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A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization. We studied the structural plasticity and oligomerization of M1NT in solution using NMR spectroscopy. We show that the isolated domain can be induced by sterol-containing compounds to undergo a conformational change and self-associate in a pH-dependent manner consistent with the stacked dimer oligomeric interface. Surface-exposed residues at one of the stacked dimer interfaces are most sensitive to sterols. Several perturbed residues are at the interface between the N-terminal subdomains and are also perturbed by changes in pH. The effects of sterols appear to be indirect and most likely mediated by reduction in water activity. The local changes are centered on strictly conserved residues and consistent with a priming of the N-terminal domain for polymerization. We hypothesize that M1NT is sensitive to changes in the aqueous environment and that this sensitivity is part of a mechanism for restricting polymerization to the membrane surface. Structural models combined with information from chemical shift perturbations indicate mechanisms by which conformational changes can be transmitted from one polymerization interface to the other.

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity., How mechanosensitive ion channels, such as MscL, are activated by lipids and physical properties of the membrane remains unclear. Here authors use PELDOR/DEER spectroscopy and identify a single site which generated an allosteric structural response in the absence of membrane tension.

The cytoplasmic C-terminal tail of the matrix protein 2 (M2) from influenza A virus has a well conserved sequence and is involved in interactions with several host proteins as well as the influenza matrix protein 1 (M1). Whereas the transmembrane domain of M2 has been well characterised structurally and functionally, high resolution information about the distal cytoplasmic tail is lacking. Here we report the chemical shifts of the cytoplasmic tail of M2 and the chemical shift perturbations at low pH and in the presence of membrane mimetics. The cytoplasmic tail residues are mostly disordered but an extended backbone conformation is adopted by the LC3 binding motif and the putative M1 interaction site has partial helical content with a small pH-dependence. The chemical shift assignments provide a basis for further investigations into interactions of the M2 cytoplasmic tail with viral and host cell factors.

The hepatitis C virus (HCV) viroporin p7 oligomerizes to form ion channels, which are required for the assembly and secretion of infectious viruses. The 63-amino acid p7 monomer has two putative transmembrane domains connected by a cytosolic loop, and has both N- and C- termini exposed to the endoplasmic reticulum (ER) lumen. NMR studies have indicated differences between p7 structures of distantly related HCV genotypes. A critical question is whether these differences arise from the high sequence variation between the different isolates and if so, how the divergent structures can support similar biological functions. Here, we present a side-by-side characterization of p7 derived from genotype 1b (isolate J4) in the detergent 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and p7 derived from genotype 5a (isolate EUH1480) in n-dodecylphosphocholine (DPC). The 5a isolate p7 in conditions previously associated with a disputed oligomeric form exhibits secondary structure, dynamics, and solvent accessibility broadly like those of the monomeric 1b isolate p7. The largest differences occur at the start of the second transmembrane domain, which is destabilized in the 5a isolate. The results show a broad consensus among the p7 variants that have been studied under a range of different conditions and indicate that distantly related HCVs preserve key features of structure and dynamics.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.Rationalin silicodesign andin vitroscreens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance., Author summary "Swine flu" illustrated that the spread of influenza pandemics in the modern era is rapid, making antiviral drugs the best way of limiting disease. One proven influenza drug target is the M2 proton channel, which plays an essential role during virus entry. However, resistance against licensed drugs targeting this protein is now ubiquitous, largely due to an S31N change in the M2 sequence. Understandably, considerable effort has focused on developing M2-N31 inhibitors, yet this has been hampered by controversy surrounding two potential drug binding sites. Here, we show that both sites can in fact be targeted by new M2-N31 inhibitors, generating synergistic antiviral effects. Developing such drug combinations should improve patient outcomes and minimise the emergence of future drug resistance.

The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.

The Hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations1-4. We are curious as to what structural solution has the viroporin adopted to afford selective cation conduction because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The p7 activity can be inhibited by amantadine and rimantadine2,5, which also happen to be potent blockers of the influenza M2 channel6 and licensed drugs against influenza infections7. The adamantane derivatives were subjects of HCV clinical trials8, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here, we determined the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbors, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also alludes to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter while an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording showed that these residues are indeed critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

International audience; Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

Background: Sigma-1 receptor is a ligand-regulated membrane protein chaperone involved in BiP regulation and the ER stress response. Results: The chaperone domain of human sigma-1 receptor is mostly helical with short extended regions. Conclusion: Regions of the sigma-1 receptor chaperone domain implicated in ligand and cholesterol binding can be mapped to separate helices. Significance: A structural framework for delineating sigma-1 receptor BiP and ligand interactions is presented., The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198–206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.

Permeation of drugs across lipid bilayers is a key factor in dictating how effective they will be. In vivo, the issue is compounded by the presence of drug-exporter proteins such as P-glycoprotein. However, despite intense effort, exactly what controls permeation and susceptibility to export is still poorly understood. In this work we examine two well-studied drugs for which interaction with P-glycoprotein has been studied before: amitriptyline, a known substrate and clozapine, which is not a substrate. Extensive MD simulations, including potential of mean force (PMF) profiles of the compounds in all possible protonation states, reveal that the preferred location of the compounds in different bilayers in different protonation states is remarkably similar. For both molecules in charged states, there is a substantial barrier to crossing the bilayer. Clozapine however, shows an energetic barrier to movement across the bilayer even in a protonation state that results in an uncharged molecule. For amitriptyline there is only a very small barrier of approximately 1.3 kcal mol(-1). Further analysis revealed that the conformational and orientational behavior of the two compounds was also similar, with the sidechain interacting with the lipid headgroups. This effect was much stronger if the sidechain was charged (protonated). These interactions with lipid bilayers were confirmed by NMR ROESY experiments. The results are discussed in terms of their potential interactions with export proteins like P-glycoprotein.

The Ndc80 complex, a kinetochore component conserved from yeast to humans, is essential for proper chromosome alignment and segregation during mitosis. It is an approximately 570 A long, rod-shaped assembly of four proteins--Ndc80p (Hec1), Nuf2p, Spc24p, and Spc25p--with globular regions at either end of a central shaft. The complex bridges from the centromere-proximal inner kinetochore layer at its Spc24/Spc25 globular end to the microtubule binding outer kinetochore layer at its Ndc80/Nuf2 globular end. We report the atomic structures of the Spc24/Spc25 globular domain, determined both by X-ray crystallography at 1.9 A resolution and by NMR. Spc24 and Spc25 fold tightly together into a single globular entity with pseudo-2-fold symmetry. Conserved residues line a common hydrophobic core and the bottom of a cleft, indicating that the functional orthologs from other eukaryotes will have the same structure and suggesting a docking site for components of the inner kinetochore.

Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP + cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30°C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.

▪ Abstract Molecular motions are widely regarded as contributing factors in many aspects of protein function. The enzyme dihydrofolate reductase (DHFR), and particularly that from Escherichia coli, has become an important system for investigating the linkage between protein dynamics and catalytic function, both because of the location and timescales of the motions observed and because of the availability of a large amount of structural and mechanistic data that provides a detailed context within which the motions can be interpreted. Changes in protein dynamics in response to ligand binding, conformational change, and mutagenesis have been probed using numerous experimental and theoretical approaches, including X-ray crystallography, fluorescence, nuclear magnetic resonance (NMR), molecular dynamics simulations, and hybrid quantum/classical dynamics methods. These studies provide a detailed map of changes in conformation and dynamics throughout the catalytic cycle of DHFR and give new insights into the role of protein motions in the catalytic activity of this enzyme.

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.

Highlights • The second transmembrane domain (TM2) of S1R includes residues 91–107. • The S1R cytosolic region contains three α-helices. • The chaperone domain structure is largely unperturbed by the addition of TM2., The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation.

Background: Endoglycosidase S (EndoS) is an immune evasion factor from Streptococcus pyogenes that impedes IgG effector functions by deglycosylation. Results: Analysis of fragments of enzyme and substrate identify components of each that contribute to catalysis. Conclusion: Regions outside of the catalytic domain of EndoS contribute to IgG deglycosylation, and catalysis does not require all antibody subunits. Significance: Engineering of EndoS specificity requires consideration of both catalytic and non-catalytic regions., Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.

The mechanism for catalysis of DNA alkylation by the potent antitumor antibiotic duocarmycin SA (DSA) has been probed by determining the structure of a DNA adduct of the indole analogue (DSA-indole, DSI) lacking three methoxy functional groups. The three-dimensional structure of DSI covalently bound to A19 in d-(G1ACTAATTGAC11)·d-(G12TCAATTAGTC22) was determined by 1H NMR spectroscopy using a total of 935 experimental distance and dihedral angle constraints. The representative ensemble of 20 conformers has no distance restraint violations greater than 0.03 A, no torsional restraint violations greater than 0.7°, and a pairwise rmsd over all atoms in the binding site of 0.48 A. Comparison of the structures of the DSA and DSI adducts reveals a structural basis for the critical role of one of the trimethoxy-indole functional groups in alkylation reactivity. A deeper penetration into the DNA minor groove in the vicinty of the indole subunit is observed for the DSI versus the DSA adduct, along with some variati...

The protein M2 from influenza is a tetrameric membrane protein with several roles in the viral life cycle. The transmembrane helix (TMH) of M2 has proton channel activity that is required for unpackaging the viral genome. Additionally a C-terminal juxtamembrane region includes an amphipathic helix (APH) important for virus budding and scission. The APH interacts with membranes and is required for M2 localization to the site of viral budding. As a step toward obtaining high resolution information on the structure and lipid interactions of the M2 APH, we sought to develop a fast tumbling bicelle system, which would make studies of M2 in a membrane-like environment by solution NMR possible. Since M2 is highly sensitive to the solubilizing environment, an M2 construct containing the APH was studied under micelle and bicelle conditions while maintaining the same detergent and lipid headgroup chemistry to facilitate interpretation of the spectroscopic results. The sequence from a human H1N1 "swine flu" isolate was used to design an M2 construct (swM2) similar in amino acid sequence to currently circulating viruses. Comparison of swM2 solubilized in either the diacyl detergent 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or a mixture of DHPC and the lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (q = 0.4) indicated that the largest changes were a decrease in helicity at the N-terminus of the TMH and a decrease in dynamics for the juxtamembrane linker residues connecting the TMH and the APH. Whereas the linker region is very dynamic and the amide protons are rapidly exchanged with water protons in micelles, the dynamics and water exchange are largely suppressed in the presence of lipid. Chemical shift changes and relaxation measurements were consistent with an overall stabilization of the linker region, with only modest changes in conformation or environment of the APH itself. Such changes are consistent with differences observed in structures of M2 in lipid bilayers and detergent micelles, indicating that the bicelle system provides a more membrane-like environment.

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture.

Advances in solution nuclear magnetic resonance (NMR) methodology that enable studies of very large proteins have also paved the way for studies of membrane proteins that behave like large proteins due to the added weight of surfactants. Solution NMR has been used to determine the high-resolution structures of several small, membrane proteins dissolved in detergent micelles and small bicelles. However, the usual difficulties with membrane proteins in producing, purifying, and stabilizing the proteins away from native membranes remain, requiring intensive screening efforts. Low levels of heterologous expression can be the most detrimental aspect to studying membrane proteins. This is exacerbated for NMR studies because of the costs of isotopically enriched media. Thus, solution NMR studies have tended to focus on relatively small, membrane proteins that can be expressed into inclusion bodies and refolded. Here, we describe the methods used to produce, purify, and refold the proton channel M2 into detergent micelles, and the procedures used to determine chemical shift assignments and the atomic level structure of the closed form of the homotetrameric channel.

Advances in solution nuclear magnetic resonance (NMR) methodology that enable studies of very large proteins have also paved the way for studies of membrane proteins that behave like large proteins due to the added weight of surfactants. Solution NMR has been used to determine the high-resolution structures of several small, membrane proteins dissolved in detergent micelles and small bicelles. However, the usual difficulties with membrane proteins in producing, purifying, and stabilizing the proteins away from native membranes remain, requiring intensive screening efforts. Low levels of heterologous expression can be the most detrimental aspect to studying membrane proteins. This is exacerbated for NMR studies because of the costs of isotopically enriched media. Thus, solution NMR studies have tended to focus on relatively small, membrane proteins that can be expressed into inclusion bodies and refolded. Here, we describe the methods used to produce, purify, and refold the proton channel M2 into detergent micelles, and the procedures used to determine chemical shift assignments and the atomic level structure of the closed form of the homotetrameric channel.

Human leukotriene C4 synthase (LTC4S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC4S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC4S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.

The influenza A virus M2 proton channel equilibrates pH across the viral membrane during entry and across the trans-Golgi membrane of infected cells during viral maturation. It is an important target of adamantane-family antiviral drugs, but drug resistance has become a critical problem. Two different sites for drug interaction have been proposed. One is a lipid-facing pocket between 2 adjacent transmembrane helices (around Asp-44), at which the drug binds and inhibits proton conductance allosterically. The other is inside the pore (around Ser-31), at which the drug directly blocks proton passage. Here, we describe structural and functional experiments on the mechanism of drug inhibition and resistance. The solution structure of the S31N drug-resistant mutant of M2, a mutant of the highly pathogenic avian influenza subtype H5N1, shows that replacing Ser-31 with Asn has little effect on the structure of the channel pore, but dramatically reduces drug binding to the allosteric site. Mutagenesis and liposomal proton flux assays show that replacing the key residue (Asp-44) in the lipid-facing binding pocket with Ala has a dramatic effect on drug sensitivity, but that the channel remains fully drug sensitive when replacing Ser-31 with Ala. Chemical cross-linking studies indicate an inverse correlation between channel stability and drug resistance. The lipid-facing pocket contains residues from 2 adjacent channel-forming helices. Therefore, it is present only when the helices are tightly packed in the closed conformation. Thus, drug-resistant mutants impair drug binding by destabilizing helix–helix assembly.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

The T cell receptor (TCR) alphabeta heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3gammaepsilon, CD3deltaepsilon, and zetazeta dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the zetazeta signaling module is known to require one basic TCRalpha and two zetazeta aspartic acid TM residues. We report the NMR structure of the zetazeta(TM) dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for zetazeta dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the zetazeta interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling.

Coiled-coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing structural and functional studies. However, owing to the dynamic nature of many of the smaller coiled-coil domains, crystallization for X-ray studies is very challenging. Determination of elongated structures using standard NMR approaches is inefficient and usually yields low-resolution structures due to accumulation of small errors over long distances. Here we describe a solution NMR approach based on residual dipolar couplings (RDCs) for rapid and accurate structure determination of coiled-coil dimers. Using this approach, we were able to determine the high-resolution structure of the coiled-coil domain of cGMP-dependent protein kinase Ialpha, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled-coil structures with higher order assemblies.

Quantification of membrane partition potential of drug compounds is of great pharmaceutical interest. Here, a novel approach combining liquid-state NMR diffusion measurements and fast-tumbling lipid/detergent bicelles is used to measure accurately the partition coefficient K(p) of amantadine in phospholipid bilayers. Amantadine is found to have a strong membrane partition potential, with K(p) of 27.6 in DMPC and 37.8 in POPC lipids. Electrostatic interaction also plays a major role in the drug's affinity towards biological membrane as introduction of negatively charged POPG dramatically increases its K(p). Saturation transfer difference experiments in small bicelles indicate that amantadine localizes near the negatively charged phosphate group and the hydrocarbon chain of bilayer lipid. The approach undertaken in this study is generally applicable for characterizing interactions between small molecules and phospholipid membranes.

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.

Coiled-coil motifs are ubiquitous in biology and play essential roles in protein assembly and molecular recognition. Here, we show that the relative orientation and stoichiometry of coiled-coil proteins in solution can be determined by comparison of residual dipolar couplings (RDCs) measured in charged liquid-crystalline medium with values predicted from the three-dimensional charge distribution of the protein. Comparison of charge-predicted RDCs with a small set of one-bond 1DNH dipolar couplings, measured in the negatively charged liquid-crystalline Pf1 bacteriophage medium, identified the coiled-coil region of the cGMP-dependent protein kinase I as a parallel homodimer in solution and ruled out an antiparallel dimeric or monomeric state. The method is very rapid, applicable to a wide variety of liquid crystals used in biological NMR to date, and can be applied to coiled-coil structures and other proteins with higher order assembly.

Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.

Telomere repeat sequence (TRS) DNA is found at the termini of most eukaryotic chromosomes. The sequences are highly repetitive and G-rich (e.g., [C1–3A/TG1–3]n for the yeast Saccharomyces cerevisiae) and are packaged into nonnucleosomal protein-DNA structures in vivo. We have used total intensity light scattering and electron microscopy to monitor the effects of yeast TRS inserts on in vitro DNA condensation by cobalt (III) hexaammine. Insertion of 72 bp of TRS into a 3.3-kb plasmid depresses condensation as seen by light scattering and results in a 22% decrease in condensate thickness as measured by electron microscopy. Analysis of toroidal condensate dimensions suggests that the growth stages of condensation are inhibited by the presence of a TRS insert. The depression in total light scattering intensity is greater when the plasmid is linearized with the TRS at an end (39–49%) than when linearized with the TRS in the interior (18–22%). Circular dichroism of a 95-bp fragment containing the TRS insert gives a spectrum that is intermediate between the A-form and B-form, and the anomalous condensation behavior of the TRS suggests a noncanonical DNA structure. We speculate that under conditions in which the plasmid DNA condenses, the telomeric insert assumes a helical geometry that is similar to the A-form and is incompatible with packing into the otherwise B-form lattice of the condensate interior.

pH-activated, proton selective channels are required during viral entry and replication of influenza viruses. We determined the high resolution structures of proton channels from influenza A and B viruses by solution NMR spectroscopy. The channel structures reveal pore features that are important for proton gating and proton relay. Structural details of the anti-influenza drug, rimantadine, bound to the channel suggests an unexpected allosteric mechanism of drug inhibition and drug resistance, which has been verified by thorough functional and mutagenesis experiments.The work is supported by NIH grant AI067438.

Signal transduction is essential for many biological processes, such as mitosis, cell regeneration, and muscle contraction. Muscle-Specific Kinase (MuSK) is in a complex with low-density lipoprotein receptor-related protein 4 (LRP4), and upon binding of the substrate Agrin to LRP4, conformational changes of these proteins initiate a signalling cascade, stimulating aggregation of acetylcholine receptors (AChRs), which results the downstream organization of neuromuscular synapses. Mutations and/or autoantibodies against MuSK affects its ability to aggregate acetylcholine receptors, resulting in the disease myasthenia gravis, which is characterised by impaired muscle contractions. Approximately 70% of the structure of the full-length MuSK protein has been determined (consisting of the most of the extracellular domain and the intracellular kinase domain). However, crucially the transmembrane domain has not been characterised, and the structure of the transmembrane dimer that MuSK forms has not been determined. Here we apply a multi-scale MD simulation method to examine at the behaviour of MuSK TM helices in a lipid bilayer vs. a detergent micelle environment. Coarse-grained (CG) simulations, which make an extended timescale readily accessible, are used to examine initial TM helix dimerisation events. Subsequent atomistic simulations serve to refine the model of the interactions observed in CG simulations. These computational studies highlight the effect of lipids vs. detergents on the interactions of the transmembrane helices within the dimer.