Dataset Information

Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA).

ABSTRACT: Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase (Kb) pathogenicity island (FPI) that is regulated by the macrophage locus protein A (MglA) and the stringent starvation protein A (SspA). These proteins interact with both RNA polymerase (RNAP) and the pathogenicity island gene regulator (PigR) to activate FPI transcription. However, the molecular mechanisms involved are not well understood. Indeed, while most bacterial SspA proteins function as homodimers to activate transcription, F. tularensis SspA forms a heterodimer with the MglA protein, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family. Unexpectedly, MglA also formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that MglA is able to self-associate in solution to form a dimer but that it preferentially heterodimerizes with SspA. Finally, the MglA structure revealed malate, which was used in crystallization, bound in an open pocket formed by the dimer, suggesting the possibility that this cleft could function in small molecule ligand binding. The location of this binding region relative to recently mapped PigR and RNAP interacting sites suggest possible roles for small molecule binding in MglA and SspA•MglA function.

SUBMITTER: Cuthbert BJ

PROVIDER: S-EPMC4488300 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA).

Cuthbert Bonnie J BJ Brennan Richard G RG Schumacher Maria A MA

PloS one 20150629 6

Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase (Kb) pathogenicity island (FPI) that is regulated by the macrophage locus protein A (MglA) and the stringent starvation protein A (SspA). These proteins interact with both RNA polymerase (RNAP) and the pathogenicity island gene regulator (PigR) to activate FPI transcription. However, the molecular mechanisms involved are not well understood. ...[more]

PMID: 26121147

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Project description:The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, β = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

Dataset Information

Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA).

Publications

Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA).

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