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Abstract: The type VI secretion systems (T6SS) have emerging roles in interspecies competition. In order to have an advantage in defense against other organisms, this system in Pseudomonas aeruginosa delivers a peptidoglycan amidase (Tse1) to the periplasmic space of a competitor. An immune protein (Tsi1) is also produced by the bacterium to protect itself from damage caused by Tse1. Tsi1 directly interacts with Tse1. We report that the crystal structure of Tse1 displays a common CHAP protein fold. Strikingly, our structures showed that the third residue in the catalytic triad may be novel as this residue type has not been observed previously. [Copyright &y& Elsevier]

p90 ribosomal S6 kinases (RSKs) respond to various mitogen stimuli and comprise two distinct protein kinase domains. The C-terminal kinase domain (CTKD) receives signal from ERK1/2 and adopts an autoinhibitory mechanism. Here, the crystal structure of human RSK1 CTKD is reported at 2.7 Å resolution. The structure shows a standard kinase fold, with the catalytic residues in the ATP-binding cleft orientated in optimal conformations for phosphotransfer. The inactivation of the CTKD is conferred by an extra α-helix (αL), which occupies the substrate-binding groove. In combination with previous knowledge, this structure indicates that activation of RSK1 involves the removal of αL from the substrate-binding groove induced by ERK1/2 phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract: The COP9 signalosome (CSN) is a multiprotein complex containing eight subunits and is highly conserved from fungi to human. CSN is proposed to widely participate in many physiological processes, including protein degradation, DNA damage response and signal transduction. Among those subunits, only CSN5 and CSN6 belong to JAMM family. CSN5 possesses isopeptidase activity, but CSN6 lacks this ability. Here we report the 2.5Å crystal structure of MPN domain from Drosophila melanogaster CSN6. Structural comparison with other MPN domains, along with bioinformation analysis, suggests that MPN domain from CSN6 may serve as a scaffold instead of a metalloprotease. Structured summary of protein interactions: CSN6 and CSN6 bind by x-ray crystallography ( View interaction ) CSN6 and CSN6 bind by x ray scattering ( View interaction ) [Copyright &y& Elsevier]

Abstract: Microtubules are composed of polymerized α/β-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures β-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with β-tubulin in plant. Furthermore, mutagenesis studies indicated that the α-helical regions of KIS participate in β-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to β-tubulin in plants. Structured summary: MINT-7968902, MINT-7968915, MINT-7968951, MINT-7968966: KIS (uniprotkb:O04350) physically interacts (MI:0915) with Tub9 (uniprotkb:P29517) by anti tag coimmunoprecipitation (MI:0007) MINT-7968928: KIS (uniprotkb:O04350) and Tub9 (uniprotkb:P29517) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809) [ABSTRACT FROM AUTHOR]

Members of the Sua5_YciO_YrdC protein family are found in both eukaryotes and prokaryotes and possess a conserved α/β twisted open-sheet fold. The Escherichia coli protein YrdC has been shown to be involved in modification of tRNA. The crystal structure of smu.1377c, a hypothetical protein from Streptococcus mutans, has been determined to 2.25 Å resolution. From structure analysis and comparison, it is shown that smu.1377c is a member of the Sua5_YciO_YrdC family and that it may play the same role as E. coli YrdC. [ABSTRACT FROM AUTHOR]

Abstract: Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. Structured summary: MINT:6700314: GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114) [Copyright &y& Elsevier]

Enolase is a multifunctional enzyme that plays important roles in many biological and disease processes. α-Enolase from human liver (hENO1) was expressed as a soluble protein and purified by affinity column chromatography and gel filtration. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.5 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 72.85, b = 66.02, c = 79.43 Å, β = 94.54°. [ABSTRACT FROM AUTHOR]

Abstract 3′3′-cyclic GMP–AMP (3′3′-cGAMP) belongs to a family of the bacterial secondary messenger cyclic dinucleotides. It was first discovered in the Vibrio cholerae seventh pandemic strains and is involved in efficient intestinal colonization and chemotaxis regulation. Phosphodiesterases (PDEs) that degrade 3′3′-cGAMP play important regulatory roles in the relevant signaling pathways, and a previous study has identified three PDEs in V. cholerae , namely, V-cGAP1, V-cGAP2, and V-cGAP3, functioning in 3′3′-cGAMP degradation. We report the crystal structure, biochemical, and structural analyses of V-cGAP3, providing a foundation for understanding the mechanism of 3′3′-cGAMP degradation and regulation in general. Our crystal and molecular dynamic (MD)-simulated structures revealed that V-cGAP3 contains tandem HD-GYP domains within its N- and C-terminal domains, with similar three-dimensional topologies despite their low-sequence identity. Biochemical and structural analyses showed that the N-terminal domain plays a mechanism of positive regulation for the catalytic C-terminal domain. We also demonstrated that the other homologous Vibrio PDEs, V-cGAP1/2, likely function via a similar mechanism. Graphical Abstract Unlabelled Image Highlights • Three PDEs for 3′3′-cGAMP have been identified in Vibrio cholera (V-cGAP1, 2, 3). • V-cGAP3 contains tandem HD-GYP domains of N and C domains with similar three-dimensional topology. • The N domain plays an important regulatory role for the catalytic C domain. • The dimeric regulation of V-cGAP3 indicates a rule for HD-GYP domain activity. [ABSTRACT FROM AUTHOR]

Abstract: Host-derived macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of malaria infection, especially in malarial anemia. Although two Plasmodium parasite-derived MIF orthologs, Plasmodium falciparum MIF and P. berghei MIF were identified recently, the crystal structure and the precise roles of Plasmodium-derived MIFs, particularly in combination with the host MIF, remain unknown. In this study, we identified another MIF ortholog from a rodent-specific P. yoelii (PyMIF). This molecule shares a conserved three-dimensional structure with murine MIF (MmMIF), but with a different substrate binding pattern and much lower tautomerase activity. It could activate host cells via several signaling pathways in vitro, and inhibiting macrophage apoptosis, also similarly to MmMIF. However, we found that PyMIF and MmMIF acted synergistically to activate the MAPK-ERK1/2 signaling pathway at very low concentration but acted antagonistically at higher concentration. Furthermore, we detected PyMIF in the sera of infected mice and found that injection of recombinant PyMIF (rPyMIF) during infection could up-regulate several pro-inflammatory cytokines in vivo and slightly delay the death of infected mice. These data suggest that PyMIF modulates host immune responses together with host MIF and has potential to prolong parasitemia or the chronicity of malaria infection. [Copyright &y& Elsevier]

Abstract: The 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC) catalyses the decarboxylation of 3-keto-l-gulonate 6-phosphate to l-xylulose in the presence of magnesium ions. The enzyme is involved in l-ascorbate metabolism and plays an essential role in the pathway of glucuronate interconversion. Crystal structures of Streptococcus mutans KGPDC were determined in the absence and presence of the product analog d-ribulose 5-phosphate. We have observed an 8Å αB-helix movement and other structural rearrangements around the active site between the apo-structures and product analog bound structure. These drastic conformational changes upon ligand binding are the first observation of this kind for the KGPDC family. The flexibilities of both the α-helix lid and the side chains of Arg144 and Arg197 are associated with substrate binding and product releasing. The open–closed conformational changes of the active site, through the movements of the α-helix lid and the arginine residues are important for substrate binding and catalysis. [Copyright &y& Elsevier]

Abstract: C4-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C4-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB–DctD system is the first identified TCS for C4-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C4 succinate, and a complex with C3 malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per–Arnt–Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C4-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases. [Copyright &y& Elsevier]

Abstract: Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme–substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme–substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring. [Copyright &y& Elsevier]

Abstract: 2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn2+, a new conserved motif, (G43YNG46), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme–regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation. [Copyright &y& Elsevier]