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Abstract
BackgroundMembers 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 O2 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 FindingsWhiB1 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 β-turn (58GVWGG62) followed by two amino acid motifs (72KRRN75 and 78TKAR81) that are conserved in WhiB1 proteins. Gly residues (Gly58, 61 and 62) in the β-turn and positively-charged residues (Lys72, Arg73, Arg74, Lys79 and Arg81) in the downstream conserved regions were required for binding of WhiB1 DNA.ConclusionsSite-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 β-turn and two conserved positively-charged motifs in facilitating DNA-binding, but not iron-sulfur cluster acquisition, by WhiB1.
Highlights
M. tuberculosis is the causative agent of tuberculosis (TB) and is a major worldwide healthcare problem [1]
Site-directed mutagenesis of M. tuberculosis whiB1 and characterization of the corresponding proteins has been used to explore structure-function relationships of the nitric oxide (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 DNAbinding
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
Summary
M. tuberculosis is the causative agent of tuberculosis (TB) and is a major worldwide healthcare problem [1]. As is the case for many bacterial pathogens, the ability of M. tuberculosis to cause disease requires efficient and effective gene regulation to survive within the hostile environment of the host. Nitric oxide (NO) production by lung macrophages (the preferred niche for M. tuberculosis) is a major component of the host defenses and M. tuberculosis has evolved mechanisms to sense NO and reprogram gene expression to counteract the deleterious effects of reactive nitrogen species [2,3,4]. Upon exposure to NO M. tuberculosis initiates a complex reprogramming of gene expression to counteract the deleterious effects of reactive nitrogen species [2,3]. Members of the WhiB-like (Wbl) protein family possess iron-sulfur clusters and are implicated in the regulation of developmental processes in Actinomycetes. Nitric oxide nitrosylates the WhiB1 iron-sulfur cluster and promotes DNA-binding; the apo-forms of WhiB1 bind DNA. The molecular requirements for iron-sulfur cluster acquisition and for DNA-binding by WhiB1 are poorly characterized
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