A two-domain elevator mechanism for sodium/proton antiport

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1, 3, for which both electron microscopy and crystal structures are available4, 5, 6. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein1, 4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur7. The only reported NhaA crystal structure so far is of the low pH inactivated form4. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding1, 8, 9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general

Structural and Functional Analysis of Validoxylamine A 7′-phosphate Synthase ValL Involved in Validamycin A Biosynthesis

Validamycin A (Val-A) is an effective antifungal agent widely used in Asian countries as crop protectant. Validoxylamine A, the core structure and intermediate of Val-A, consists of two C7-cyclitol units connected by a rare C-N bond. In the Val-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF valL was initially annotated as a validoxylamine A 7′-phosphate(V7P) synthase, whose encoded 497-aa protein shows high similarity with trehalose 6-phosphate(T6P) synthase. Gene inactivation of valL abolished both validoxylamine A and validamycin A productivity, and complementation with a cloned valL recovered 10% production of the wild-type in the mutant, indicating the involvement of ValL in validoxylamine A biosynthesis. Also we determined the structures of ValL and ValL/trehalose complex. The structural data indicates that ValL adopts the typical fold of GT-B protein family, featuring two Rossmann-fold domains and an active site at domain junction. The residues in the active site are arranged in a manner homologous to that of Escherichia coli (E.coli) T6P synthase OtsA. However, a significant discrepancy is found in the active-site loop region. Also noticeable structural variance is found around the active site entrance in the apo ValL structure while the region takes an ordered configuration upon binding of product analog trehalose. Furthermore, the modeling of V7P in the active site of ValL suggests that ValL might have a similar SNi-like mechanism as OtsA

A simple randomised algorithm for convex optimisation: Application to two-stage stochastic programming

We consider maximising a concave function over a convex set by a simple randomised algorithm. The strength of the algorithm is that it requires only approximate function evaluations for the concave function and a weak membership oracle for the convex set. Under smoothness conditions on the function and the feasible set, we show that our algorithm computes a near-optimal point in a number of operations which is bounded by a polynomial function of all relevant input parameters and the reciprocal of the desired precision, with high probability. As an application to which the features of our algorithm are particularly useful we study two-stage stochastic programming problems. These problems have the property that evaluation of the objective function is #P-hard under appropriate assumptions on the models. Therefore, as a tool within our randomised algorithm, we devise a fully polynomial randomised approximation scheme for these function evaluations, under appropriate assumptions on the models. Moreover, we deal with smoothing the feasible set, which in two-stage stochastic programming is a polyhedron

The structure of the KtrAB potassium transporter

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na+ or K+ channels and possibly as cation/K+ symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K+ transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB-ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters.We are grateful for access to ID14-1/ID14-4/ID-29 at ESRF (through the Portuguese BAG), PXII at SLS, XRD1 at ELETTRA and PROXIMA1 at SOLEIL and thank the respective support staff. A.S. was supported by FEBS (Long term fellowship). This work was funded by EMBO (Installation grant), by FEDER funds through the Operational Competitiveness Program-COMPETE and by National Funds through FCT-Fundacao para a Ciencia e a Tecnologia under the projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011), FCOMP-01-0124-FEDER-009028 (PTDC/BIA-PRO/099861/2008) and FCOMP-01-0124-FEDER-010781 (PTDC/QUI-BIQ/105342/2008). We also thank G. Gabant and M. Cadene at the 'Plateforme de Spectrometrie de Masse' at CBM, CNRS, Orleans for mass spectrometry analysis, and C. Harley for critical reading of the manuscript

How to do an evaluation: pitfalls and traps

The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used

How to do an evaluation: pitfalls and traps

The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs

Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements

Homology modelling and spectroscopy, a never-ending love story

Homology modelling is normally the technique of choice when experimental structure data are not available but three-dimensional coordinates are needed, for example, to aid with detailed interpretation of results of spectroscopic studies. Herein, the state of the art of homology modelling will be described in the light of a series of recent developments, and an overview will be given of the problems and opportunities encountered in this field. The major topic, the accuracy and precision of homology models, will be discussed extensively due to its influence on the reliability of conclusions drawn from the combination of homology models and spectroscopic data. Three real-world examples will illustrate how both homology modelling and spectroscopy can be beneficial for (bio)medical research

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund