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Ligand binding reduces conformational flexibility in the active site of tyrosine phosphatase related to biofilm formation A (TpbA) from Pseudomonasaeruginosa.

ABSTRACT: Tyrosine phosphatase related to biofilm formation A (TpbA) is a periplasmic dual-specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA adopts a typical DUSP fold, it also possesses distinct structural features that distinguish it from eukaryotic DUSPs. These include additional secondary structural elements, ?0 and ?6, and unique conformations of the variable insert, the ?4-?5 loop and helix ?5 that impart TpbA with a flat active-site surface. In the absence of ligand, the protein tyrosine phosphatase loop is disordered and the general acid loop adopts an open conformation, placing the catalytic aspartate, Asp105, more than 11Å away from the active site. Furthermore, the loops surrounding the active site experience motions on multiple timescales, consistent with a combination of conformational heterogeneity and fast (picosecond to nanosecond) timescale dynamics, which are significantly reduced upon ligand binding. Taken together, these data structurally distinguish TpbA and possibly other bacterial DUSPs from eukaryotic DUSPs and provide a rich picture of active-site dynamics in the ligand-free state that are lost upon ligand binding.

SUBMITTER: Koveal D

PROVIDER: S-EPMC3665656 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Ligand binding reduces conformational flexibility in the active site of tyrosine phosphatase related to biofilm formation A (TpbA) from Pseudomonasaeruginosa.

Koveal Dorothy D Clarkson Michael W MW Wood Thomas K TK Page Rebecca R Peti Wolfgang W

Journal of molecular biology 20130321 12

Tyrosine phosphatase related to biofilm formation A (TpbA) is a periplasmic dual-specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that ...[more]

PMID: 23524133

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Project description:With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

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Dataset Information

Ligand binding reduces conformational flexibility in the active site of tyrosine phosphatase related to biofilm formation A (TpbA) from Pseudomonasaeruginosa.

Publications

Ligand binding reduces conformational flexibility in the active site of tyrosine phosphatase related to biofilm formation A (TpbA) from Pseudomonasaeruginosa.

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