Structure-Function Relationships of the Mycobacterium tuberculosis Transcription Factor WhiB1

Background: Members of the WhiB-like (Wbl) protein family possess iron-sulfur clusters and are implicated in the regulation of developmental processes in Actinomycetes. Mycobacterium tuberculosis possesses seven Wbl proteins. The [4Fe-4S] cluster of M. tuberculosis WhiB1 is relatively insensitive to O-2 but very sensitive to nitric oxide (NO). Nitric oxide nitrosylates the WhiB1 iron-sulfur cluster and promotes DNA-binding; the apo-forms of WhiB1 also bind DNA. However, the molecular requirements for iron-sulfur cluster acquisition and for DNA-binding by WhiB1 are poorly characterized. Methods and Findings: WhiB1 variants were created by site-directed mutagenesis and the abilities of the corresponding proteins to acquire an iron-sulfur cluster and/or bind to whiB1 promoter DNA were assessed. All four Cys residues (Cys9, 37, 40, and 46) in the N-terminal region of WhiB1 were required for incorporation of a [4Fe-4S] cluster, whereas a possible alternative cluster ligand Asp13 (by analogy with M. smegmatis WhiB2) was not. The C-terminal region of WhiB1 is predicted to house the DNA-binding domain of the protein consisting of a predicted beta-turn ((58)GVWGG(62)) followed by two amino acid motifs ((KRRN75)-K-72 and (78)TKAR(81)) that are conserved in WhiB1 proteins. Gly residues (Gly58, 61 and 62) in the b-turn and positively-charged residues (Lys72, Arg73, Arg74, Lys79 and Arg81) in the downstream conserved regions were required for binding of WhiB1 DNA. Conclusions: Site-directed mutagenesis of M. tuberculosis whiB1 and characterization of the corresponding proteins has been used to explore structure-function relationships of the NO-responsive transcription factor WhiB1. This showed that all four conserved Cys residues in the N-terminal region are required for incorporation of iron-sulfur clusters but not for DNA-binding. Analysis of variants with amino acid substitutions in the C-terminal region revealed the crucial roles played by a predicted b-turn and two conserved positively-charged motifs in facilitating DNA-binding, but not iron-sulfur cluster acquisition, by WhiB1.