Mechanistic insights from functional characterization of an unnatural His37 mutant of the influenza A/M2 protein.

The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH8 in bilayers and is virtually inactive at external pH7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Correction: Dynein and Dynactin Leverage Their Bivalent Character to Form a High-Affinity Interaction.

[This corrects the article DOI: 10.1371/journal.pone.0059453.].

Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel.

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 pK(a) 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.

Structural basis for ligand-mediated mouse GITR activation.

Glucocorticoid-induced TNF receptor ligand (GITRL) is a member of the TNF super family (TNFSF). GITRL plays an important role in controlling regulatory T cells. The crystal structure of the mouse GITRL (mGITRL) was determined to 1.8-A resolution. Contrary to the current paradigm that all ligands in the TNFSF are trimeric, mGITRL associates as dimer through a unique C terminus tethering arm. Analytical ultracentrifuge studies revealed that in solution, the recombinant mGITRL exists as monomers at low concentrations and as dimers at high concentrations. Biochemical studies confirmed that the mGITRL dimer is biologically active. Removal of the three terminal residues in the C terminus resulted in enhanced receptor-mediated NF-kappaB activation than by the wild-type receptor complex. However, deletion of the tethering C-terminus arm led to reduced activity. Our studies suggest that the mGITRL may undergo a dynamic population shift among different oligomeric forms via C terminus-mediated conformational changes. We hypothesize that specific oligomeric forms of GITRL may be used as a means to differentially control GITR receptor signaling in diverse cells.

A pH-dependent conformational ensemble mediates proton transport through the influenza A/M2 protein.

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) values. 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 pH values 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 nuclear magnetic resonance and crystallographic structures, optical spectroscopy, and molecular dynamics calculations.

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex.

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus.

We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2's specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.

Experimental and computational evaluation of forces directing the association of transmembrane helices.

The forces that define the interactions of transmembrane helices have been evaluated using a model membrane-soluble peptide (MS1), whose packing is modeled on the two-stranded coiled-coil from GCN4. The thermodynamic stability of water-soluble coiled-coils depends on the side chain at the buried "a" position of the repeat, favoring large hydrophobic residues over small side chains. Here we show that just the opposite is true for the membrane-soluble peptide. Analytical ultracentrifugation and equilibrium disulfide interchange show that the stability of MS1 is greatest when Gly is at each "a" position of the heptad repeat (MS1-Gly), followed by Ala > Val > Ile. Moreover, MS1-Gly has a strong tendency to form antiparallel dimers, MS1-Ala forms a mixture of parallel and antiparallel dimers, while MS1-Val and MS1-Ile have a preference to form parallel dimers. Calculations based on exhaustive conformational searching and rotamer optimization were in excellent agreement with experiments, in terms of the overall stability of the structures and the preference for parallel vs antiparallel packing. The MS1-Gly helices are able to achieve more favorable and uniform packing in an antiparallel dimer, while MS1-Val and MS1-Ile have more favorable van der Waals interactions in a parallel dimer. Finally, the electrostatic component arising from the partial charges of the backbones become significant in the antiparallel MS1-Gly and MS1-Ala conformations, due to close packing of the helices. Thus, van der Waals interactions and electrostatic interactions contribute to the stability and orientational preferences of the dimers.

Using alpha-helical coiled-coils to design nanostructured metalloporphyrin arrays.

We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides.

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function.

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability.

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.

Relationship between salt-bridge identity and 14-helix stability of beta3-peptides in aqueous buffer.

We report a systematic analysis of the relationship between salt bridge composition and 14-helix structure within a family of model beta-peptides in aqueous buffer. We find an inverse relationship between side-chain length and the extent of 14-helix structure as judged by CD. Introduction of a stabilizing salt bridge pair within a previously reported beta-peptide ligand for hDM2 led to changes in structure that were detectable by NMR.

A hierarchic approach to the design of hexameric helical barrels.

The design of large macromolecular assemblies is an endeavor with implications for protein engineering as well as nanotechnology. A hierarchic approach was used to design an antiparallel hexameric, tubular assembly of helices. In previous studies, a domain-swapped, dimeric three-helix bundle was designed from first principles. In the crystal lattice, three dimers associate around a 3-fold rotational axis to form a hexameric assembly. Although this hexameric assembly was not observed in solution, it was possible to stabilize its formation by changing three polar residues per monomer to hydrophobic (two Phe and one Trp) residues. Molecular models based on the crystallographic coordinates of DSD (PDB accession code 1G6U) show that these side-chains pack in the central cavity (the "supercore") of the hexameric bundle. Analytical ultracentrifugation, fluorescence spectroscopy, CD spectroscopy, and guanidine-HCl denaturation were used to determine the assembly of the hexamer. To probe the requirements for stabilizing the hexamer, we systematically varied the polarity and steric bulk of one of the Phe residues in the supercore of the hexamer. Depending on the nature of this side-chain, it is possible to modulate the stability of the hexamer in a predictable manner. This family of hexameric proteins may provide a useful framework for the construction of proteins that change their oligomeric states in response to binding of small molecules.

Computational de novo design, and characterization of an A(2)B(2) diiron protein.

Diiron proteins are found throughout nature and have a diverse range of functions; proteins in this class include methane monooxygenase, ribonucleotide reductase, Delta(9)-acyl carrier protein desaturase, rubrerythrin, hemerythrin, and the ferritins. Although each of these proteins has a very different overall fold, in every case the diiron active site is situated within a four-helix bundle. Additionally, nearly all of these proteins have a conserved Glu-Xxx-Xxx-His motif on two of the four helices with the Glu and His residues ligating the iron atoms. Intriguingly, subtle differences in the active site can result in a wide variety of functions. To probe the structural basis for this diversity, we designed an A(2)B(2) heterotetrameric four-helix bundle with an active site similar to those found in the naturally occurring diiron proteins. A novel computational approach was developed for the design, which considers the energy of not only the desired fold but also alternatively folded structures. Circular dichroism spectroscopy, analytical ultracentrifugation, and thermal unfolding studies indicate that the A and B peptides specifically associate to form an A(2)B(2) heterotetramer. Further, the protein binds Zn(II) and Co(II) in the expected manner and shows ferroxidase activity under single turnover conditions.

How do helix-helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo-oligomeric helical bundles.

The final, structure-determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo-oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins-all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water-soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen-bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix-helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices.

Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange.

Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.

Computational design of a water-soluble analog of phospholamban.

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.

Proton conduction through the M2 protein of the influenza A virus; a quantitative, mechanistic analysis of experimental data.

The M2 proton channel from influenza A virus forms proton-selective ion channels, which are the target of the drug amantadine. Here, existing experimental data are quantitatively examined for insights into mechanisms to account for the pH- and voltage-dependences of M2 proton conduction. The analysis shows that a model involving protonation equilibria of His37, including pH-dependent changes in the relative rates of diffusion on either side of the pore, is quantitatively able to account for recently reported electrophysiological data examining the pH- and voltage-dependences of Rostock and Weybridge strain M2 proton conduction.

Volatile anesthetic modulation of oligomerization equilibria in a hexameric model peptide.

To determine if occupancy of interfacial pockets in oligomeric proteins by volatile anesthetic molecules can allosterically regulate oligomerization equilibria, variants of a three-helix bundle peptide able to form higher oligomers were studied with analytical ultracentrifugation, hydrogen exchange and modeling. Halothane shifted the oligomerization equilibria towards the oligomer only in a mutation predicted to create sufficient volume in the hexameric pocket. Other mutations at this residue, predicted to create a too small or too polar pocket, were unaffected by halothane. Inhaled anesthetic modulation of oligomerization interactions is a novel and potentially generalizable biophysical basis for some anesthetic actions.