scholarly journals Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY

1999 ◽  
Vol 339 (2) ◽  
pp. 223-226 ◽  
Author(s):  
Ivo S. RIDDER ◽  
Bauke W. DIJKSTRA
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Phosphate Binding ◽  
Haloacid Dehalogenase ◽  
Regulator Protein ◽  
P Type ◽  
Response Regulator Protein

The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and l-2-haloacid dehalogenases. A comparison of the three-dimensional structure of l-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the assignment of a conserved pair of aspartate residues as the Mg2+-binding site in the P-type ATPase and phosphatase members of the superfamily. From the resulting model of the active site, a conserved serine/threonine residue is suggested to be involved in phosphate binding, and a mechanism comprising a phosphoaspartate intermediate is postulated.

scholarly journals Switched or Not?: the Structure of Unphosphorylated CheY Bound to the N Terminus of FliM

2006 ◽  
Vol 188 (21) ◽  
pp. 7354-7363 ◽  
Author(s):  
Collin M. Dyer ◽  
Frederick W. Dahlquist
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Active Role ◽  
Crystallographic Structure ◽  
Coupling Mechanism ◽  
Allosteric Communication ◽  
Regulator Protein ◽  
Target Binding ◽  
Response Regulator Protein

ABSTRACT Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF3 −-CheY, a phosphorylated CheY (CheY∼P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY∼P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM16]). While the peptide conformation and binding site are similar to those of the BeF3 −-CheY-FliM16 complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg2+-CheY-FliM16 complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF3 −-activated CheY. This structure makes clear the active role that the β4-α4 loop plays in the Tyr87-Tyr106 coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.

scholarly journals Candida albicans Response Regulator Gene SSK1 Regulates a Subset of Genes Whose Functions Are Associated with Cell Wall Biosynthesis and Adaptation to Oxidative Stress

2003 ◽  
Vol 2 (5) ◽  
pp. 1018-1024 ◽  
Author(s):  
Neeraj Chauhan ◽  
Diane Inglis ◽  
Elvira Roman ◽  
Jesus Pla ◽  
Dongmei Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Cell Wall ◽  
Response Regulator ◽  
Cell Wall Biosynthesis ◽  
Regulator Protein ◽  
Two Component ◽  
Response Regulator Protein ◽  
Mutant Cells

ABSTRACT Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

scholarly journals Three-dimensional Structure of l-2-Haloacid Dehalogenase fromXanthobacter autotrophicusGJ10 Complexed with the Substrate-analogue Formate

1997 ◽  
Vol 272 (52) ◽  
pp. 33015-33022 ◽  
Author(s):  
Ivo S. Ridder ◽  
Henriëtte J. Rozeboom ◽  
Kor H. Kalk ◽  
Dick B. Janssen ◽  
Bauke W. Dijkstra
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Haloacid Dehalogenase ◽  
Three Dimensional Structure ◽  
Substrate Analogue

THE THREE-DIMENSIONAL STRUCTURE OF THE ANTIGEN BINDING SITE OF McPC 603 PROTEIN

1974 ◽  
pp. 7-14 ◽  
Author(s):  
E.A. Padlan ◽  
D.M. Segal ◽  
G.H. Cohen ◽  
D.R. Davies ◽  
S. Rudikoff ◽  
...  
Keyword(s):  
Binding Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Three Dimensional Structure

Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin

1998 ◽  
Vol 4 (S2) ◽  
pp. 462-463
Author(s):  
P. Zhang ◽  
C. Toyoshima ◽  
K. Yonekura ◽  
G. Inesi ◽  
M. Green ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Large Family ◽  
Calcium Pump ◽  
Dimensional Structure ◽  
Transmembrane Helices ◽  
Site Mutagenesis ◽  
P Type

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

A murrel cysteine protease, cathepsin L: bioinformatics characterization, gene expression and proteolytic activity

Biologia ◽  
2014 ◽  
Vol 69 (3) ◽  
Author(s):  
Venkatesh Kumaresan ◽  
Prasanth Bhatt ◽  
Rajesh Palanisamy ◽  
Annie Gnanam ◽  
Mukesh Pasupuleti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Infections ◽  
Cathepsin L ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Data Bank ◽  
Minimum Free Energy ◽  
Dimensional Structure ◽  
Peptide Bonds ◽  
Gene Expression Studies

AbstractCathepsin L, a lysosomal endopeptidase, is a member of the peptidase C1 family (papain-like family) of cysteine proteinases that cleave peptide bonds of lysosomal proteins. In this study, we report a cathepsin L sequence identified from the constructed cDNA library of striped murrel Channa striatus (designated as CsCath L) using genome sequencing FLXTM technology. The full-length CsCath L contains three eukaryotic thiol protease domains at positions 134-145, 278-288 and 299-318. Phylogenetic analysis revealed that the CsCath L was clustered together with other cathepsin L from teleosts. The three-dimensional structure of CsCath L modelled by the I-Tasser program was compared with structures deposited in the Protein Data Bank to find out the structural similarity of CsCath L with experimentally identified structures. The results showed that the CsCath L exhibits maximum structural identity with pro-cathepsin L from human. The RNA fold structure of CsCath L was predicted along with its minimum free energy (−471.93 kcal/mol). The highest CsCath L gene expression was observed in liver, which was also significantly higher (P < 0.05) than that detected in other tissues taken for analysis. In order to investigate the mRNA transcription profile of CsCath L during infection, C. striatus were injected with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila) and its expression was up-regulated in liver at various time points. Similar to gene expression studies, the highest CsCath L enzyme activity was also observed in liver and its activity was up-regulated by fungal and bacterial infections.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

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