creators_name: Das, Arijit Kumar
creators_name: Pathak, Anuj
creators_name: Sinha, Akesh
creators_name: Datt, Manish
creators_name: Singh, Balvinder
creators_name: Karthikeyan, Subramanian
creators_name: Sarkar, Dibyendu
type: article
datestamp: 2012-02-28 16:08:13
lastmod: 2012-02-28 16:08:13
metadata_visibility: show
title: A single-amino-acid substitution in the C terminus of PhoP determines DNA-binding specificity of the virulence-associated response regulator from Mycobacterium tuberculosis.
ispublished: pub
subjects: QR
full_text_status: restricted
keywords: direct repeats; DNA recognition specificity; M. tuberculosis PhoP; recognition helix; response regulator
note: Copyright of this article belongs to Elsevier Science.
abstract: The Mycobacterium tuberculosis PhoP-PhoR two-component system is essential for virulence in animal models of tuberculosis. Genetic and biochemical studies indicate that PhoP regulates the expression of more than 110 genes in M. tuberculosis. The C-terminal effector domain of PhoP exhibits a winged helix-turn-helix motif with the molecular surfaces around the recognition helix (alpha 8) displaying strong positive electrostatic potential, suggesting its role in DNA binding and nucleotide sequence recognition. Here, the relative importance of interfacial alpha 8-DNA contacts has been tested through rational mutagenesis coupled with in vitro binding-affinity studies. Most PhoP mutants, each with a potential DNA contacting residue replaced with Ala, had significantly reduced DNA binding affinity. However, substitution of nonconserved Glu215 had a major effect on the specificity of recognition. Although lack of specificity does not necessarily correlate with gross change in the overall DNA binding properties of PhoP, structural superposition of the PhoP C-domain on the Escherichia coli PhoB C-domain-DNA complex suggests a base-specific interaction between Glu215 of PhoP and the ninth base of the DR1 repeat motif. Biochemical experiments corroborate these results, showing that DNA recognition specificity can be altered by as little as a single residue change of the protein or a single base change of the DNA. The results have implications for the mechanism of sequence-specific DNA binding by PhoP.
date: 2010-05-21
date_type: published
publication: Journal of molecular biology
volume: 398
number: 5
publisher: Elsevier Science
pagerange: 647-56
refereed: TRUE
issn: 1089-8638
official_url: http://www.sciencedirect.com/science/article/pii/S0022283610003311
related_url_url: http://www.sciencedirect.com/science/article/pii/S0022283610003311
related_url_type: pub
citation:   Das, Arijit Kumar and Pathak, Anuj and Sinha, Akesh and Datt, Manish and Singh, Balvinder and Karthikeyan, Subramanian and Sarkar, Dibyendu  (2010) A single-amino-acid substitution in the C terminus of PhoP determines DNA-binding specificity of the virulence-associated response regulator from Mycobacterium tuberculosis.  Journal of molecular biology, 398 (5).  pp. 647-56.  ISSN 1089-8638     
document_url: http://crdd.osdd.net/open/1114/1/sarkar2010.pdf