Metal specificity and mechanism of regulation by the toxic metal ion sensor S. aureus p1258 CadC /
Staphylococcus aureus pI258 CadC is a metalloregulatory protein from the SmtB/ArsR family that represses the expression of the cad resistance operon. This operon encodes CadC and a Cd(II)/Pb(II)/Bi(III)/Zn(II)-specific efflux ATPase, CadA. To gain insight into metal ion selectivity and allosteric...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2003.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=765867101&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Staphylococcus aureus pI258 CadC is a metalloregulatory protein from the SmtB/ArsR family that represses the expression of the cad resistance operon. This operon encodes CadC and a Cd(II)/Pb(II)/Bi(III)/Zn(II)-specific efflux ATPase, CadA. To gain insight into metal ion selectivity and allosteric regulation of DNA binding, wild-type and cysteine-substitution CadCs have been characterized. Two distinct metal binding sites exist within homodimeric-CadC, denoted α3N and α5. The α3N site is composed entirely of cysteine ligands (Cys7, Cys11, Cys58, and Cys60) and preferentially coordinates toxic metal ions such as Cd(II), Pb(II), and Bi(III) with high equilibrium affinities. Biologically required metal ions such as Zn(II) and Co(II) also bind to this site, as well as to the α5 site via imidazole/carboxylate ligation. Optical, X-ray absorption, ¹¹³Cd(II) NMR, and perturbed angular correlation spectroscopies reveal that the coordination geometry of the α3N site can vary between distorted tetrahedral (Cys₄) and trigonal (Cys₃). Kinetic experiments support that the α3N site forms metal complexes with very fast association kinetics (1x10⁷ M⁻¹s⁻¹) and slow dissociation kinetics (~10⁻⁵-10⁻⁶ s⁻¹). These properties are proposed to play the major role in the selectivity of metal ions. Dimeric apo-CadC binds with high affinity to a 34-base pair cad operator/promoter. Stoichiometric binding of Cd(II), Pb(II), or Bi(III) to the α3N site results in strong negative allosteric regulation the affinity of dimeric CadC for the cad operator/promoter. Non-inducing metal ions can also regulate DNA binding, although to a lesser degree. Of the cysteine thiolates that coordinate the α3N metal ion, Cys7 in the unstructured N-terminal "arm" and Cys60 in the α3 helix of the helix-turn-helix DNA binding are indispensable for the allosteric regulation of DNA binding, despite the ability of C7G and C60G substitution mutants of CadC to form stable coordination complexes. Further experiments confirm that only the α3N metal binding site is required for metalloregulation. Equilibrium fluorescence anisotropy experiments reveal that metal binding to the CadC-DNA complex results in significant dissociation of the metallated-protein from the operator/promoter sequence, providing a plausible mechanism of metalloregulation. A model of DNA binding proposed for CadC is discussed in the context of other SmtB/ArsR metal sensor proteins. |
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| Item Description: | Vita. In title numerals are used. "Major Subject: Biochemistry". |
| Physical Description: | xii, 210 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 197-209). |