Investigation of proton conductance in the matrix 2 protein of the influenza virus by
solution NMR spectroscopy
© Daniel Turman
Emmanuel College Class 0[2012 Abstract
The Influenza Matrix 2 (M2) protein is a homo-tetrameric integral membrane protein that forms a proton selective transmembrane channell Its recognized function is to equilibrate
pH across the viral envelope following endocytosis and across the trans-golgi membrane
during viral maturation2 Its function is vital for viral infection and proliferation but the mechanism and selectivity of proton conductance is not well understood. Mutagenesis
studies have identified histidine 37 as the pH sensing element and tryptophan 41 as the
gating selectivity filter3 This study uses solution nuclear magnetic resonance
spectroscopy and an M2 transmembrane protein construct to elucidate key interactions
between the aromatic residues believed to confer proton selectivity and pH dependent
conduction of M2 in the low pH open and high pH closed states. PH dependent 13 C_1 H
HSQC-Trosy experiments were completed in the pH range of 8.0 - 4.0 and the 13 C, and
13 Co2 chemical shift perturbations of histidine 37 revealed multiple saturation points. The
protonation states of histidine 37 suggest a shuttling mechanism for proton conduction.
Introduction
Influenza is a pathogenic virus that has reached pandemic status four times in the twentieth century. The latest pandemic occurred in 2009 from the influenza A HINI
strain (figure 1t The World Health Organization (WHO) commented in July of 2009,
"this outbreak is unstoppable." Following this event, significant research has been
allocated to understand all aspects of the influenza virus in an effort to produce effective vaccines and medications to prevent and control another pandemic. Pandemic (H1N1) 2009, Status as of 06 July 2009 Number of laboratory confirmed cases as reported to WHO 09:00 GMT
Cumulative deaths • 1 -10 • 11 -50 • 51 -100 ,~,. ., • 101 and more Cumulative cases 0 1-10 _ . 11-50 . 51-500 Chinese Taipei has report ed 61 confi rmed cases of pandemic (H1 N1 ) 2009 w ith 0 deaths. Cases fr om .. 501 and more Chinese Taipei are included in the cu mulati ve totals
Figure 1: World Health Organization global publication of the 2009 HINI pandemic.
Unfortunately, influenza experiences antigenic drift as a result of its seasonal nature making longstanding antiviral therapy difficult to produce.
Influenza viruses are comprised of a negative-sense RNA core encapsulated in a lipid envelope that contains necessary protein machinery for virus infection and
2 replication . Upon infection, the viral particle incorporates into the endosome pocket of endothelial respiratory cells by receptor-mediated endocytosis. The pH within the endosome decreases from ~6 to ~5 prior to membrane fusion. This leads to acidification of the influenza vi ron by the M2 protein and Hemigluttinin (HA) mediated membrane fusion. Viral ribonucleoproteins (RNP) are then ejected into the host cell cytosol (figure
2). M2 also plays a crucial role in viral maturation equilibrating the pH across the trans- golgi network prior to virus budding\figure 3). The M2 protein has been the focus of significant research due to its presence and function in all variants of influenza viruses. The medications rimantidine and amantidine were widely used to combat influenza until the early 1990's. Following significant overuse of rim anti dine in poultry, evolutionary
pressure led to mutations in the M2 protein rendering rimantidine ineffective for future
anti viral therapy5
Endosomal M2 Activity (Earl y Role)
Endosome
ri bonucleoprotein complexes
Figure 2: Role ofM2 in the early stage of the influenza life cycle.
trans-Go igi net work M2 Activity (Late Ro le)
Goigi ves icle
Figure 3: Role ofM2 in the late stage of the influenza life cycle prior to viral budding.
The M2 protein is a 97 amino acid membrane protein that is crucial to virulence
in influenza6 It is comprised of an N-terminal extra-viral region (residues 1-23), trans-
membrane segment (residues 24-46) and an intra-viral C-terminal region (residues 47-97)
and functions as a highly selective proton transporter across the viral membrane\Figure
4). The transmembrane section is believed to contain necessary amino acid elements that
provide proton selecti vity. - N-Terminal Extra-Viral (23 Residues)
Transmembrane [ -~ A -tJ: (19 Residues)
C-Terminal Intra-Viral (54 Residues) -
Figure 4: Graphical representation ofthe M2 protein. M2 has an integral membrane protein with intra and extra viral domains.
The activity of M2 was discovered by electrophysiology studies in xenopus
oocytes, where it was found that M2 is proton selective2 The ftrst structural
characterization ofM2 was completed in detergent micelles and calculated from solution nuclear magnetic resonance spectra 3(Figure 5). The structure of M2 revealed a homo-
tetrameric four right-hand helices bundle with a clearly defined pore. This study also proposed a radical allosteric mechanism to rirnantidine inhibition that was heavily
debated. In addition, this structure was completed at pH 7.5 and attempts to determine a
low pH structure were unsuccessful due to loss of signal and protein construct
. 3 compromlse . Figure 5: Solution NMR structure of the influenza M2 protein in complex with rimantidine.
In response to the publication of this structure several additional solution and solid-state NMR structures were completed to assist in understanding the mechanism of
3 inhibition and proton conduction ofM2 , 7-10. Figure 6 was the first crystal structure published for M2 and it showed electron density in the pore, which was attributed to amantidine. Interestingly, the resolution of the crystal structure was 3.5 angstroms, while the amantidine cage is under 3.5 angstroms in diameter10.
Figure 6: Crystal structure ofM2 at pH 7.5 showing amantidine complexed in the pore. Nevertheless, much study into the drug binding site in the M2 protein has
ll revealed that the primary, physiological relevant, binding site is within the pore . The final determining study relied on data collected from an AM2-BM2 chimera protein that
contains structural characteristics of the pore binding site and no allosteric binding site
characteristics 12 This protein did not exhibit drug resistance leading to the hypothesis that the drug does in fact bind in the pore.
From these structures it was also clear that the presence of a conserved
H37XXXW41 motif in the transmembrane region coupled with mutagenesis studies
confirm that histidine 37 and Tryptohan 41 are responsible for unidirectional pH
dependent proton conductance across the virus membrane3 These studies of the transmembrane region ofM2 in solution and solid state NMR demonstrate a four-helix
9 bundle with a hydrophobic core occluded by histidine 37 and tryptophan 41 • 13. Histidine
37 has been characterized as the channel gate initiating conductance at pH ~5.4 while tryptophan 41 appears to occlude the C-terminus of M2 promoting unidirectional proton
conduction through possible cation-n interactions between histidine 37 and tryptophan 41
upon protonation of histidine 3i4 However, the precise role of histidine 37 has been heavily debated.
Two models have been proposed for the mechanism of proton conductance in M2.
The "shuttle" model has been described as a Michaelis-Menten like kinetic mechanism with a rate-limiting step attributed to protonation and subsequent tautomerization of histidine 3i4In comparison, the "shutter" model attributes proton conduction to electrostatic pore widening as a result of histidine 37 protonation and formation of an
intermittent water wire indicative of a Grotthuss type mechanism7 (Figure 7). The
Grotthuss water wire mechanism is based on hydrogen bond chains that result in a rate of
proton conductance far faster than calculated proton diffusion rates in water15 Studies of the voltage-gated proton channel Hvl, which forms a Grotthuss water-wire for proton
40 transport, resulted in a rate of conductance near 10 protons/second 16 In contrast, M2
exhibits proton conductance of 10 protons/second/channee· 17. 18 The slow rate of proton
conductance in M2 supports the "shuttle" mechanism, which depends on protonation of histidine 37 in the channel pore18 (Figure 8).
Vonll_
Figure 7: A water wire Grothuss type mechanism for proton conduction. This mechanism is diffusion limited only. r d )"i H H' H" ~.l H ..H Y E ' IY • ~, . • '='- •I Ir • I •I •r N61-H IMJtOtnlfl ~ N. 1-H~ • • l intff'tM'di.to! I• I 0• 0
Figure 8: Shuttle type mechanism representation where histidine 37 would convert between tautomers and an imidiazolium intermediate.
3 19 The mechanism of M2 proton selective and conduction is still heavily debated .
Outstanding results support both a water wire type mechanism as well as a histidine
3 shuttle mechanism . 7. 8 The lack of structural data in the low pH conducting range hampers efforts to pinpoint the exact role of histidine 37 and tryptophan 4l.
Determination of their role in atomic detail would offer necessary information to infer the
exact role of these key amino acids. Publication of the structure of an A1!2-BM2 chimeraprote!n m 2011, which sett! ed th e debate about the ct-ug -binding Sl te , op ens the door to detenmmng these key features m a low pH env!ronmentll The AM2-BM2 Chim era 1)J:2-chimera) structure was solvedm solution NMR at high pH (figure 9). This system is surpnSlngly stable atlow pH and offers the opportunity to study the conduction of protons through this channelm atomic det,.,l with solution NMR
Figure 9: M2-Cbimera structure solved by solution NMR. Insets show a supenmposition of the AM2 and BM2 elements and their structural Similarity between the wild type and chimera structure
D eterm!nati on of the atomic !nteracti on, of the key histidine 37 and tryptophan 41 will offer !nSlght mto the role of these amino ac!ds!n th e conduction ofM2. The outstanding qu esti ons of how protons ar e sel ecti vely shuttled through thi s transmembrane pore fonning protein will fill a crucial gap in knowledge within this field. The use of
solution NMR spectroscopy is an ideal platfonn to complete these studies due to the
ability to address dynamic pH dependent chemical shift perturbations. Functional assays
are also presented to prove the concept of the chosen model system. It is believed that histidine 37 has a specific chemical environment within the pore due to the unique
packing. The electrostatic environment is believed to change upon protonation of one,
possibly two, histidines leading to multiple saturation points in the channel histidines.
Detennination of these saturation points is key in choosing the correct pH to carry out rigorous structural characterizations of the pore-lining aromatic amino acids.
Methods
Protein preparation
Recombinant expression of the M2-Chimera protein
C8RSNDSSDPL VV AASIIGIe7HFIA WTIGHLNQIKR52G) construct was completed in
Escherichia coli. A 9 histidine c-tenninal tag as well as Trp-LE c-tenninal fusion in a
pMM-LR6 vector was incorporated to target the M2-Chimera complex to inclusion
3 bodies Minimal media for cell culture contained 50 mM Na2HP04, 10 mM KH2P04, 5 mM NaCl, 100 mM 15NH4Cl, 2 mM MgS04, 2 mM 13C6H1206 and ImM CaCho E. coli
cultures were induced with IPTG at an OD600 of 0.75 at 37"C and temperature was reduced to 18°C and allowed to grow overnight. E.coli cultures were then harvested by
centrifugation at 18,000 rpm for 30 minutes. From this point forward Beta mercaptoethanol at 0.01% was incorporated to all buffers for reducing conditions. Cell pellets were resuspended in lysis buffer (50 mM Na2HP04, 200mM NaCl) and sonicated thoroughly. Celllysates were then centrifuged at 18,000 rpm for 30 minutes to pellet
inclusion bodies. Cell pellets were resuspended in 6 M guanidine, 200 mM NaCl, 50 mM
Na2HP04 and 0.1 % Tritton-X overnight. Resuspended cell pellets were then purified on a nickel-NTA column and M2-Chimera was eluted with 50 mM Na2HP04, 6 M guanidine,
200 mM NaCI and 400 mM imidiazole at pH 6.8. Eluted M2-Chimera was then dialyzed
against 0.01 % BME and water overnight at 4°C. Dialyzed material is then centrifuged at
18,000 rpm for 30 minutes to pellet precipitated protein. Pelleted protein is then digested
in 70% formic acid and cyanogens bromide to remove the Trp-LE and 9 histidine tag from the M2-Chimera protein. Reaction is quenched with dialysis against water and
lyophilized in 50% acetonitrile. Lyophilized protein is then reconstituted in hexafluoroisopropanol, formic acid and water in a 2: 1: 1 ratio and the M2-Chimera is
purified from reaction mixture on a (Grace-Vydac) C4 column by reverse-phased HPLC.
Final M2-Chimera peptide is lyophilized and stored at _80°C. Characterization of purity was completed by SDS-Page and labeling efficiency and molecular weight of monomer was characterized by MALDI-TOF mass spectrometry.
Functional analysis
Functional analysis was completed in a liposome assay as published20 Liposomes were
prepared as documented in withE.Coli polar lipids (Avanti) in an internal buffer (50 mM
(C3HsO(COO)3), 50 mM Na2P04, 122 mM NaCl, 122 mM KCl). Thin films were
dissolved in inner buffer noted above,S [tM M2-Chimera and 0.1 [tM valanomycin and
extruded through a 0.2 [tm filter (Avanti) and analyzed by dynamic light scattering for ull1fonn S1 ze. Buffer exchange Into external buffer (122 mM NaCl, 122 mM KQ and 5 mM N""Po.) was completed on a PD-l0 salt exchange column (Sigma) Finalliposomes m external buffer were ali cp oted In 1 5 mL eppenoorfs and stored at 4° C for no more than 12 hours pnor to assay. The assay was compl eted by moru tonng pH as afuncti on of tim e (3 second mcrements) W1th a mlcro-pH electrode (Sigma). 1 M 2nd 0.5 M HQ was us ed to titrated mto assay to decrease th e pH of the external buffer under cootinuoos m1Xmg. The change m pH of the external buffer was then us ed to detennme proton flux from M2-Chimera
pH ...... _ _
/stronglY bllff.. ed (Internal) ~. ./ wukly , buff.r .... "- (1t"ltrn.l) Upo>Ome ~. Iv.II .... "'l'dnl
Figure 10: Graphical reJXe sentation ofliposome assay
Solution NMR spectroscopy
&lmple Prqaration
All chemicals wer e purchased from :igma Aldrich at th e highest aVailable purity unless othe!W1 '" sp ecified. Lycphilized M2-Chimera pepti de In 2 mg allquotS was r esuspended m andrefolded to a tetramer state In 6 M guall1 din e and 150 mM 1,2-diheptanoyJ-.sn- glycero-3-phosjllocholine (DHPC) and dialyzed agai nst 40 mM Na,PO, and 40 mM C3HsO(COO)3 overnight to remove guanidine. The sample was then concentrated to 275
[tL in an Ambicon 3000 Dalton cutoff concentrator. 25 [tL of D20 was then added to
sample and 300 [tL of sample was then loaded to a Shigemi NMR tube. pH titration
experiments were completed by titrating 0.1 M HCI to desired pH. A total of 29 separate
pH points were recorded.
NMR Experiments
All pH titration experiments were conducted on a bruker 600 MHz spectrometer
equipped with cryogenic probes. Sample temperatures were at 30°C for duration of each
experiment. 13 C_l H transverse optimized heteronuclear single quantum coherence (trosy
HSQC) experiments were completed at each pH point and the root mean squared change
in chemical shift was graphed as a function of pH21 Assignment of histidine 37 and tryptophan 41 resonances was completed from published and un-published data 3. 11. 22
Results
Protein Purification
M2-Chimera was successfully expressed in E. coli in minimal media as noted in figure
1l. Pre-induction (lane 2) shows no protein at ~ 18 kDa (fusion protein monomer) while
post induction (lane 3) does show a clear band at ~ 18 kDa. Nickel-TA purification noted
in lanes 4 thru 6 show clearly the wash step that removes unwanted protein and the
elution step with imidiazole that liberates the M2-Chimera fusion peptide from the nickel beads resul ling ln pure M 2- Chimer a fus10n pro tem. Reverse Fhase high-pressure liqU1d
chromato graphy resulted m sep aralion of the cl eave d M 2-Chimer a peptide from th e
cyano gens bromlde reoction mlXture as noted ln figure 1 2 The M 2- Chimer a p epli de was th en characterlzed by MALD J-TOF mass sp ectrometry (fi gure 13). The parent p eak at
4 00 9. 7974 mlz represents a 99% lab eling efficlency Mmor p e2l
possibly double charge d sp ecle, The Yl el d of this punficalion was -1 0 mg/L of final
M 2-Chimera peptide
u ' ,.. .. - ./ ,•
Fi gure 11 SDS -Page gel from 13 C_" N lab ele d M2 -Chimera punfi cali on A _ """"' A ""r...... lIi10 1 ~aJ25 ll15 4145511 2 ~'00$00i.~~.'--r~------TS~~~~~==~~ Chaperone 1~ Protein l!il 11l Uncleaved II. 01 O~ Protein OlO Oil ------M2 Chimera .Il
0.1l JJ1Jl 9l1Jl AU ..... r.... AU
Figure 12: HPLC chromatogram depicting separation of Trp-LE from M2 Chimera peptide. The black line indicates the solvent gradient, pnrple line depicts absorbance at 214 nm and the green line depicts absorbance at 280 nm.
Applied Blosyst&ms 4700 Proteom/cs An_/yzer ZOOS "_~.p.'" .C(Bp · ...... '."S]
4009.7974 '"I "' I
8018.5874 ,I "
",
12023.0498
1 ". 16031 .6484 ,',..,. 1 ., ,.""' .... 2003B.8555
Figure 13: MALDI -TOF mass spectrum of 13C, 15N labeled M2-Chimera peptide. Parent peak at m/z 4009.7974 representing a 99% labeling efficiency. functional Analysis ofM2-Chimru.
Analy":s 0 fthe function of the M2-Chimru. peptide was completed in a liposome as",y as describ ed in the methods section. figure 14 depicts a ":ngle liposome as",y of the M2-
Chimru. protein. Conduction rates were measured at 10 +/- 1 H*/sltetramer based on the
following equation and the derivation that can be found in reference"
The as=ption of 50 % orientation oftetramers in the liposome is noted in the final term
of the equation
6.09
~ 6.07 ~
•0 6.05 S" 6.03 If w• 6.01
5.99 -15 18' 2" " Time (s)
figure 14 Liposome assay analysis ofM2-Chimru. shows conduction at a rate of 10 H+/s/tetramer Solution NMR pH Titration
Table one shows the pH, chemical shift in parts per million and the change in chemical
shift as determined by the euclidean root mean square distance noted in the following
equation for histidine 37.
Where 0 denoted chemical shift in parts per million and 0) is a weighting factor (114 or
23 13 C) that corrects for the gyromagnetic ratio ofheteronuclear atoms Table 2 shows the
same data for histidine 45. Chemical shift data was tabulated from nmrDraw software
(nmrPipe). The resulting titration curves and spectra are noted in figure 15. Both titration
curves were fit to the Hill equation as noted below18
Where A is a fitting constant representing the max change in shift and H is the hill
coefficient. In this application of the Hill equation the hill coefficient is a fitting
parameter with no physical meaning. The multi-modal titration curve noted in histidine
37 was graphed with a linear combination of the Hill equation resulting in an addition fitting factor B as well as a second hill coefficient. The resulting pKa of histidine 45 was
6.33 ± 0.03 and the pKa's of histidine 37 were 4.93 ± 0.03 and 6.20 ± 0.03. Table 3 has
all fitting parameters determined by non-linear least squares regression in Prism. Table I: Histidine 37 chemical shift and change in chemical shift data.
Histidine 37 lHe1Y Ce1 pH l'C (ppm) lH (ppm) Chan2e in Shift 7.95 115.9109 7.5014 0.00 7.76 115.9213 7.5043 0.01 7.52 116.1974 7.5029 0.29 7.5 116.3798 7.5117 0.47 7.29 116.0181 7.5295 0.11 7.14 115.9186 7.5517 0.05 6.93 115.8942 7.5687 0.07 6.72 115.9382 7.6373 0.14 6.6 115.8166 7.6387 0.17 6.57 115.4764 7.6653 0.46 6.36 115.8998 7.7141 0.21 6.34 115.8308 7.7089 0.22 6.12 115.7379 7.7776 0.33 6.09 115.4967 7.6629 0.44 5.95 115.7174 7.8261 0.38 5.8 115.4377 7.7862 0.55 5.63 115.4881 7.997 0.65 5.53 115.4946 8.1096 0.74 5.37 115.4121 8.1582 0.82 5.12 115.311 8.3692 l.05 5.04 115.3982 8.3116 0.96 4.98 115.2162 8.4676 l.l9 4.86 115.1761 8.5206 l.26 4.63 115.0757 8.6306 1.40 4.44 115.013 8.6806 1.48 4.22 115.0887 8.6129 l.38 4.22 114.9959 8.7141 l.52 3.87 114.9644 8. 7573 l.57 Table 2: Histidine 45 chemical shift and change in chemical shift data.
Histidine 45 IHe1Y Ce1 pH 13C (ppm) IH (ppm) Chan2e in Shifts 7.95 ll6.4274 7.4562 0 7.76 ll6.398 7.4693 0.03 7.52 ll5.9737 7.5268 0.46 7.5 ll5.8443 7.513 0.59 7.29 ll6.0181 7.5295 0.42 7.14 ll5.9186 7.5517 0.52 6.93 ll5.6702 7.6154 0.77 6.72 ll5.471 7.7012 0.99 6.6 ll5.2635 7.744 l.20 6.57 ll5.8794 7.5814 0.56 6.36 ll5.0281 7.8049 l.44 6.34 ll4.9212 7.802 l.55 6.12 ll4.6502 7.8742 l.83 6.09 ll4.4392 7.8374 2.02 5.95 ll4.4977 7.8886 l.98 5.8 ll4.2178 7.8889 2.25 5.63 ll4.1431 7.9457 2.34 5.53 ll4.1704 8.0027 2.32 5.37 ll4.0681 8.0ll9 2.42 5.12 ll4.0344 8.0316 2.46 5.04 ll4.0756 8.0448 2.42 4.98 ll3.9961 8.0676 2.51 4.86 ll3.982 8.0502 2.52 4.63 ll3.8719 8.0922 2.63 4.44 ll3.8666 8.0729 2.63 4.22 ll3.923 8.0652 2.58 4.22 ll3.8519 8.0811 2.65 3.87 ll3.8571 8.0865 2.65 Discussion
It is shown that the influenza Ml [Kotein is an integral [Kotein in the life cycle of the influenza virus and it fllllction is vital to all variants. M2 selective conwctance of protoo.s is a llllique fllllction in a viral system in the rate and selectivity in which it perronns. Past
antiviral therapy has targeted this [Kotein but mutations have conferred resistance to these medications. Elucidation of the role of the key anxnatic amino acids histidine 37 and tIYPtophan 45 is key in determining the mechanism of proton conductioo. and future
antiviral therapy. It is also shown that the Ml-C1timera protein is a valid experimental
system to study proton conwctance much like it was used to settle the debate regarding inhibition!!. - • His 37
Histidine 45 1He1-1 3Ce1 Histidine 37 l He1-1 3Ce1
r i~ 1.5 . . ,., "~ •< •, ,• ,., ,, 1.' • ! .! '-' ", '.5 "£ ,.,
~ O· ~.9 4.4 4.9 5.4 • . .. ,.. •• ,.. ,.. ~ o . ~. g '" ,H • ••• • .. ,.. '-' ••• ,.. ,.. " " ,H
Figure 15: pH titratioo. data foc both histidine 37 and 45 with spectra cocresponding to each point. An experimental system should be readily available for study. In the case of the
M2-Chimera much of the wet chemistry involved is in the purification, characterization
and sample preparation steps. It is shown that this system can be purified as evidenced by the presence of clear expression bands in figure 12, lane 3. Further processing of the
protein results in an M2-Chimera peptide that is separated from its purification
chaperones Trp-LE and the 9 histidine tag liberating a monomer of preferred product.
Characterization by MALDI-TOF mass spectrometry ensures that the predominant
species in the sample is in fact isotopically labeled and very close to the theoretical monomer weight. However, there are oligomeric species in the mass spectrum that are
difficult to explain, as the sample is prepared in 50 % acetonitrile. This is a relatively
polar organic solvent that should dissociate hydrophobic helix-helix weak interactions.
Yet another key determination of an accurate biochemical system is the mimicking of native activity. This is also shown in the in vitro liposome assay data
collected during this study. The liposome assay is an accepted method for proton
conductance in the influenza M2 protein18 The liposome assay is limited by the presence
of proton leakage in some samples. This is corrected by preparing control assays that are
used to baseline correct for assumed proton leakage as a result of a loosely oriented
protein-liposome population in the sample that may not be stable in a rapidly mixed
environment. Nevertheless, subsequent studies of the M2-Chimera protein in the
18 liposome assay has led to confirmed proton flux of 10 ± I H+ /s/tetramer This aligns well with previously published data for M2 wild type albeit very slow conduction
(decoursey, schnell, lamb pinto). Interestingly, the presence of very slow proton
conduction is strong evidence of a histidine shuttle mechanism. Diffusion limited Grothuss mechanism based proton conductance through water is much faster on the order
3 of 1 x 10 H+ /s 15 Studies on the deuterium isotope effect in water diffusion have also
showed that D+ introduction into in vitro systems does not lead to a change in the
conduction rate of M2 wild type24
Previous studies noted above indicate a clear need to understand in atomic detail the mechanism of proton conductance in the influenza M2 protein and the role of histidine 37 and tryptophan 4l. A low pH picture of M2 in its state of conductance is
crucial in understanding how this protein performs its function. This study provides pH
dependent characterization of histidine 37 in an effort to determine saturation points to
guide further study. This characterization is completed using the M2-Chimera system with solution NMR spectroscopy. Solution NMR, while intrinsically limited by size due to low tumbling rates in large proteins, is an excellent method to characterize pH
dependence of histidine 37 in this system.
It is shown in this study that histidine 37 exhibits multiple pKa's in the pH range
of proton conductance. In addition, the presence of histidine 45 offers an internal control that titrates as a free histidine in solution with a pKa of 6.33 ± 0.03. This is a striking
example of the relative chemical environment of a packed histidine within a pore of the
M2-Chimera protein and the chemical environment of a lipid facing histidine. The
channel histidine 37 experiences multiple pKa's as noted in figure 65. The
physiologically relevant closed state at pH ~ 7.5 is believed to be characterized by a
shared hydrogen between the histidine 37 tetrameric complex. As the endosomal pH
decreases below ~6.00 protonation of one of the histidines in the complex releases the
shared proton creating an imidiazolium ion in two of the histidines. As the pH continues to decrease this state is saturated at pH ~5.5. This agrees very well with published data regarding the endosomal environment25 This state of saturation is considered the "open"
state that is of utmost importance for further structural characterization. Further decrease
of pH leads to the protonation of the second hydrogen bonded histidine moiety and most
likely a greatly increased pore size due to electrostatic repulsion created by the four
imidiazolium's in the pore.
The next crucial step is the atomic characterization of the interactions between histidine 37 and tryptophan 41 in the saturate state determined during this study at pH
5.5. Nuclear Overhauser Spectroscopy will be used to identify distance restraints between these residues in the aromatic regions. Also, an open-state structure will be calculated to
determine the difference in the closed and open structures of the influenza M2 protein
leading to a complete structural characterization of the proton conductance in this protein.
Table 3: Best fit values from non-linear least squares regression analysis.
Best-fit values histidine 45 Parameter Value A l.568 H l.26 PK1 6.335 Std. Error A 0.02595 H 0.083 PK1 0.02621 95% Confidence Intervals A l.513 to l.622 H l.085 to l.434 PK1 6.280 to 6.390 Goodness of Fit R squared 0.9913 Table 4: Best fit values from non-linear least squares regression analysis.
Best-fit values histidine 37 Parameter Value A l.l52 H 2.909 PKI 4.929 B l.lll PK2 6.201 Std. Error A 0.05536 H 0.4041 PKI 0.03253 B 0.04414 PK2 0.02944 95% Confidence Intervals A l.033 to l.270 H 2.042 to 3.776 PKI 4.860 to 4.999 B l.016 to l.206 PK2 6.138 to 6.264 Goodness of Fit R squared 0.9957 w " .t~ ~~,) ~3-J ~ ~~,) , • " "8 " - ", ", pKa, ,, 6.02 ~/- ,"' , " <""j" J~) , J~" .«NJ! " " " .~~ , " pKa,,, 4.93 +1· 0,03 " H, . y;'J! • "" Figure 16 Proposed model of the multiple protonation states of hdidine 37 as evidence by multiple pKa's noted in the pH titration data
Conclusion
The findings presmted in this study have implications for the detmnination of the role of hdidine in the function of the inlluema M2 protein. This is a key piece of data that will
drive the elucidation of the open state structl.!u of the inftuenza M2 protein leading to
complete structural characterization of the influenza M2 proton conduction ,,-., chani= References
1. Sugrue RJ, H. A., Structural Characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 1991, (180), 6l7- 624. 2. Rossman JS, L. R., Influenza virus assembly and budding. Virology 2011,411, (2), 229-36.
3. Schnell JR, C. J., Structure and mechanism of the M2 proton channel of influenza A virus. Nature 2008,7178, (451),591-5. 4. Prevention, C. f. D. C. a., 2009 HINI Flu. In 2011. 5. Abed Y, G. N., Boivin G, Generation and characterization of recombinant influenza A (HINl) viruses harboring amantidine resistance mutations. Antimicrobial Age 2005. 6. Cox NJ, N. G., Donis RO, Kawaoka Y, Orthomyxoviruses: influenza. In Wilson's Microbiological and Microbial Infections, Wiley: 1998; Vol. 2003. 7. Acharya R, C. V., Fiortin G, Levine BG, Polishchuk AL, Balannik V, Samish I, Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus. Proceedings of the National Academy ofSciences 2010,107, (34),15075-15080. 8. Hu, F.; Luo, W.; Hong, M., Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR. Science 2010,330, (6003), 505. 9. Sharma, M.; Yi, M.; Dong, H.; Qin, H.; Peterson, E.; Busath, D.; Zhou, H.-X.; Cross, T., Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer. Science 2010,330, (6003), 509. 10. Stouffer A, A. R., Salom D, Levine A, Costanzo L, Soto C, Tereshko V, Degrado W, Structural basis for the function and inhibition of an influenza virus proton channel. Nature 2008. 11. Pielak RM, O. K., Chou JJ, Structural investigation of rimantidine inhibition of the AM2-BM2 chimera channel of influenza virus. Cell Structure 2011. 12. Ohigashi, Y.; Ma, C.; Jing, x.; Balannick, V.; Pinto, L.; Lamb, R., An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition. Proceedings of the National Academy ofSciences of the United States ofAmerica 2009, 106, (44), 18775. 13. Pielak, R.; Schnell, J.; Chou, J., Mechanism of drug inhibition and drug resistance of influenza A M2 channel. Proceedings of the National Academy ofSciences of the United States ofAmerica 2009,106, (18), 7379. 14. Shimahara, H.; Yoshida, T.; Shibata, Y.; Shimizu, M.; Kyogoku, Y.; Sakiyama, F.; Nakazawa, T.; Tate, S.-i.; Ohki, S.-y.; Kato, T.; Moriyama, H.; Kishida, K.-i.; Tano, Y.; Ohkubo, T.; Kobayashi, Y., Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase. The Journal of biological chemistry 2007,282, (13), 9646. 15. DeCoursey, T., Voltage-gated proton channels. Cellular and molecular life sciences: CMLS 2008,65, (16), 2554. 16. Ramsey IS, M. Y., Carvacho I, Sands Z, Sansom MSP, Clapham DE, An aqueous H+ permeation pathway in the voltage-gated proton channel Hv-l. Nature Structural and Molecular Biology 2010, l7, (7), 869-75. 17. Lin T, S. C., Definitive Assignment of Proton Selectivity and Attoampere Unitary Current to the M2 Ion Channel Protein of Influenza A Virus. Virology 2000. 18. Pielak RM, C. J., Kinetic Analysis of the M2 proton conduction of the influenza virus. Journal ofthe American Chemical Society 2010, 132, (50), 17695-7. 19. Andreas LB, E. M., Pielak RM, Chou JJ, Griffin RG, Magic Angle Spinning NMR investigation of influenza A (18-60): support for an allosteric mechanism of inhibition. Journal oftheAmerican Chemical Society 2010. 20. Olson F, H. C., Szoka F, Vail W, Paphadjopoulos D, Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochimica et biophysica acta 1979. 2l. Riek R, K. P., Wuthrich K, TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution. TIBS 2000. 22. Wang, J.; Pielak, R.; McClintock, M.; Chou, J., Solution structure and functional analysis of the influenza B proton channel. Nature structural & molecular biology 2009, 16, (12), 1267. 23. Hass MA, Y. A., Christenson H, Led J, Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion. Journal of Biomolecular NMR 2009. 24. Zhou, H.-X., Mechanistic insight into the H201D20 isotope effect in the proton transport fo the influena virus M2 protein. Journal ofMembrane Biology 2011. 25. Geishow MJ, E. W., pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis. Experimental Cell 1984.