ATP binding by an F1Fo ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second-shell residues thus the function of the ε subunit changes with growth conditions.

Conformational triggers associated with influenza matrix protein 1 polymerization.

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.

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

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.

pH-dependent secondary structure propensity of the influenza A virus M2 cytoplasmic tail.

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.

Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features.

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.

A conserved amphipathic helix is required for membrane tubule formation by Yop1p.

The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization.

Fragments of bacterial endoglycosidase s and immunoglobulin g reveal subdomains of each that contribute to deglycosylation.

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 ß 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.

Isotropic bicelles stabilize the juxtamembrane region of the influenza M2 protein for solution NMR studies.

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.

Bacterial production and solution NMR studies of a viral membrane ion 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.

Structural, dynamic, and chemical characterization of a novel S-glycosylated bacteriocin.

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two ß-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.

Two-state conformational equilibrium in the Par-4 leucine zipper domain.

Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par-4 at neutral pH. Further, we have investigated the properties of the Par-4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment-dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium.

Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

Intrinsic disorder and coiled-coil formation in prostate apoptosis response factor 4.

Prostate apoptosis response factor-4 (Par-4) is an ubiquitously expressed pro-apoptotic and tumour suppressive protein that can both activate cell-death mechanisms and inhibit pro-survival factors. Par-4 contains a highly conserved coiled-coil region that serves as the primary recognition domain for a large number of binding partners. Par-4 is also tightly regulated by the aforementioned binding partners and by post-translational modifications. Biophysical data obtained in the present study indicate that Par-4 primarily comprises an intrinsically disordered protein. Bioinformatic analysis of the highly conserved Par-4 reveals low sequence complexity and enrichment in polar and charged amino acids. The high proteolytic susceptibility and an increased hydrodynamic radius are consistent with a largely extended structure in solution. Spectroscopic measurements using CD and NMR also reveal characteristic features of intrinsic disorder. Under physiological conditions, the data obtained show that Par-4 self-associates via the C-terminal domain, forming a coiled-coil. Interruption of self-association by urea also resulted in loss of secondary structure. These results are consistent with the stabilization of the coiled-coil motif through an intramolecular association.

A picornaviral loop-to-loop replication complex.

Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3C(pro)) and the 5'-UTR region of the viral genome. The 3C(pro) catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3C(pro) and a region of the 5'-UTR, stem-loop D (SLD). Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3C(pro) and SLD components. The results clearly identify a 1:1 binding stoichiometry, with pronounced loops from each molecule providing the key binding determinants for the interaction. Binding between SLD and 3C(pro) induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein.

NMR structure of stem-loop D from human rhinovirus-14.

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3Cpro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine-pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3Cpro and SLD.