Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H and Na. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na and Li at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.     

Identification of a Mechanical Rheostat in the Hydrophobic Core of Protein L

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔF_I60V-WT = − 36 pN at 447 nm s^− 1, Δx_uI60V-WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s^− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s^− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.     

Erratum to “Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States” [J. Mol. Biol. 386 (2009) 351-385]: Conformations of NhaA, the Na/H Exchanger from Escherichia coli, in the pH-Activated and Ion-Translocating States

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H⁺ and Na⁺. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na⁺ and Li⁺ at pH above 7 and involves a 7-Å displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.     

The c13 Ring from a Thermoalkaliphilic ATP Synthase Reveals an Extended Diameter Due to a Special Structural Region

We have structurally characterized the c-ring from the thermoalkaliphilic Bacillus sp. strain TA2.A1 F₁F_o-ATP synthase. Atomic force microscopy imaging and cryo-electron microscopy analyses confirm previous mass spectrometric data indicating that this c-ring contains 13 c-subunits. The cryo-electron microscopy map obtained from two-dimensional crystals shows less closely packed helices in the inner ring compared to those of Na⁺-binding c₁₁ rings. The inner ring of α-helices in c₁₁ rings harbors a conserved GxGxGxGxG motif, with glycines located at the interface between c-subunits, which is responsible for the close packing of these helices. This glycine motif is altered in the c₁₃ ring of Bacillus sp. strain TA2.A1 to AxGxSxGxS, leading to a change in c-c subunit contacts and thereby enlarging the c-ring diameter to host a greater number of c-subunits. An altered glycine motif is a typical feature of c-subunit sequences in alkaliphilic Bacillus species. We propose that enlarged c-rings in proton-dependent F-ATP synthases may represent an adaptation to facilitate ATP synthesis at low overall proton-motive force, as occurs in bacteria that grow at alkaline pH.     

Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

Transient protein interactions mediate many vital cellular processes such as signal transduction or intermolecular electron transfer. However, due to difficulties associated with their structural characterization, little is known about the principles governing recognition and binding in weak transient protein complexes. In particular, it has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP). Using isothermal titration calorimetry and NMR spectroscopy, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Furthermore, we employ a double-mutant cycle to illustrate that Cc R13 interacts with CcP Y39. The present results, in combination with those of earlier mutational studies, have enabled us to outline the extent of the energetically important Cc-CcP binding region. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions.     

Characterization of the In Vitro HIV-1 Capsid Assembly Pathway

During the morphogenesis of mature human immunodeficiency virus-1 cores, viral capsid proteins assemble conical or tubular shells around viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which capsid proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period, followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle preexisting tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature human immunodeficiency virus-1 core assembly and avenues for antiviral inhibition.     

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.     

Contributions of Conserved TPLH Tetrapeptides to the Conformational Stability of Ankyrin Repeat Proteins

Ankyrin repeat (AR) proteins are one of the most abundant classes of repeat proteins and are involved in numerous physiological processes. These proteins are composed of various numbers of AR motifs stacked in a nearly linear fashion to adopt an elongated and nonglobular architecture. One salient feature prevalent in such a structural unit is the TPLH tetrapeptide or a close variant, T/SxxH, which initiates the helix-turn-helix conformation and presumably contributes to conformational stability through a hydrogen-bonding network. In the present study, we investigated the roles of T/SxxH motif in the stability, structure, and function of AR proteins by a systematic and rationalized mutagenic study on, followed by biochemical and biophysical characterization of, gankyrin, an oncogenic protein composed of seven ARs and six T/SxxH tetrapeptides, and P16, a tumor suppressor with four ARs but no TPLH tetrapeptide. Our results showed that this tetrapeptide is ineffectual on global structure and function, but contributes significantly to conformational stability when its stabilizing potentials are fully realized in the local conformation, including (1) the intra-AR hydrogen bonding involving the hydroxyl group; (2) the intra-AR and inter-AR hydrogen bonds involving the imidazole ring; and (3) the hydrophobic interaction associated with the Thr-methyl group. Considering that the capping and close-to-capping units tend to have more sequence diversity and more conformational variation, it could be also generally true that a T/SxxH motif close to the terminal repeats contributes little or even negatively to stability with respect to Ala substitution, but substantially stabilizes the global conformation when located in the middle of a long stretch of ARs.     

Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling

Understanding how epithelial organs form during morphogenesis is a major problem in developmental biology. In the present paper, we provide a detailed analysis of vang-1, the only homolog of the planar cell polarity protein Strabismus/Van Gogh in Caenorhabditis elegans. We demonstrate that during organogenesis of the intestine, (i) VANG-1 specifically interacts with PDZ 2 domain of DLG-1 (Discs large) and becomes phosphorylated by the kinase domain of the FGF-like receptor tyrosine kinase EGL-15; (ii) VANG-1 is predominantly restrained to the cell cortex but relocates to the apical junction; and (iii) in vang-1 embryos epithelial cells of the intestine are not correctly arranged along the anterior-posterior axis. To investigate what determines the disposition of the VANG-1 protein, either truncated protein forms were expressed in the intestine or RNAi was used to remove the functions of gene products previously shown to be involved in apical junction formation. Removal of the VANG-1 PDZ binding motif “− ESAV” and depletion of dlg-1 or let-413 gene functions interferes with the localization of VANG-1. In addition, egl-15 embryos show a premature relocation of VANG-1 to the apical junction, causing defects that resemble those observed in mutant vang-1 embryos and after intestine-specific overexpression of full-length vang-1. Finally, the localization of VANG-1 depends on DSH-2, a homolog of the planar cell polarity protein Dishevelled and depletion phenocopies vang-1 and egl-15 phenotypes in the embryonic intestine.     

DNA Packaging-Associated Hyper-Capsid Expansion of Bacteriophage T3

Evidence that in vivo bacteriophage T3 DNA packaging includes capsid hyper-expansion that is triggered by lengthening of incompletely packaged DNA (ipDNA) is presented here. This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNA-containing capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids are based on two-dimensional, non-denaturing agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDS-PAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The following hypotheses are presented: (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.     

A Single Destabilizing Mutation (F9S) Promotes Concerted Unfolding of an Entire Globular Domain in γS-Crystallin

Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. γS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse γS-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected β-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at ∼ 1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils.     

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pK_a of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pK_a 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pK_a, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pK_a from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pK_a from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325 cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pK_a shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3 kcal/mol to set the enzyme into a catalytically competent form.     

Modulating the Mechanical Stability of Extracellular Matrix Protein Tenascin-C in a Controlled and Reversible Fashion

Stretching force can induce conformational changes of proteins and is believed to be an important biological signal in the mechanotransduction network. Tenascin-C is a large extracellular matrix protein and is subject to stretching force under its physiological condition. Regulating the mechanical properties of the fibronectin type III domains of tenascin-C will alter its response to mechanical stretching force and thus may provide the possibility of regulating the biological activities of tenascin-C in living cells. However, tuning the mechanical stability of proteins in a rational and systematic fashion remains challenging. Using the third fibronectin type III domain (TNfn3) of tenascin-C as a model system, here we report a successful engineering of a mechanically stronger extracellular matrix protein via engineered metal chelation. Combining steered molecular dynamics simulations, protein engineering and single-molecule atomic force microscopy, we have rationally engineered a bihistidine-based metal chelation site into TNfn3. We used its metal chelation capability to selectively increase the unfolding energy barrier for the rate-limiting step during the mechanical unfolding of TNfn3. The resultant TNfn3 mutant exhibits enhanced mechanical stability. Using a stronger metal chelator, one can convert TNfn3 back to a state of lower mechanical stability. This is the first step toward engineering extracellular matrix proteins with defined mechanical properties, which can be modulated reversibly by external stimuli, and will provide the possibility of using external stimuli to regulate the biological functions of extracellular matrix proteins.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.     

Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.     

Ostreopexin: A hemopexin fold protein from the oyster mushroom, Pleurotus ostreatus

Proteins with hemopexin repeats are widespread in viruses, prokaryotes and eukaryotes. We report here for the first time the existence of a protein in fungi with the four-bladed β-propeller fold that is typical for hemopexin-like proteins. This protein was isolated from the edible basidiomycetous fungus Pleurotus ostreatus and is named ostreopexin. It binds to Ni^2 +-NTA-agarose, and is structurally and functionally very similar to PA2 albumins isolated from legume seeds and the hemopexin fold protein from rice. Like these plant proteins, ostreopexin shows reversible binding to hemin with moderate affinity, but does not bind to polyamines. We suggest that ostreopexin participates in intracellular management of metal (II or III)-chelates.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.