کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2190189 | 1096243 | 2006 | 13 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Individual Metal Ligands Play Distinct Functional Roles in the Zinc Sensor Staphylococcus aureus CzrA Individual Metal Ligands Play Distinct Functional Roles in the Zinc Sensor Staphylococcus aureus CzrA](/preview/png/2190189.png)
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 α5 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 (KMe) by ≥103 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 (ΔGcoupling≈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 (ΔGcoupling≥+3.0 kcal mol−1). 1H–15N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4° 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° structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of KMe.
Journal: Journal of Molecular Biology - Volume 356, Issue 5, 10 March 2006, Pages 1124–1136