographic results (Fig. TFP structures merging back at Glu221. In this 53-residue di- vergent region, about 40 residues from Asp170 to Pro209 form the 2B) show that the protein exists in solution as a mixture of α6β6 putative membrane binding helices, H4-H5 in the hTFP structure and α4β4, with the latter in the majority, consistent with pre- viously published results of recombinant human TFP (12). In- (SI Appendix, Figs. S3A and S4A), while in both bacterial TFPs, terestingly, in the presence of 24 mM β-octyl glucoside (OBG), this region is shorter, has a different secondary structure, and is the protein was eluted as a sharp peak, corresponding to an apparent molecular mass of ∼320 kDa (close to the calculated value for α2β2, 260 kDa). However, in the presence of 0.4 mM n- dodecyl-β-maltoside (DDM), the protein was eluted as a mixture of α2β2 and α4β4, which is again consistent with the oligomer distribution observed in the cryo-EM studies (9).

Crystal Symmetry, Oligomeric States, and Membrane-Binding of Human TFP. The crystal structure of hTFP has been determined at 3.6-Å resolution (13). The asymmetric unit of the hTFP crystal contains three α2β2 units forming a large ball with a diameter of ∼150 Å and has a 32 symmetry (three twofold axes perpendicular to the threefold axis) (Fig. 3 and Movie S1). The inside of the ball is occupied by six helix (H4)–loop–helix (H5) segments (see SI Appendix, Fig. S2 for assignment of the secondary structures, hereafter referred to as H4-H5) corresponding to residues Asp170-Val214, from each of the six β-subunits, and another six helix (H10) segments from the α-subunits. In addition, at the north and south poles of the ball, where three α-subunits meet, lie two hydrophobic residues, Phe277 and Ile275, from the end of H10 of the α-subunit, making hydrophobic interactions (Fig. 3C). Fig. 2. (A) Enzymatic activity of various TFP proteins. The overall reaction Similar hydrophobic interactions were also observed at the dimer activity is in milliunit/mg (purified TFP), Numbers in parentheses are the numbers of repeated measurements. (B) Chromatographic elution profile of interface in the α4β4 species found in the recent cryo-EM studies wild type (green), Δβ(170-209) (blue), and F277K (cyan). Purified proteins of hTFP (9). In the cryo-EM α4β4 structure, the hydrophobic residues (275-IleProPhe-277) of one α β heterotetramer in- were applied to a size-exclusion column (Bio-Rad Enrich SEC 650) running 2 2 buffer containing 25 mM Tris·HCl, pH 7.5, 150 mM NaCl and 5% at a teract with the corresponding part of the other tetramer making “ ” flow rate of 0.4 mL/min using a Shimadzu Prominence HPLC system. Wild- a skewed clam shell like structure. However, size-exclusion type TFP in the same running buffer plus 24 mM octyl-β-D-glucopyranoside chromatographic results (Fig. 2B) showed that the mutant pro- (black) or plus 0.4 mM n-dodecyl-β-D-maltoside (red) is also shown. The ap- α β tein (F277K) was still mainly 4 4, suggesting that, in the absence parent molecular mass of the major peaks 1 and 2; and the shoulder peak 3 of detergent or bilayer, the TFP protein exists as α4β4 or are calculated at about 317, 554, and 813 kDa, respectively, corresponding to α β α β α β α β α β 6 6, not 2 2. TFP 2 2 (258 kDa), 4 4 (516 kDa), and 6 6 (774 kDa) oligomeric states.

6070 | www.pnas.org/cgi/doi/10.1073/pnas.1816317116 Xia et al. Downloaded by guest on September 30, 2021 Fig. 3. Surface representation of the structure of the

α6β6 form. One asymmetric unit of the human TFP crystal contains three α2β2 units, which form a ball with a 32 symmetry (three twofold axes are perpendicular to the threefold axis as marked). All α-subunits are shown in green or orange/yellow shades, and β-subunits are shown in blue or magenta. (A) View down a twofold axis lo-

cated at the middle of a α2β2 unit. (B)Viewdownthe threefold axis, where three α-subunits meet. (C)Enlarged view of the hydrophobic interactions among Ile275 and Phe277 of the three α-subunits at the threefold axis.

involved in making an interface with the ECH domain of their Importance of the Membrane Integrity. Judging from the distances corresponding α-subunits (SI Appendix,Fig.S4A). between the membrane-binding helices in the α2β2 structure, the To confirm the membrane affinity of H4-H5, we made a curvature of the bend is ∼50 Å (i.e., a circle of 50-Å radius). This mutant, in which the 40 residues between Asp170 and Pro209 in suggests that, in the absence of membrane, large micelles (di- the β-subunit were deleted [Δβ(170-209)], and the resulting TFP ameter of ∼100 Å) would be required to stabilize the curved shape mutant was expressed in the same manner as the wild-type of the α2β2 structure. Interestingly, in the presence of 24 mM OBG protein. Compared with the wild-type protein, which was [1× critical micelle concentration (CMC)], only the α2β2 form expressed nearly 100% in the membrane fraction, the mutant exists, while in the presence of 0.4 mM DDM (3× CMC), about protein was expressed in and purified from both soluble and two-thirds exists as α4β4 and the remaining one-third as α2β2 (Fig. membrane fractions with a 2:3 ratio, respectively. Presumably, 2B). OBG forms only very small size micelles, while DDM can the remaining membrane-binding affinity of the deletion mutant form stable 16 × 28 Å oblate-shaped micelles (14). This is con- must lie in the α-subunit. Unlike the β-subunit, for the α-subunit, sistent with the EM results that in the presence of OBG, no stable there is no apparent amino acids insertion between Asp253 and hTFP image can be recorded since OBG does not form the large Phe277. However, this region’s secondary structure and spatial stable micelles required to stabilize the membrane curvature to fit BIOCHEMISTRY arrangement are very different from the bacterial TFPs. In the α2β2 structure. However, in the presence of DDM, both α2β2 hTFP, this part extrudes out of the main body of the ECH do- and α4β4 forms were observed in the EM images (9). main of the α-subunit, forming another membrane-binding helix, H10 (SI Appendix, Figs. S3A and S4B), consistent with the Catalytic Residues in All Three Active Sites Are Conserved. In the membrane-binding property of the α-subunit. hTFP structure, as in the two bacterial TFPs, the α-subunit is The spatial arrangement of these membrane-binding regions is composed of two large domains. The N-terminal domain con- consistent with the ball-shaped structure of the α6β6 form de- tains enoyl-CoA hydratase activity (ECH) and the C-terminal scribed above. These membrane-binding regions are situated domain contains 3-hydroxyacyl-CoA dehydrogenase activity inside the “ball” and sequestered from the outside aqueous so- (HAD). The β-subunit contains the keto thiolase activity (KT). lution. Furthermore, these membrane regions are likely inter- The structures of these three active sites are well conserved from acting with each other directly or indirectly via detergent those of the corresponding mitochondrial soluble enzymes. For molecules to further stabilize the α4β4 and α6β6 forms. There are each of the three active sites in the hTFP structure, the corre- some patches of electron density inside the ball that are scattered sponding substrate can be modeled in, demonstrating that each in between the membrane-binding helices, especially between active site has a large/long enough cavity to accommodate long the α-subunit helix H10 and the β-subunit helix H5. These are fatty acyl chains, and that the catalytic residues for each active most likely either from detergent molecules that remain bound site are all preserved (SI Appendix, Fig. S5). to the protein during the protein purification (Tween 20) or α C8E5 detergent molecules included in the crystallization media. Subunit Interfaces. In hTFP, each -subunit makes two interfaces β α β As with the F277K mutant, the amount of the α6β6 form in the with the 2 dimer: one between the ECH domain of 1 and 1 β-subunit deletion mutant [Δβ(170-209)] was decreased, but the and the other between the HAD domain of α1 and β2 (Fig. 4, α β α β α4β4 amount remained the same as the wild type (Fig. 2B). This is Right; note the assignment of 1, 1, 2, and 2). There are due probably to the fact that an exquisite arrangement of the short-sequence insertions in both α-and β-subunits: one with three Ile275/Phe277 interactions among the three α-subunits is residues Leu225-Ile237 in the α-subunit (loop between S7 and required to assemble the α6β6 ball structure. The deletion of H4- H9 and extending into part of H9 in SI Appendix, Fig. S2B) and H5 of the β-subunit would eliminate the interactions between the Ala392-Lys408 in the β-subunit forming a short helix–loop–helix membrane-binding helices in both subunits, resulting in making structure (SI Appendix, Fig. S2A). These two regions together the H10 of the α-subunit more mobile and weakening the pivotal with helix H6 in the β-subunit (three red-highlighted regions in Ile275/Phe277 interactions among the three α-subunits. SI Appendix, Fig. S2) form the first subunit interface between the

Fig. 4. Cartoon representation of the α2β2 structure. The ECH domain (Thr37-Thr333) and HAD domain (Lys334-Gln763) of α1 are shown in light and dark green, respectively. The corresponding domains of α2 are in light and dark orange, respectively. β1isin blue and β2 in magenta. The shaded area in Left rep- resents possible hydrophobic interactions with a curved membrane, and four red dashed ellipses in Right in- dicate interfaces between the α-andβ-subunits.

Xia et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6071 Downloaded by guest on September 30, 2021 ECH domain of α1- and the β1-subunit (Fig. 4, Right and SI sites would be beneficial for TFP enzyme efficiency. Indeed, Appendix, Fig. S3B, Left) with an interface area of 820 Å2, con- Schulz and coworkers (10) and Eaton et al. (2) have provided taining a significant amount of hydrophobic interaction as evidence for channeling between the active sites of the TFP characterized by the PISA interface calculation (www.ebi.ac.uk/ protein. Since TFP contains three enzyme activities utilizing a pdbe/pisa). In the bacterial TFPs, there is only one interface channeling mechanism, we consider that TFP is a mini metab- between the two subunits, i.e., between the ECH domain of the olon of the fatty acid β-oxidation (2). Here, we propose a α-subunit and the β-subunit. The HAD domain of the α-subunit structure-based channeling pathway from the ECH active site to makes no other contact with the β2-dimer, allowing some flexi- HAD to KT active sites. bility between the β2 dimer and the HAD domain (6). This flexibility in the PfTFP is necessary for the substrate channeling From the ECH Site to the HAD Site. Ishikawa et al. (6) suggested that between the HAD active site in the α-subunit to the KT active in PfTFP, the substrate channeling from the ECH to HAD active site in the β-subunit (6, 15). However, in hTFP there is a second sites occurs by flipping the hydrophobic acyl group of the sub- interface between the α1-subunit and the β2 dimer, which is strate, while fixing the AMP-PPi moiety of the acyl-CoA sub- formed between helices H16 and H17 of the α1 HAD domain strate to the common binding pocket, thus avoiding the substrate and H1, H2, S6, and S7 of the β2 subunit (area, 607 Å2) (six pink- diffusing into the solvent. Although the substrate binding modes highlighted regions in SI Appendix, Fig. S2, also Fig. 4, Right and that have been modeled in the ECH and HAD sites of the hTFP SI Appendix, Fig. S3B, Right). With both the ECH and HAD structure (SI Appendix, Figs. S5 and S6) do not share a common domains fixed to the β2 dimer, the hTFP α2β2 structure is more binding pocket for the AMP-PPi moiety, the substrate channel- rigid than the bacterial TFPs. This also infers that the curved ing mechanism between these two active sites appears similar to membrane-binding domain arrangement observed in the α2β2 that suggested for PfTFP. In hTFP, the channel from the ECH to structure is indeed that of the hTFP structure in mitochondria. HAD active sites is also located in the cleft between the ECH and HAD domains, as shown in Fig. 5. Along both edges of the Substrate Channeling Between Active Sites and the Concept of a cleft, there are arrays of positively charged residues stretching Metabolon. The concept of a metabolon was first proposed by out to the middle of the cleft, and of particular interest is a Welch (16) over 40 y ago and heavily promoted by Srere (17). cluster of positively charged residues, including Arg165, Arg205, The concept is that many groups of enzymes within common Arg208, Arg211, Lys411, Lys414, and Lys415, that are situated metabolic pathways are physically associated together into about halfway between the two active sites (Fig. 5 and SI Ap- “metabolons,” e.g., TCA cycle enzymes, glycolytic enzymes, or pendix, Fig. S6). In addition, a series of hydrophobic residues are fatty acid oxidation enzymes. The idea makes good sense, but no lining the “bottom” of the cleft, which could make hydrophobic stable such complexes have been observed in vitro, indicating interactions with the fatty acyl group of the substrate during the that these complexes are weakly associated and perhaps their substrate channeling. The distance between the two active sites, assembly needs to be regulated. Recently, there have been many ECH and HAD, is about 27 Å. The distance was measured be- observations that support the existence of metabolons. For ex- tween the pyrophosphate oxygen atoms (-P-O-P-) of each sub- ample, Benkovic and coworkers (18) demonstrated by fluores- strate bound to the active sites, since the highly negatively cent labeling techniques that the enzymes in purine biosynthetic charged pyrophosphate group plays an important role in the enzymes colocalize in vivo (“purinosome”). The large separa- electrostatic retention of the substrate, preventing it from dif- tions between the ECH, HAD, and KT active sites strongly fusing into the bulk aqueous solvent. The depth of the cleft, suggests that a sequential substrate channeling between the three measured from the hydrophobic residues lying at the bottom to

Fig. 5. Proposed substrate channels between the three active sites. The same color scheme is used as in Fig. 4, unless stated otherwise. (A) Proposed channel from the ECH to HAD binding sites in the α-subunit (pink) and from the HAD to KT binding sites in the β-subunit (gray). The channels were created using the program HOLLOW (hollow.sourceforge.net). When creating the channel from HAD/α1toKT/β1, a set of lipid bilayer molecules was also included to mimic the bound membrane. The dashed line represents the other possible substrate pathway from HAD/α1toKT/β2, that has a large portion open to the solvent (see text for details). Substrates/intermediates are shown with cyan sticks. For clarity, the substrate acyl binding cavities are not included. (B) A different view from A with a 90° rotation along the x axis. For clarity, the α2-subunit is not shown. The alternative channel from HAD/α1toKT/β2 (dashed line) clearly shows the solvent-exposed passage. (C) Schematic drawing of the substrate channeling between the three active sites. The negatively charged 3′- AMP-PPi moiety (shown as red octagons labeled as AMP-PPi) and the fattyacyl group (black zigzags) are linked by a pantetheine group. The channel between the ECH and HAD sites (pink) has a length of about 27 Å, a width of ∼12 Å at the widest section, and a depth of ∼20 Å (receding into the plane of the diagram). Its volume is ∼2,900 Å3. The channel between the HAD and KT sites (gray) is ∼49 Å long with a cross-section of ∼10 × 10 Å, excluding the hydrophobic membrane bilayer (beneath the plane of the diagram). Its volume is ∼6,000 Å3. The overall substrate/product channeling events, after 2-enoyl-CoA binds to the ECH active site and 3-hydroxyacyl-CoA is formed, are as follows: (1) The negatively charged AMP-PPi is relocated from the ECH active site to the positively charged area situated at the interface between the ECH/HAD domains (dotted red octagon). (2) The hydrophobic acyl group is released from the ECH active site and relocated to the HAD active site. (3) The AMP-PPi then binds to the HAD active site. The above three steps complete the channeling from the ECH to HAD sites. (4) Then, the negatively charged AMP-PPi moves fromthe HAD binding site back to the positively charged area (dotted red octagon). (5) The hydrophobic ketoacyl group is released from the HAD active site and moves into the hydrophobic membrane bilayer. (6) The 3-ketoacyl-CoA intermediate wades through the HAD-to-KT channel and finally binds to the KT active site.

6072 | www.pnas.org/cgi/doi/10.1073/pnas.1816317116 Xia et al. Downloaded by guest on September 30, 2021 the cluster of the positive residues covering the cleft is about computational analysis of simulation of substrate transfer, will be 20 Å. Therefore, when the negatively charged AMP-PPi moiety of required to establish how the substrates/intermediates are the substrate is fixed by the cluster of positively charged residues transferred from one active site to the other. at the ceiling of the channel/tunnel, there is enough room for the acyl group to change its orientation with respect to the AMP-PPi Membrane Is Required for Channel Formation. Since the membrane group from that found in the ECH active site to that observed at is an essential part of substrate channeling, any disruption of the HAD active site. Thus, the landscape and the topology of the interactions between the membrane and the membrane-binding ECH-to-HAD pathway appears to be well-suited for channeling regions of the protein would affect the channel integrity as well the fattyacyl-CoA intermediate, 3-hydroxyacyl-CoA, from the as the overall enzyme activity. Our activity data (Fig. 2A) show ECH site to the HAD site. that in the presence of OBG, there is no activity, while only 66% activity was observed in the presence of DDM. These activity From the HAD Site to the KT Site. Although the structures of each data appear to coincide with the percentage amount of the α4β4 individual domain/subunit in hTFP are very similar to the cor- form determined by our size-exclusion chromatographic results responding domains in two known bacterial TFP structures (Fig. 2B), suggesting that the α2β2 form has no activity. At first [MtTFP (PDB ID code 4B3I) and PfTFP (PDB ID code glance, this contradicts the fact that in mitochondria, hTFP exists 1WDK)], the arrangements of the two α-subunits relative to the as the α2β2 form and must be active. There are two possible β2 dimer are very different. Therefore, the distances between the explanations for this inconsistency: (i) OBG is an inhibitor by HADactivesiteoftheα-subunit and the KT active site in binding to the active site and (ii) OBG disrupts membrane in- the β-subunit are very different among the three TFPs. There are tegrity. For the following reasons, the latter is a more plausible α two possible pathways from the HAD active site of the -subunit explanation. When hTFP exists in the α2β2 form in the presence to the KT active site of the β-subunit: α1toβ1 and α1toβ2. (For of OBG or DDM, their micelles are not large enough to form bacterial TFPs, α1, β1, α2, and β2 are defined such that α1toβ1 stable interactions with the TFP’s membrane-binding regions, and α2toβ2 are closer than the distance between α1to β2 and α2 resulting in the formation of an unstable (OBG) or partially to β1. For hTFP, they are defined as shown in Fig. 4.) For both stable (DDM) curved membrane, not with the curvature re- bacterial TFPs, one pathway is obviously preferred to the other. quired to form a stable α2β2 heterotetramer. In the case of the α In PfTFP, the distance from the HAD active site of the 1 α4β4 form, the curved membrane is conserved by combining two β subunit to the KT active site of 1 is 21 Å, while the distance α2β2 tetramers that results in a skewed clam shell (see Importance from α1toβ2 is 52 Å (6). In MtTFP, the distance from the HAD of the Membrane Integrity above). Apparently, DDM micelles are α β active site of the 1 subunit to the KT active site of the 1 capable of stabilizing the curved membrane structure required BIOCHEMISTRY α β subunit is 31 Å, while the distance for 1to 2 is 58 Å (7). In for the formation of the clam shell structure. Thus, the α4β4 form addition, this α1-to-β2 passage is also blocked by the ECH do- in the presence of DDM is active. Similarly, when the enzyme main. However, in hTFP, the situation is very different. First, the assay was performed in the presence of dimyristoyl-sn-glycero-3- distances between the two active sites (for both α1toβ1 and α1 (DMPC) liposomes, which can provide the to β2) are much longer than those of the preferred pathways in curved membrane, an increased overall activity was observed both MtTFP and PfTFP; thus, they would require a more effi- (Fig. 2A), again consistent with the second explanation. cient, tighter channeling. Second, at first glance, there is no preferable pathway: The distance from the HAD/α1 active site to Pathogenic Mutations. Inherited deficiency of TFP or LCHAD is a the KT/β1 active site is 49 Å, while that to the β2-subunit is 52 Å, recessive genetic disorder. Among over 60 missense mutations not significantly different. However, a careful inspection of the found in patients, the most common TFP mutation is the E510Q environment near the two pathways revealed that they are very mutation in the α-subunit (c.1528G > C), accounting for about 87% different. As shown in Fig. 5A, considering the presence of a of the alleles in LCHAD deficiency and a cause of AFLP (20). When membrane bilayer at the bottom, a relatively well-protected we expressed the E510Q mutant in E. coli, the enzyme is stable but channel can be formed between the HAD/α1 and the KT/β1 has drastically decreased activity (14% of wild type, Fig. 2A and SI active sites. However, the pathway from the HAD/α1 to the KT/ Appendix,Fig.S1), consistent with the results from IJlst et al. (20), β2 active sites is almost completely exposed to the bulk solvent which showed diminished HAD activity (∼25% of wild type) when the on one side of the pathway (dashed line in Fig. 5 A and B). mutant was expressed in Saccharomyces cerevisiae. This result is not Another significant feature for the α1toβ1 pathway is the surprising, since Glu510 is a part of the catalytic dyad for the HAD abundance of positively charged residues along this pathway (SI activity of TFP (see section on CatalyticResiduesinAllThreeActive Appendix, Fig. S6). Most noticeably, there are two clusters of Sites Are Conserved,alsoSI Appendix,Fig.S5). Other mutations in positively charged residues next to each other, Arg205, Arg208, both the α-andβ-subunits have been shown to result in the loss of all Arg211, Lys213, and Lys214 of α1 in one cluster and Lys411, three enzyme activities, resulting in complete TFP deficiency. A Lys413, Lys414, and Lys415 in the other, located at about one- mapping on the α2β2 structure of the 46 mutation sites currently third of the way into the channel (SI Appendix, Fig. S6C). The identified in patients of TFP deficiency has been reported (9). first three residues in each cluster are shared with the ECH- HAD channel. Similar to the channel between the ECH and The Linkage Between hTFP α-Subunit and Cardiolipin Remodeling. HAD active sites, the negatively charged AMP-PPi can be Cardiolipin is a major component of mammalian attracted by the clusters of positively charged residues at the mitochondrial inner membrane and supports the mitochondrial ceiling (opposite side of the membrane) of the channel. Because respiratory chain complexes (21). It has a characteristic acyl the mitochondrial membrane is composed mainly of neutral chain composition that depends on the function of phospholipid- (44% phosphatidyl and 34% phosphatidyl etha- lysophospholipid transacylases, including and the newly nolamine) (19), it is unlikely that the negatively charged AMP- identified monolysocardiolipin acyltransferase-1 (MLCLAT-1). PPi group of the substrate would be attracted to membrane head MLCLAT-1 is identical to the TFP α-subunit (TFPα) that groups. More importantly, the hydrophobic fatty acyl group can lacks the first N-terminal 191 residues (not counting the mito- be easily directed to the hydrophobic membrane bilayer and chondrial transit peptide) (22). Based on the structure of TFPα, reorient itself from the HAD active site toward the KT active we propose a plausible catalytic mechanism of MLCL acyl- site. For these reasons, we conclude that the pathway from the transferase (SI Appendix, Fig. S7). From the inspection of the α1 HAD active site to the β1 KT active site is preferred to the HAD active site and binding cavities of the substrate (3- + one from the α1 HAD active site to the β2 KT active site, con- hydroxyacyl-CoA) and cofactor NAD , we concluded that the + trary to the conclusions made from the cryo-EM studies (9). A MLCL molecule would bind at the NAD -binding groove in the schematic model of substrate channeling is shown in Fig. 5C (see HAD active site of TFPα. For the HAD activity in TFPα, resi- legend for description of the model). Further studies, including dues His498 and Glu510 act as a catalytic dyad to abstract the

Xia et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6073 Downloaded by guest on September 30, 2021 3-hydroxyl proton from the substrate 3-hydroxyacyl-CoA (23). In Milwaukee. These cDNAs were subcloned into E. coli expression vectors, MLCLAT-1, this pair of residues would activate the hydroxyl pET21b and pET28a, resulting in TFPα/pET21b and TFPβ/pET28a, respectively. group of MLCL by forming an H bond between MLCL-OH and Both expression plasmids were cotransformed with groEL/ES plasmid into Ne2 of His498. The activated MLCL-OH then undergoes a nucleo- E. coli BL21(DE3) competent cells. The protein was purified using Ni-NTA philic attack of the C1 of linoleoyl-CoA, and the tetrahedral in- affinity chromatography. Detailed description of these experimental pro- termediate is stabilized by an oxyanion hole consisting of the hydroxyl cedures is available in SI Appendix. sidechains of Thr548 and/or Ser477, in a similar manner found in a squash glycerol-3-phosphate (1)-acyltransferase (G3PAT, ref. 24). Enzyme Activity Assay. The TFP-specific activity for the overall reaction (i.e., three reactions coupled) was assayed as described (10), Briefly, 5 μLofTFP Although TFPα does not contain a conserved HisX4Asp motif as solution containing about 1–10 μg of TFP protein was added to 0.5 mL of assay observed in classical acylglycerophosphate transferases, the constel- + lation of His-Asp/Glu in TFPα, together with the C1 atom of mod- solution, containing 0.1 M potassium phosphate buffer (pH 7.6), 1 mM NAD , μ eled linoleoyl-CoA, is identical to that observed in the squash G3PAT 0.2 mM CoA, and 20 M 2-transhexadecenoyl-CoA, to initiate the overall en- zyme reaction at 23 °C. The product NADH was monitored by the absorbance structure. Ser477 lies in close proximity to the C1-carbonyl group of μ linoleoyl-CoA and is conserved in all monofunctional HADs, bi- increase at 340 nm. One unit of enzyme activity is defined as 1 mole of NADH functional enzymes, and TFPs. However, for the MLCLAT-1 activity, produced/min. To obtain the TFP activities in the presence of detergents, Thr548 might be in a better position to stabilize the tetrahedral in- 0.2 mM DMPC, 24 mM OBG, or 1 mM DDM was included in the assay solution. termediate. Thr548 is not conserved in most HADs, bifunctional enzymes, and soluble bacterial TFPs, but is conserved in all known Crystallization, Data Collection, and Structure Determination. Crystals were obtained by hanging drop vapor-diffusion method, with crystallization dips mitochondrial TFP sequences, including human, pig, bovine, rat, + composed of 2 μL of the protein solution (0.1 mM hTFP, 20 mM NAD ,1mM mouse, and zebrafish. Further studies including site-directed mu- acetoacetyl-CoA, and 0.5% C8E5) and 2 μL of reservoir solution (0.1 M Hepes tagenesis are necessary to confirm the exact role of each residue in buffer pH 7.0, 12% PEG3350, and 0.2 M MgCl2). Diffraction data were col- the active site. lected at Beamline IMCA-CAT 17-ID-B at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL. Data were processed by pro- Concluding Remarks. The structure of human TFP has been de- grams Mosflm and Scala in the CCP4 program package (25). The initial termined by X-ray crystallography at 3.6-Å resolution. The struc- structure was determined using the Phaser program (26), as detailed in SI ture reveals how the substrate can travel through the three active Appendix, Methods. Refinement was carried out using iterative cycles of CNS sites. The pathway between the ECH to the HAD sites is similar to refinement followed by manual fitting and rebuilding using the COOT those found in two bacterial TFPs. However, the pathway from the graphics software (27). Chains A and B (β2) and G and H (α2) have the most HAD to the KT sites is novel in that the acyl-CoA substrate can be residues modeled in and, therefore, unless otherwise stated, Chains A, B, G,

transferred between the two active sites by wading along the mi- and H corresponding to one α2β2 heterotetramer were used for structural tochondrial inner membrane using the hydrophobic nature of the interpretations. Data collection and processing statistics and the final re- acyl chain, while the AMP-PPi moiety is guided by the positively finement statistics are given in SI Appendix, Table S1. charged residues located along the ceiling of the channel. Thus, the membrane integrity is an essential part of the channel and is ACKNOWLEDGMENTS. We thank Dr. Suresh Kumar for his generous gift of required for the overall activity of the enzyme. It is likely that cDNAs encoding hTFP. Use of the Industrial Macromolecular Crystallography VLCAD and TFP would associate closely on the mitochondrial Association-Collaborative Access Team (IMCA-CAT) beamline 17-ID at the membrane and form a metabolon of fatty acid β-oxidation. Advanced Photon Source was supported by the companies of the IMCA through a contract with the Hauptman-Woodward Medical Research In- Materials and Methods stitute. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the Protein Cloning, Expression, and Purification. Two cDNAs each encoding the α- DOE Office of Science by Argonne National Laboratory under Contract DE- (TFPα/pcDNA3.3) or β-subunit (TFPβ/pcDNA3.3) of human TFP protein were AC02-06CH11357. This work was supported by National Institutes of Health the generous gift of Suresh Kumar at the Medical College of Wisconsin, Grant GM29076.

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