Unique GTP-Binding Pocket and Allostery of Uridylate Kinase from a Gram-Negative Phytopathogenic Bacterium

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

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Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520300113 ◽

2016 ◽

Vol 113 (6) ◽

pp. 1648-1653 ◽

Cited By ~ 30

Author(s):

Lu Li ◽

Qiyao Wang ◽

Hui Zhang ◽

Minjun Yang ◽

Mazhar I. Khan ◽

...

Keyword(s):

Cell Wall ◽

Histidine Kinase ◽

Vibrio Parahaemolyticus ◽

Response Regulator ◽

Strong Support ◽

Binding Pocket ◽

Single Amino Acid ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Direct Recognition

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogenVibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

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Cell Individuality: The Bistable Gene Expression of the Type III Secretion System in Dickeya dadantii 3937

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-11-0105 ◽

2012 ◽

Vol 25 (1) ◽

pp. 37-47 ◽

Cited By ~ 8

Author(s):

Quan Zeng ◽

Michael D. Laiosa ◽

Douglas A. Steeber ◽

Eulandria M. Biddle ◽

Quan Peng ◽

...

Keyword(s):

Gene Expression ◽

Type Iii Secretion ◽

Expression Patterns ◽

Type Iii Secretion System ◽

Secretion System ◽

Expression Level ◽

Dickeya Dadantii ◽

Phytopathogenic Bacterium ◽

Gram Negative ◽

Type Iii

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.

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Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

10.1101/388546 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gareth W. Hughes ◽

Stephen C. L. Hall ◽

Claire S. Laxton ◽

Pooja Sridhar ◽

Amirul H. Mahadi ◽

...

Keyword(s):

Crystal Structure ◽

Outer Membrane ◽

Binding Pocket ◽

Periplasmic Protein ◽

Inner Membrane ◽

Gram Negative ◽

Membrane Atpase ◽

Phospholipid Transport ◽

First Time ◽

Β Sheet

AbstractThe Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

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Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family

10.1101/2021.04.20.21254596 ◽

2021 ◽

Author(s):

Kevin M. Brown ◽

Mai Xu ◽

Michael Sargen ◽

Hyunbum Jang ◽

Mingzhen Zhang ◽

...

Keyword(s):

Mapk Pathway ◽

Binding Pocket ◽

Pi3k Pathway ◽

Melanoma Risk ◽

Mediterranean Populations ◽

Risk Genes ◽

1000 Genomes ◽

Gtp Binding ◽

The Us

AbstractWhile several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations, we identified a novel NRAS variant (c.170A>C, p.D57A) in a melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11 273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2+ binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A>C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

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Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00825-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6248-6255 ◽

Cited By ~ 90

Author(s):

M. Berrazeg ◽

K. Jeannot ◽

Véronique Yvette Ntsogo Enguéné ◽

I. Broutin ◽

S. Loeffert ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Reference Strain ◽

Binding Pocket ◽

Amino Acid Substitutions ◽

Substrate Binding Pocket ◽

Strain Pao1 ◽

Gram Negative ◽

Content Type ◽

Omega Loop ◽

New Mutations

ABSTRACTMutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains ofPseudomonas aeruginosato antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficientP. aeruginosa4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show thatP. aeruginosais able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.

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Unusual Interplay of Two Types of Ras Activators, RasGRP and SOS, Establishes Sensitive and Robust Ras Activation in Lymphocytes

Molecular and Cellular Biology ◽

10.1128/mcb.01882-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2732-2745 ◽

Cited By ~ 120

Author(s):

Jeroen P. Roose ◽

Marianne Mollenauer ◽

Mary Ho ◽

Tomohiro Kurosaki ◽

Arthur Weiss

Keyword(s):

B Lymphocytes ◽

Feedback Loop ◽

Binding Pocket ◽

Lymphocyte Development ◽

Ras Signaling ◽

Receptor Stimulation ◽

Gtp Binding ◽

Ras Activation ◽

Low Levels

ABSTRACT Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition, RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here, we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1, initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)—hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation.

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Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525143113 ◽

2016 ◽

Vol 113 (5) ◽

pp. 1405-1410 ◽

Cited By ~ 41

Author(s):

Alfred D. Kinana ◽

Attilio V. Vargiu ◽

Thithiwat May ◽

Hiroshi Nikaido

Keyword(s):

Efflux Pump ◽

Hydrolysis Product ◽

Binding Pocket ◽

Multidrug Resistant ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Short Period ◽

Resistance Nodulation Division ◽

Dynamics Simulations

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.

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SAR1 paralogs differ biochemically in assembly of the COPII coat

10.1101/2020.02.09.940833 ◽

2020 ◽

Author(s):

David B. Melville ◽

Sean Studer ◽

Randy Schekman

Keyword(s):

Binding Site ◽

Membrane Surface ◽

Binding Pocket ◽

Small Gtpase ◽

The Other ◽

The Novel ◽

Vesicular Traffic ◽

Gtp Binding ◽

Binding Preference ◽

Lipoprotein Secretion

ABSTRACTCOPII-coated vesicles are the primary mediators of vesicular traffic from the ER to the Golgi apparatus. SAR1 is a small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. In mammals, there are two SAR1 paralogs which genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion for SAR1B. We identified two clusters of amino acids that have conserved, paralog-specific sequences. One cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site of COPII components SEC31 and SEC23. We found that the latter cluster confers a SEC23A binding preference to SAR1B over SAR1A. In contrast to SAR1B, SAR1A is prone to oligomerize on a membrane surface. Importantly, in relation to its physiological function, SAR1B, but not SAR1A, can compensate for loss of SAR1B in lipoprotein secretion. The SEC31/SEC23-binding site-adjacent divergent cluster is critical for this function. These data identify the novel paralog-specific function for SAR1B, and provide insights into the mechanisms of large cargo secretion and COPII related diseases.

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1,8-Naphthalimide derivatives: new leads against dynamin I GTPase activity

Organic & Biomolecular Chemistry ◽

10.1039/c5ob00751h ◽

2015 ◽

Vol 13 (29) ◽

pp. 8016-8028 ◽

Cited By ~ 5

Author(s):

Mohammed K. Abdel-Hamid ◽

Kylie A. Macgregor ◽

Luke R. Odell ◽

Ngoc Chau ◽

Anna Mariana ◽

...

Keyword(s):

In Silico ◽

Binding Pocket ◽

Gtpase Activity ◽

Gtp Binding ◽

Dynamin I

Fragment-basedin silicoscreening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI.

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