Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers.

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22-46) showed electron densities attributed to a single amantadine in the amino-terminal half of the pore, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein-amantadine distances resulted in a 0.3 A-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by (2)H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

Assembly of the transmembrane domain of E. coli PhoQ histidine kinase: implications for signal transduction from molecular simulations.

The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM) domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.

Structural basis for the function and inhibition of an influenza virus proton channel.

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Species differences in small molecule binding to alpha IIb beta 3 are the result of sequence differences in 2 loops of the alpha IIb beta propeller.

Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat alphaIIbbeta3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the alphaIIb beta propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human alphaIIbbeta3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the alphaIIbbeta3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with alphaIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of alphaIIbbeta3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the alphaIIb beta-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.

Molecular basis of ChvE function in sugar binding, sugar utilization, and virulence in Agrobacterium tumefaciens.

ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners.

pH-induced conformational change of the influenza M2 protein C-terminal domain.

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.

Loop modeling: Sampling, filtering, and scoring.

We describe a fast and accurate protocol, LoopBuilder, for the prediction of loop conformations in proteins. The procedure includes extensive sampling of backbone conformations, side chain addition, the use of a statistical potential to select a subset of these conformations, and, finally, an energy minimization and ranking with an all-atom force field. We find that the Direct Tweak algorithm used in the previously developed LOOPY program is successful in generating an ensemble of conformations that on average are closer to the native conformation than those generated by other methods. An important feature of Direct Tweak is that it checks for interactions between the loop and the rest of the protein during the loop closure process. DFIRE is found to be a particularly effective statistical potential that can bias conformation space toward conformations that are close to the native structure. Its application as a filter prior to a full molecular mechanics energy minimization both improves prediction accuracy and offers a significant savings in computer time. Final scoring is based on the OPLS/SBG-NP force field implemented in the PLOP program. The approach is also shown to be quite successful in predicting loop conformations for cases where the native side chain conformations are assumed to be unknown, suggesting that it will prove effective in real homology modeling applications.

Using multiple structure alignments, fast model building, and energetic analysis in fold recognition and homology modeling.

We participated in the fold recognition and homology sections of CASP5 using primarily in-house software. The central feature of our structure prediction strategy involved the ability to generate good sequence-to-structure alignments and to quickly transform them into models that could be evaluated both with energy-based methods and manually. The in-house tools we used include: a) HMAP (Hybrid Multidimensional Alignment Profile)-a profile-to-profile alignment method that is derived from sequence-enhanced multiple structure alignments in core regions, and sequence motifs in non-structurally conserved regions. b) NEST-a fast model building program that applies an "artificial evolution" algorithm to construct a model from a given template and alignment. c) GRASP2-a new structure and alignment visualization program incorporating multiple structure superposition and domain database scanning modules. These methods were combined with model evaluation based on all atom and simplified physical-chemical energy functions. All of these methods were under development during CASP5 and consequently a great deal of manual analysis was carried out at each stage of the prediction process. This interactive model building procedure has several advantages and suggests important ways in which our and other methods can be improved, examples of which are provided.

A photon-free approach to transmembrane protein structure determination.

The structures of membrane proteins are generally solved using samples dissolved in micelles, bicelles, or occasionally phospholipid bilayers using X-ray diffraction or magnetic resonance. Because these are less than perfect mimics of true biological membranes, the structures are often confirmed by evaluating the effects of mutations on the properties of the protein in their native cellular environments. Low-resolution structures are also sometimes generated from the results of site-directed mutagenesis when other structural data are incomplete or not available. Here, we describe a rapid and automated approach to determine structures from data on site-directed mutants for the special case of homo-oligomeric helical bundles. The method uses as input an experimental profile of the effects of mutations on some property of the protein. This profile is then interpreted by assuming that positions that have large effects on structure/function when mutated project toward the center of the oligomeric bundle. Model bundles are generated, and correlation analysis is used to score which structures have inter-subunit C(ß) distances between adjoining monomers that best correlate with the experimental profile. These structures are then clustered and refined using energy-based minimization methods. For a set of 10 homo-oligomeric TM protein structures ranging from dimers to pentamers, we show that our method predicts structures to within 1-2 Å backbone RMSD relative to X-ray and NMR structures. This level of agreement approaches the precision of NMR structures solved in different membrane mimetics.

Determination of the physiological dimer interface of the PhoQ sensor domain.

PhoQ is the transmembrane sensor kinase of the phoPQ two-component system, which detects and responds to divalent cations and antimicrobial peptides and can trigger bacterial virulence. Despite their ubiquity and importance in bacterial signaling, the structure and molecular mechanism of the sensor kinases is not fully understood. Frequently, signals are transmitted from a periplasmic domain in these proteins to the cytoplasmic kinase domains via an extended dimeric interface, and the PhoQ protein would appear to follow this paradigm. However, the isolated truncated periplasmic domain of PhoQ dimerizes poorly, so it has been difficult to distinguish the relevant interface in crystal structures of the PhoQ periplasmic domain. Thus, to determine the arrangement of the periplasmic domains of Escherichia coli PhoQ in the physiological homodimer, disulfide-scanning mutagenesis was used. Single cysteine substitutions were introduced along the N-terminal helix of the periplasmic region, and the degree of cross-linking in each protein variant was determined by Western blotting and immunodetection. The results were subjected to periodicity analysis to generate a profile that provides information concerning the C(beta) distances between corresponding residues at the interface. This profile, together with a rigid-body search procedure, side-chain placement, and energy minimization, was used to build a model of the dimer arrangement. The final model proved to be highly compatible with one of the PhoQ crystal structures, 3BQ8, indicating that 3BQ8 is representative of the physiological arrangement. The model of the periplasmic region is also compatible with a full-length PhoQ protein in which a four-helix bundle forms in the membrane. The membrane four-helix bundle has been proposed for other sensor kinases and is thought to have a role in the mechanism of signal transduction; our model supports the idea that signaling through a membrane four-helix bundle is a widespread mechanism in the transmembrane sensor kinases.

Identification of the pore-lining residues of the BM2 ion channel protein of influenza B virus.

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser(9), Ser(12), Phe(13), Ser(16), His(19), and Trp(23) to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser(9), Ser(12), Ser(16), or Trp(23). Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

Evaluating conformational free energies: the colony energy and its application to the problem of loop prediction.

In this paper, we introduce a method to account for the shape of the potential energy curve in the evaluation of conformational free energies. The method is based on a procedure that generates a set of conformations, each with its own force-field energy, but adds a term to this energy that favors conformations that are close in structure (have a low rmsd) to other conformations. The sum of the force-field energy and rmsd-dependent term is defined here as the "colony energy" of a given conformation, because each conformation that is generated is viewed as representing a colony of points. The use of the colony energy tends to select conformations that are located in broad energy basins. The approach is applied to the ab initio prediction of the conformations of all of the loops in a dataset of 135 nonredundant proteins. By using an rmsd from a native criterion based on the superposition of loop stems, the average rmsd of 5-, 6-, 7-, and 8-residue long loops is 0.85, 0.92, 1.23, and 1.45 A, respectively. For 8-residue loops, 60 of 61 predictions have an rmsd of less than 3.0 A. The use of the colony energy is found to improve significantly the results obtained from the potential function alone. (The loop prediction program, "Loopy," can be downloaded at http://trantor.bioc.columbia.edu.)