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1.9 A structure of the signal receiver domain of the putative response regulator NarL from Mycobacterium tuberculosis.

ABSTRACT: NarL from Mycobacterium tuberculosis is a putative nitrate response regulator that is involved in the regulation of anaerobic metabolism in this pathogen. The recombinant purified N-terminal signal receiver domain of NarL has been crystallized in space group C222(1), with unit-cell parameters a = 85.6, b = 90.0, c = 126.3 A, and the structure was determined by molecular replacement to 1.9 A resolution. Comparisons with related signal receiver domains show that the closest structural homologue is an uncharacterized protein from Staphylococcus aureus, whereas the nearest sequence homologue, NarL from Escherichia coli, displays larger differences in three-dimensional structure. The largest differences between the mycobacterial and E. coli NarL domains were found in the loop between beta3 and alpha3 in the proximity of the phosphorylation site. The active site in response regulators is similar to that of members of the haloacid dehalogenase (HAD) family, which also form a phospho-aspartyl intermediate. In NarL, the aspartic acid that acts as catalytic acid/base in several HAD enzymes is replaced by an arginine residue, which is less likely to participate in steps involving proton abstraction. This substitution may slow down the breakdown of the phospho-aspartyl anhydride and allow signalling beyond the timescales defined by a catalytic reaction intermediate.

SUBMITTER: Schnell R

PROVIDER: S-EPMC2593691 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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1.9 A structure of the signal receiver domain of the putative response regulator NarL from Mycobacterium tuberculosis.

Schnell Robert R Agren Daniel D Schneider Gunter G

Acta crystallographica. Section F, Structural biology and crystallization communications 20081128 Pt 12

NarL from Mycobacterium tuberculosis is a putative nitrate response regulator that is involved in the regulation of anaerobic metabolism in this pathogen. The recombinant purified N-terminal signal receiver domain of NarL has been crystallized in space group C222(1), with unit-cell parameters a = 85.6, b = 90.0, c = 126.3 A, and the structure was determined by molecular replacement to 1.9 A resolution. Comparisons with related signal receiver domains show that the closest structural homologue is ...[more]

PMID: 19052358

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Project description:BackgroundThe Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function.ResultsNarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric.ConclusionsThe NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD.

Dataset Information

1.9 A structure of the signal receiver domain of the putative response regulator NarL from Mycobacterium tuberculosis.

Publications

1.9 A structure of the signal receiver domain of the putative response regulator NarL from Mycobacterium tuberculosis.

Similar Datasets

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