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.