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In the c-ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c-ring from the alkaliphile B acillus pseudofirmus OF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X-ray structures of the wild-type c13 ring at pH 9.0 and a 'neutralophile-like' mutant ( P51 A) at pH 4.4, at 2.4 and 2.8 Å resolution, respectively, reveal a dependency of the conformation and protonation state of the proton-binding glutamate ( E54) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide ( DCCD) demonstrate a greater flexibility of E54 in the mutant due to reduced water occupancy within the H+ binding site. A second 'neutralophile-like' mutant ( V21 N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E54 carboxylate. The study directly connects subtle structural adaptations of the c-ring ion binding site to in vivo effects of alkaliphile cell physiology. [ABSTRACT FROM AUTHOR]

The secreted pore-forming toxin listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes is a member of the family of cholesterol-dependent cytolysins (CDC) with broad properties in pathogenesis. Its role as a virulence factor is enigmatic: it disrupts membranes and acts as an inductor of both pro- and anti-inflammatory responses in infected cells. In addition, LLO is also a potent target for immunogenicity during infection. Natively secreted LLO from a recombinant L. innocua strain was crystallized in its water-soluble monomeric form. The crystals obtained belonged to the orthorhombic space group P212121, with unit-cell parameters a = 26.7, b = 85.1, c = 230.0 Å, and diffracted to beyond 2.2 Å resolution. The Matthews coefficient and the solvent content were estimated to be 2.4 Å3 Da−1 and 49.2%, respectively. The structure with one molecule in the asymmetric unit was solved using Phaser employing the structure of the previously characterized CDC toxin perfringolysin O as a search model. [ABSTRACT FROM AUTHOR]

Multi-disciplinary methods reveal a novel type of ion binding in the rotor ring of the F1Fo-ATP synthase from the opportunistic pathogen Fusobacterium nucleatum. [ABSTRACT FROM AUTHOR]

Existing quality models focus on some specific diseases, clinics or clinical areas. Although they contain structure, process, or output type measures, there is no model which measures quality of health care processes comprehensively. In addition, due to the not measured overall process quality, hospitals cannot compare quality of processes internally and externally. To bring a solution to above problems, a new model is developed from software quality measures. We have adopted the ISO/IEC 9126 software quality standard for health care processes. Then, JCIAS (Joint Commission International Accreditation Standards for Hospitals) measurable elements were added to model scope for unifying functional requirements. Assessment (diagnosing) process measurement results are provided in this paper. After the application, it was concluded that the model determines weak and strong aspects of the processes, gives a more detailed picture for the process quality, and provides quantifiable information to hospitals to compare their processes with multiple organizations. [ABSTRACT FROM AUTHOR]

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning α-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound α-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed. [ABSTRACT FROM AUTHOR]

The microscopic mechanism of coupled c-ring rotation and ion translocation in F1Fo-ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the Fo complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c15 rotor from crystals grown at low pH, soaked at high pH and, after N,N′-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 Å, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient. [ABSTRACT FROM AUTHOR]

We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle. [ABSTRACT FROM AUTHOR]

In this study, we investigated the adoptability of software quality measures for healthcare process measurement. Quality measures of ISO/IEC 9126 are redefined from a process perspective to build a generic healthcare process quality measurement model. Case study research method is used, and the model is applied to a public hospital's Entry to Care process. After the application, weak and strong aspects of the process can be easily observed. Access audibility, fault removal, completeness of documentation, and machine utilization are weak aspects and these aspects are the candidates for process improvement. On the other hand, functional completeness, fault ratio, input validity checking, response time, and throughput time are the strong aspects of the process. [ABSTRACT FROM AUTHOR]

This paper examines the role of non-governmental organizations in relation to question of "street children." For the last two decades, the number of street children in developing countries has been on the rise dramatically. Behind this rapid increase are poverty and social exclusion that impact children, the old and women most adversely. Poverty, social deprivation and violence not only force children to participate in informal economy to contribute to the household economy, but also result in weakening of family ties, living on the streets, and substance and drug abuses among young children. In this context, NGOs have important roles to play in bringing up solution to the problem. Indeed, in the past decade many NGOs emerged with an agenda to help street children. While the activities of NGOs are important and must be appreciated, civil society organizations often tend to assume uncritically the role neo-liberal agenda set for them. However, we argue that the most important role NGOs could play is to challenge the very neo-liberal agenda that destroys the social policies and responsibilities of the welfare state. [ABSTRACT FROM AUTHOR]

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The ubiquitous mammalian Na+/H+ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca2+-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1CaMBR) in complex with CaM and Ca2+. The C- and N-lobes of CaM bind the first and second helix of NHE1CaMBR, respectively. Both the NHE1 helices and the Ca2+-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca2+ regulates NHE1 activity. [ABSTRACT FROM AUTHOR]

The channel activity of the outer-membrane protein G (OmpG) from Escherichia coli is pH-dependent. To investigate the role of the histidine pair His231/His261 in triggering channel opening and closing, we mutated both histidines to alanines and cysteines. Fourier transform infrared spectra revealed that the OmpG mutants stay—independent of pH—in an open conformation. Temperature ramp experiments indicate that the mutants are as stable as the open state of wild-type OmpG. The X-ray structure of the alanine-substituted OmpG mutant obtained at pH 6.5 confirms the constitutively open conformation. Compared to previous structures of the wild-type protein in the open and closed conformation, the mutant structure shows a difference in the extracellular loop L6 connecting β-strands S12 and S13. A deletion of amino acids 220–228, which are thought to block the channel at low pH in wild-type OmpG, indicates conformational changes, which might be triggered by His231/His261. [Copyright &y& Elsevier]

Abstract: A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes. [Copyright &y& Elsevier]

Lipopolysaccharides are central elements of the outer leaflet of the outer membrane of Gram-negative bacteria and as such, of cyanobacteria. In the past, the structural analysis of the system in proteobacteria like Escherichia coli has contributed to a deep understanding of the transport of lipopolysaccharides from plasma membrane to the outer membrane. While many components of the transport system are conserved between proteobacteria and cyanobacteria, the periplasmic LptC appears to be distinct. The cyanobacterial proteins are twice as long as the proteobacterial proteins or proteins from firmicutes. This prompted the question whether the structure of the cyanobacterial proteins is comparable the one of the proteobacterial proteins. To address this question, we expressed LptC from Anabaena sp. PCC 7120 in E. coli as truncated protein without the transmembrane segment. We purified the protein utilizing HIS-tag based affinity chromatography and polished the protein after removal of the tag by size exclusion chromatography. The purified recombinant protein was crystallized by the sitting-drop vapor diffusion technique and best crystals, despite being twinned, diffracted to a resolution of 2.6 Å. • Cyanobacterial LptC proteins are larger in size when compared to proteobacterial LptC proteins. • The LptC from the filamentous cyanobacterium Anabaena can be recombinantly expressed in E. coli. • The cyanobacterial LptC can be crystalized after growth in the presence or absence of selenomethionine. [ABSTRACT FROM AUTHOR]

Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins., (© 2022. The Author(s).)

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells., (© 2021. The Author(s).)

Cells employ membrane-embedded antiporter proteins to control their pH, salt concentration, and volume. The large family of cation/proton antiporters is dominated by Na + /H + antiporters that exchange sodium ions against protons, but homologous K + /H + exchangers have recently been characterized. We show experimentally that the electroneutral antiporter NhaP1 of Methanocaldococcus jannaschii (MjNhaP1) is highly selective for Na + ions. We then characterize the ion selectivity in both the inward-open and outward-open states of MjNhaP1 using classical molecular dynamics simulations, free energy calculations, and hybrid quantum/classical (QM/MM) simulations. We show that MjNhaP1 is highly selective for binding of Na + over K + in the inward-open state, yet it is only weakly selective in the outward-open state. These findings are consistent with the function of MjNhaP1 as a sodium-driven deacidifier of the cytosol that maintains a high cytosolic K + concentration in environments of high salinity. By combining experiment and computation, we gain mechanistic insight into the Na + /H + transport mechanism and help elucidate the molecular basis for ion selectivity in cation/proton exchangers.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae , perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane β-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning β-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Na + /H + antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na + /H + antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na + /H + exchanger NHE1, which is an important drug target. Here we resolve the Na + and H + transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

Background: The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied., Methods: Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases., Results: The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation., Conclusions: The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes., General Significance: The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC 50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z-isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

Na + /H + antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H + against Na + ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na + /H + antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na + to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s -1 We attribute the recorded currents to an electrogenic reaction that includes Na + binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H + binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na + affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na + /H + exchangers. We propose that both, electroneutral and electrogenic Na + /H + antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

OmpG is a nonselective, pH dependent outer membrane protein from Escherichia coli. It consists of 281 residues, forming a 14-stranded β-sheet structure. In this study, OmpG is extended by 38 amino acids to produce a 16-stranded β-barrel (OmpG-16S). The resulting protein is investigated by IR-spectroscopy. The secondary structure, pH-dependent opening/closing mechanism, buffer accessibility and thermal stability of OmpG-16S are compared to OmpG-WT. The results show that OmpG-16S is responsive to pH change as indicated by the Amide I band shift upon a switch from acidic to neutral pH. This spectral shift is consistent with that observed in OmpG-WT, which confirms the existence of structural differences consistent with the presence of the open or closed state. Secondary structure analysis after curve-fitting of Amide I band revealed that the additional residues do not fold into β-sheet; rather they are in the form of turns and unordered structure. In thermal stability experiments, OmpG-16S is found to be as stable as OmpG-WT. Additionally, H/D exchange experiments showed no difference in the exchange rate of OmpG-16S between the acidic and alkaline pH, suggesting that the loop L6 is no longer sufficient to block the pore entrance at acidic pH., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring's ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens.

OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity., (© 2015 Elsevier Inc. All rights reserved.)

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Purpose: The purpose of this paper is to review the literature on quality model development, validation and limitations., Design/methodology/approach: The systematic literature review used online journal indexes between January 1995 and April 2010. International studies focusing on multiple functional domains and those in which development methods were selected. Two reviewers assessed all studies and 18 were shortlisted., Findings: Literature reviews, peer reviews, questionnaires and expert panels are the most frequently used model development methods. Expert judges were widely used to validate the models. The most important limitation was that key indicators were missing., Originality/value: Existing healthcare quality models are not comprehensive and there is no consensus on targets, clinical areas or diseases.

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

The ubiquitous mammalian Na(+)/H(+) exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca(2+)-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1(CaMBR)) in complex with CaM and Ca(2+). The C- and N-lobes of CaM bind the first and second helix of NHE1(CaMBR), respectively. Both the NHE1 helices and the Ca(2+)-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca(2+) regulates NHE1 activity.