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.


RESULTS
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 β-turn ((58)GVWGG(62)) followed by two amino acid motifs ((72)KRRN(75) and (78)TKAR(81)) 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.


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 β-turn and two conserved positively-charged motifs in facilitating DNA-binding, but not iron-sulfur cluster acquisition, by WhiB1.
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom. mbp08ljs@sheffield.ac.uk
Laura J
LJ
Smith
Melanie R
MR
Stapleton
Roger S
RS
Buxton
Jeffrey
J
Green
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; C574744:WhiB1 protein, Mycobacterium tuberculosis; D003545:Cysteine