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The precise mechanism leading to profound immunodeficiency of HIV-infected patients is still only partially understood. Here, we show that more than 80% of [CD4.sup.+] T cells from HIV-infected patients have morphological abnormalities. Their membranes exhibited numerous large abnormal membrane microdomains (aMMDs), which trap and inactivate physiological receptors, such as that for IL-7. In patient plasma, we identified phospholipase A2 group IB (PLA2G1B) as the key molecule responsible for the formation of aMMDs. At physiological concentrations, PLA2G1B synergized with the HIV gp41 envelope protein, which appears to be a driver that targets PLA2G1B to the [CD4.sup.+] T cell surface. The PLA2G1B/gp41 pair induced [CD4.sup.+] T cell unresponsiveness (anergy). At high concentrations in vitro, PLA2G1B acted alone, independently of gp41, and inhibited the IL-2, IL-4, and IL-7 responses, as well as TCR-mediated activation and proliferation, of [CD4.sup.+] T cells. PLA2G1B also decreased [CD4.sup.+] T cell survival in vitro, likely playing a role in CD4 lymphopenia in conjunction with its induced IL-7 receptor defects. The effects on [CD4.sup.+] T cell anergy could be blocked by a PLA2G1B- specific neutralizing mAb in vitro and in vivo. The PLA2G1B/gp41 pair constitutes what we believe is a new mechanism of immune dysfunction and a compelling target for boosting immune responses in HIV-infected patients., Introduction [CD4.sup.+] lymphocytes play a critical role in the severe immunodeficiency that characterizes HIV-infected patients. Although fewer than 0.5% of blood [CD4.sup.+] T cells are infected, almost all are dysfunctional. [...]

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal D-Ala:D-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in D-alanyl-D-lactate which are not substrates for the [beta]-1actam-resistant penicillin binding protein PBP2'. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to [beta]-1actams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus. doi: 10.1128/JB.00613-10

Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five‐helix bundle, similar to the known structure of other HHD domains, and a three‐helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five‐helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain. Whirlin is a large multidomain scaffolding protein essential for sound and light perception. One of its domains, the second Harmonin Homology Domain (HHD2), can adopt two conformations: a monomeric five‐helix bundle and a three‐helix bundle organized as a swapped dimer. The HHD2 domain does not interact with adjacent domains of the protein, but its structural plasticity might play a role in regulating whirlin oligomerization or scaffolding function. [ABSTRACT FROM AUTHOR]

Glutathionyl-hydroquinone reductases (GHRs) catalyze the deglutathionylation of quinones via a catalytic cysteine. The two GHR genes in the Populus trichocarpa genome, Pt-GHR1 and Pt-GHR2 , are primarily expressed in reproductive organs. Both proteins are localized in plastids. More specifically, Pt-GHR2 localizes in nucleoids. At the structural level, Pt-GHR1 adopts a typical GHR fold, with a dimerization interface comparable to that of the bacterial and fungal GHR counterparts. Pt-GHR1 catalyzes the deglutathionylation of both reduced and oxidized glutathionylated quinones, but the enzyme is more catalytically efficient with the reduced forms. [ABSTRACT FROM AUTHOR]

In many γ -proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role. [ABSTRACT FROM AUTHOR]

Anti-coagulant FXa-binding Viperidae snake venom phospholipases A2 (sPLA2) have been identified as specific, exogenous, non-competitive FXa-inhibitors. Detailed knowledge of the sites of interaction of PLA2 with FXa is essential for understanding the hemostatic process at the molecular level and is important for the development of new anti-coagulant drugs. New insight was obtained by structural and biophysical studies including detailed analysis of the CA-CB-binding interface of crotoxin determined by X-ray crystallography and comparison with the CB-FXa-binding interface determined by molecular docking simulations. In this short review we describe our investigations that contribute to a better understanding of the possible role of PLA2 oligomerisation in the anti-coagulant effect of PLA2. [ABSTRACT FROM AUTHOR]

Certain snake venom phospholipases A2 (PLA2) have been identified as specific, non-competitive blood coagulation inhibitors that bind with high affinity to human activated blood coagulation factor X (hFXa). Recent determination of the three-dimensional structures of PLA2 isoforms which differ in anticoagulant activity contributes to a better understanding of their mode of binding to human FXa. Detailed analysis of the crystal structures of natural PLA2 isoforms from Viperidae snake venom which differ in binding affinity to hFXa allows us to detect local conformational changes and precisely delineate the role of critical residues in the anticoagulant function of these PLA2. We find conformational changes at conserved position Lys127 and mutated position Lys128 > Glu in the C-terminal regions of less potent anti-coagulant PLA2 (AtxC and CBa2), which contribute to the observed decrease in affinity for hFXa. The mutation His1 > Ser in less potent CBa2 is associated with a significant displacement of the side chain of Lys69 and Trp70 in the loop 65-72 and could also explain the reduced anticoagulant activity of the CBa2 -FXa complex. Knowledge of the spatial arrangement of the sites of interaction of PLA2 with hFXa is important for understanding of the hemostatic process at the molecular level and could provide new anticoagulant drug leads. [ABSTRACT FROM AUTHOR]

Abstract: Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A2 which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124>Ile and Lys128>Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding. In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128>Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124>Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal α-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model. Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here. [Copyright &y& Elsevier]

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described. [ABSTRACT FROM AUTHOR]

Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in d-alanyl- d-serine, to which vancomycin exhibits low binding affinity. VanG, a d-alanine: d-serine ligase, catalyzes the ATP-dependent synthesis of the d-Ala- d-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis of VanG in complex with ADP are reported. The crystal belonged to space group P3121, with unit-cell parameters a = b = 116.1, c = 177.2 Å, and contained two molecules in the asymmetric unit. A complete data set has been collected to 2.35 Å resolution from a single crystal under cryogenic conditions using synchrotron radiation. [ABSTRACT FROM AUTHOR]