The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2 þ and 3 þ with a pK a near 6. A 1.65 Å resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with wellordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.ion channels | M2 proton channel | membrane proteins | water clusters | histidine protonation W ater molecules confined at interfaces or in cavities behave differently from those in the bulk. Studies of water clusters have shed light not only on their fundamental properties (1-4) but also on the mechanism employed by various nano-bio systems to fine-tune water and proton transport (5-9). A relevant example from biology is the M2 protein of the influenza A virus (10, 11), which is the target of the influenza drugs amantadine and rimantadine (12-17). Tetrameric M2 (18) transports protons across the viral envelope to acidify the virion interior and trigger uncoating of the viral RNA prior to fusion of the viral envelope with the endosomal bilayer (19). M2 is one of the smallest bona fide channel/transporter proteins (96 residues), capable of pHdependent activation and highly selective conduction of protons vs. other ions (20)(21)(22)(23)(24)(25). A narrow pore leads to the highly conserved His37 and Trp41 residues (16,17,(26)(27)(28)(29), which are respectively responsible for proton selectivity (30) and asymmetry in the magnitude of conductance when the proton gradient is reversed (31). Thus the control of proton diffusion across the membrane relies on the ability of the imidazole moieties of His37 to accept and store protons from water molecules in the pore.An M2 peptide (residues 22-46), slightly longer than the transmembrane domain (32), associates into a functional four-helix bundle (33). Solid state 15 N nuclear magnetic resonance (ssNMR) experiments indicate that the first protons ...

The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pKa values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.liposomes ͉ oocyte ͉ H ϩ ͉ intracellular pH ͉ Xenopus laevis

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high and low pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.M2 is a 96-residue homotetrameric integral membrane protein with a small N-terminal ectodomain, a single transmembrane helix and a C-terminal cytoplasmic tail. Despite data from solid state NMR (1), x-ray crystallography (2) and solution NMR (3), a detailed understanding of how the M2 protein works continues to puzzle investigators and generate sharp controversy.The majority of published studies on the proton channel function of M2 have focused on the transmembrane (TM) 1 domain. However, truncation studies indicate that the cytoplasmic domain also plays a role in channel stability (4). Proteolysis of micelle-bound full length M2 revealed that a 15-20 residue segment C-terminal to the TM helix was highly protected from cleavage by proteases (5). Helical wheel analysis of the protected region (5) suggested that the segment could form an amphiphilic helix, consistent with later findings from solid state NMR on M2 protein in lipid bilayers (6). In order to further test the proposed models, we probed the conformation of the segment C-terminal to the TM domain at both high and low pH using sitedirected spin-labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy.EPR studies were performed on a series of 38-residue synthetic M2 peptides (residues 23-60; M2TMC) spanning the TM region and the beginning of the C-terminal domain. We spin- † This research was supported by R01AI57363 (LHP), GM56423 (WFD), a Henry Dreyfus Teacher Scholar Award (KPH) and R15AI074033 (KPH).

The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 μM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H 2 O to THF it decreased by 8 cm −1 . The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra-and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.

The structure and function of the Influenza A M2 proton channel have been the subject of intensive investigations in recent years because of its critical role in the life cycle of the Influenza virus. Using a truncated version of the M2 proton channel (i.e., M2TM) as a model, here we show that fluctuations in the fluorescence intensity of a dye reporter that arise from both fluorescence quenching via the mechanism of photoinduced electron transfer (PET) by an adjacent tryptophan (Trp) residue and local motions of the dye molecule can be used to probe the conformational dynamics of membrane proteins. Specifically, we find that the dynamics of the conformational transition between the N-terminally-open and C-terminally-open states of the M2TM channel occur on a timescale of about 500 μs and that binding of either amantadine or rimantadine does not inhibit the pH-induced structural equilibrium of the channel. These results are consistent with the direct occluding mechanism of inhibition which suggests that the antiviral drugs act by sterically occluding the channel pore.

The influenza A M2 protein exhibits inwardly rectifying, pH-activated proton transport that saturates at low pH. A comparison of high-resolution structures of the transmembrane domain at high and low pH suggests that pH-dependent conformational changes may facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. Here, we show that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 pK a s. We then develop and test two similar, quantitative mechanistic models of proton transport, where protonation shifts the equilibrium between structural states having different proton affinities and solvent accessibilities. The models account well for a collection of experimental data sets over a wide range of pHs and voltages and require only a small number of adjustable parameters to accurately describe the data. While the kinetic models do not require any specific conformation for the protein, they nevertheless are consistent with a large body of structural information based on high-resolution NMR and crystallographic structures, optical spectroscopy, and MD calculations.The influenza virus infects cells by receptor-mediated endocytosis, which is followed by acid-induced fusion of the viral and endosomal membranes. The fusion event is mediated by a conformational change of the influenza hemagglutinin proteins, triggered by the low pH in the endosome lumen (1). It is also necessary, upon fusion, that the viral interior be acidified in order for the viral RNA to be freed in a competent form into the cell cytoplasm (2). Viral acidification within the time of endosomal residence requires a proton transport facilitator denoted "M2" (matrix protein 2), a 97 residue homotetrameric integral membrane protein (3). In addition, it has been shown that influenza strains with higher pH of transition of hemagglutinin from the pre-fusion to the fusion-competent state also require proton transport through M2 when newly synthesized viral proteins are trafficked in the Golgi † This research was supported by NIH grants R01AI57363 to L.H.P. and U011074571 to W.F.D. and R.A.L. R.A.L., L.H.P., and W.F.D. are founders and members of the SAB of Influmedix (www.influmedix.com).

No abstract

BACKGROUND: Adolescent and adult patients with neuroblastoma appear to have a more indolent disease course but a lower survival rate compared with their younger counterparts. The majority of neuroblastoma tumors specifically accumulate the radiolabeled norepinephrine analogue iodine‐131–metaiodobenzylguanidine (131I‐MIBG). Therefore, 131I‐MIBG has become increasingly used as targeted radiotherapy for patients with recurrent or refractory neuroblastoma. The objective of the current study was to characterize the toxicity and activity of this therapy in older patients. METHODS: The authors performed a retrospective analysis of 39 consecutive patients aged ≥10 years with recurrent or refractory neuroblastoma who were treated with 131I‐MIBG monotherapy at the University of California at San Francisco under phase 1, phase 2, and compassionate access protocols. RESULTS: Sixteen patients were aged ≥18 years at the time of MIBG treatment initiation, whereas 23 patients were ages 10 to 17 years. The median cumulative administered dose of 131I‐MIBG was 17.8 millicuries (mCi)/kg. The majority of treatments led to grade 3 or 4 hematologic toxicities (graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events [version 3]) that were similar in frequency among age strata. Three patients subsequently developed a hematologic malignancy or myelodysplasia. The overall rate of complete plus partial response was 46%. Patients aged ≥18 years at the time of first MIBG treatment had a significantly higher response rate compared with patients ages 10 to 17 years (56% vs 39%; P = .023). The median overall survival was 23 months with a trend toward longer overall survival for the subgroup of patients aged ≥18 years (P = .12). CONCLUSIONS: The findings of the current study suggest that 131I‐MIBG is a highly effective salvage agent for adolescents and adults with neuroblastoma. Cancer 2011;. © 2011 American Cancer Society.