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dUTPases are key enzymes in all life kingdoms. A staphylococcal repressor protein (Stl) inhibited dUTPases from multiple species to various extents. Understanding the molecular basis underlying the inhibition differences is crucial to develop effective proteinaceous inhibitors of dUTPases. Herein, we report the complex structure of Stl N-terminal domain (Stl N-ter ) and Litopenaeus vannamei dUTPase domain (lvDUT 65-210 ). Stl inhibited lvDUT 65-210 through its N-terminal domain. The lvDUT 65-210 -Stl N-ter complex structure revealed a heterohexamer encompassing three Stl N-ter monomers bound to one lvDUT 65-210 trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT 65-210 active-site region that is contributed by motifs I-IV from its two subunits; interface II results from Stl binding to the C-terminal motif V of the third lvDUT 65-210 subunit. Structural comparison revealed both conserved features and obvious differences in Stl-dUTPase interaction patterns, giving clues about the inhibition differences of Stl on dUTPases. Noticeably, interface II is only observed in lvDUT 65-210 -Stl N-ter . The Stl-interacting residues of lvDUT 65-210 are conserved in other eukaryotic dUTPases, particularly human dUTPase. Altogether, our study presents the first structural model of Stl interaction with eukaryotic dUTPase, contributing to a more complete view of Stl inhibition and facilitating the development of proteinaceous inhibitor for eukaryotic dUTPases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protein tyrosine phosphorylation regulates the production of capsular polysaccharide, an essential virulence factor of the deadly pathogen Vibrio vulnificus. The process requires the protein tyrosine kinase Wzc and its cognate phosphatase Wzb, both of which are largely uncharacterized. Herein, we report the structures of Wzb of V. vulnificus (VvWzb) in free and ligand-bound forms. VvWzb belongs to the low-molecular-weight protein tyrosine phosphatase (LMWPTP) family. Interestingly, it contains an extra four-residue insertion in the W-loop, distinct from all known LMWPTPs. The W-loop of VvWzb protrudes from the protein body in the free structure, but undergoes significant conformational changes to fold toward the active site upon ligand binding. Deleting the four-residue insertion from the W-loop severely impaired the enzymatic activity of VvWzb, indicating its importance for optimal catalysis. However, mutating individual residues or even substituting the whole insertion with four alanine residues only modestly decreased the enzymatic activity, suggesting that the contribution of the insertion to catalysis is not determined by the sequence specificity. Furthermore, inserting the four residues into Escherichia coli Wzb at the corresponding position enhanced its activity as well, indicating that the four-residue insertion in the W-loop can act as a general activity enhancing element for other LMWPTPs. The novel W-loop type and phylogenetic analysis suggested that VvWzb and its homologs should be classified into a new group of LMWPTPs. Our study sheds new insight into the catalytic mechanism and structural diversity of the LMWPTP family and promotes the understanding of the protein tyrosine phosphorylation system in prokaryotes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

β-Lactams are the most widely used antibiotics in treating bacterial infections. However, they are rarely applied in infections caused by Vibrio parahaemolyticus, as the bacterium is intrinsically resistant to penicillins by expressing β-lactamase. Here we report structural characterization of the CARB β-lactamase from V. parahaemolyticus (CARB-20). CARB-20 is a class A β-lactamase, belonging to subclass A1 (containing 70STFKAL75, 130SDNTAANL137, 164RXEXXLN170, 231VGDKTG236, etc.), group LSBL2 (with the disulfide bridge C77-C123, motif 231IADRSGAG238 and R244). CARB-20 adopts a typical subclass A1 β-lactamase fold consisting of two domains. Its active site is constituted by four conserved motifs, similar to that of known subclass A1 β-lactamases. Analysis of the active site structure reveals its substrate preference for penicillin, ampicillin and carbenicillin but not for latterly developed cephalosporins. Meanwhile, β-lactamase inhibitors such as clavulanate and sulbactam can well fit into the active site, supporting β-lactams combined with β-lactamase inhibitors as a potential approach for treating infection of V. parahaemolyticus. The residues around the active site show certain variations, which can be useful for specific inhibitor design. In the directed evolution experiment, CARB-20 exhibited plasticity in developing significant resistance to inhibitors by accumulated residue substitutions. Therefore, careful monitoring of enzyme mutations is necessary for successfully applying β-lactam/β-lactamase inhibitor combination therapy. Taken together, our results open up an avenue of inhibitor design targeting vibrio β-lactamases, facilitating the application of β-lactams in treating vibrio infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Extracellular proteases are often produced as pre-pro-enzyme and then undergo multiple processing steps to mature into the active form. The protease Epp, a virulent factor of Vibrio anguillarum, belongs to this family. Its maturation might be regulated by Ca 2+ via its polycystic kidney disease (PKD) domain, but the molecular mechanism is unknown. Herein, we report the crystal structure of the first PKD domain from V. anguillarum Epp (Epp-PKD1) and its specific Ca 2+ -binding capacity. Epp-PKD1 exists as a monomer, consisting of seven β-strands which form two β-sheets stacking with each other. One Ca 2+ is bound by the residues Asn3, Gln4, Asp27, Asp29, Asp68 and a water molecule with a pentagonal bipyramidal geometry. Incubating the apo Epp-PKD1 with Ca 2+ but not Mg 2+ , Mn 2+ , or Zn 2+ , enhances the thermal and chemical stability of Epp-PKD1, indicating its specific binding to Ca 2+ . Epp-PKD1 shares high similarity in both sequence and overall structure with that of Vibrio cholerae PrtV, a homologous protease of Epp, however, they differ in the oligomeric state and local structure at the Ca 2+ -binding site, suggesting maturation of PrtV and Epp might be differently regulated by Ca 2+ . Likely, proteases may take advantage of the structural diversity in PKD domains to tune their Ca 2+ -regulated maturation process., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Siderophore-interacting proteins (SIPs) play an important role in iron acquisition in many bacteria. SIPs release iron from the internalized ferric siderophore complex by reducing ferric iron to ferrous iron, but how the iron is reduced is not well understood. Here, a sip gene was identified in the genome of Vibrio anguillarum 775. To further understand the catalytic mechanism of the protein, the SIP was overexpressed in Escherichia coli Rosetta (DE3) cells, purified and crystallized for X-ray diffraction analysis. The crystal diffracted to 1.113 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 64.63, b = 58.47, c = 70.65 Å, β = 114.19°.

Protein biotinylation, a rare form of post-translational modification, is found in enzymes required for lipid biosynthesis. In mycobacteria, this process is essential for the formation of their complex and distinct cell wall and has become a focal point of drug discovery approaches. The enzyme responsible for this process, biotin protein ligase, substantially varies in different species in terms of overall structural organization, regulation of function and substrate specificity. To advance the understanding of the molecular mechanism of biotinylation in Mycobacterium tuberculosis we have biochemically and structurally characterized the corresponding enzyme. We report the high-resolution crystal structures of the apo-form and reaction intermediate biotinyl-5'-AMP-bound form of M. tuberculosis biotin protein ligase. Binding of the reaction intermediate leads to clear disorder-to-order transitions. We show that a conserved lysine, Lys138, in the active site is essential for biotinylation., (© 2014 The Protein Society.)

This paper briefly reviews contemporary protein crystallography and focuses on six receptor proteins of membrane-intrinsic ATP binding cassette (ABC) transporters. Three of these receptors are specific for carbohydrates and three for amino acids. The receptor GacH of the transporter GacFGH from Streptomyces glaucescens is specific for acarbose and its homologs, and MalE of Salmonella typhimurium is specific for maltose but also forms a complex with acarbose, and the third receptor is the highly specific d-galactose receptor AcbH of the transporter AcbFGH from Actinoplanes sp. Concerning the receptors for amino acids, ArtJ belongs to the ArtJ-(MP)(2) transporter of Geobacillus stearotermophilus and recognizes and binds to positively charged arginine, lysine, and histidine with different sizes of side chains, contrasting the receptors Ngo0372 and Ngo2014 from Neisseria gonorrhaeae that are highly specific for cystine and cysteine, respectively. The differences in the rather unspecific receptors GacH, MalE and ArtJ are compared with the highly specific receptors AcbH, Ngo0372 and Ngo2014., (Copyright © 2012. Published by Elsevier GmbH.)

C(4)-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C(4)-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 C(4)-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 C(4) succinate, and a complex with C(3) 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 C(4)-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.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type. [ABSTRACT FROM AUTHOR]