Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha 5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K-Me) by >= 10(3) M-1. Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (Delta G(coupling) approximate to 0 kcal mol(-1) In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (Delta G(coupling) >= + 3.0 kcal mol(-1)). H-1-N-15 heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K-Me. (c) 2006 Elsevier Ltd. All rights reserved.