Role of Cys54 in AbrB multimerization and DNA-binding activity

Abstract

The DNA-binding, global regulatory protein AbrB from Bacillus subtilis is homotetrameric in solution. Mutation of the lone cysteine present in the protomers (C54), to either a serine, tyrosine or tryptophan, abolishes DNA-binding activity in vitro and regulatory activity in vivo. The effect of these changes is not due to abrogation of disulfide bond formation since it can be shown biochemically that none of the C54 residues participates in disulfide bond formation. It is unlikely that C54 is involved in direct contact with DNA targets. Rather, it appears that the role of C54 is to provide a nucleophilic center required for proper spatial orientation of the polypeptide subunits.

Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains

“…WT, wild-type; PGA", polyglutamate deficient; Gly", glycine auxotroph. (Neubauer et al, 2014;Phillips & Strauch, 2001;Xu et al, 1996). ORFs are depicted as arrows; dashed lines refer to the deletion in M18.…”

Independent and Interchangeable Multimerization Domains of the AbrB, Abh, and SpoVT Global Regulatory Proteins

“…(C54W was not obtained in sufficiently pure form to allow characterization.) Although there is some evidence that the sulfhydryl groups of the Cys54 residues have altered solvent accessibility upon binding DNA (18), other lines of evidence strongly imply that free Cys54 sulfhydryl groups are not involved in direct DNA contact (30). On the basis of the observations, we believe that Cys54 residues in their reduced state must play a crucial role in the stability of the tetrameric configuration needed for stable DNA binding.…”

“…The C54Y, C54S, and C54W mutations all result in an AbrB mutant phenotype in B. subtilis (17,18,29). The defect caused by the absence of a cysteine at this position cannot be due to elimination of disulfide bond formation between subunits, as it has been shown that the sulfhydryls of Cys54 residues are present in the reduced state in active AbrB tetramers (18). When present in the AbrB 48-94 segment fused to the cI DBD, the C54S and C54W mutations prevent dimerization but the C54Y mutation does not (Fig.…”

“…Previous studies of purified mutant forms of AbrB had indicated that the R23S mutant protein is tetrameric, L67P is probably a dimer, and C54Y may have a reduced, but not abolished, capacity to form tetramers (18,29). To examine these assessments further and to investigate the multimerization defect of other missense mutations, we constructed a variety of cI DBD fusions to segments of AbrB containing the mutations.…”

Substitutional Analysis of the C-Terminal Domain of AbrB Revealed Its Essential Role in DNA-Binding Activity

“…The active AbrB protein is a tetramer of identical subunits [10], [22], [23]. The N-termini (aa 1 to 53) of two monomers form dimers that in turn interact via the free C-termini (aa 54–94) to form tetramers [23], [24].…”