Characterization of the cadmium complex of peptide 49-61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA.
The synthetic peptide fragment containing residues 49-61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd7MT, forms a stable, monomeric Cd-peptide complex with 1:1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The 111Cd NMR chemical shift at 716 ppm indicates that the 111Cd2+ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, approximately 3.3, demonstrates an affinity similar to that for Cd7MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd2+ center has the same counterclockwise chirality (lambda or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS4 structure toward 5,5'-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (a-domain or holo-protein). The rate law for the reaction with DTNB is rate = (k(uf) + k(1,f) + k(2,f) [DTNB])[peptide], where k(uf) = 0.15 s(-1), k(1,f)= 2.59x10(-3) s(-1), and k(2,f) = 0.88 M(-1) s(-1). The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M7MT) and crustacean (M6MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg2+, Cd2+, and Zn2+, M2-peptide for Hg2+ and Au+, and (Et3PAu)2-peptide.
Muñoz
A
A
Department of Chemistry and the UWM-NIEHS Marine and Freshwater Biomedical Core Center, University of Wisconsin-Milwaukee 53201-0413, USA.
Laib
F
F
Petering
D H
DH
Shaw
C F
CF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D011817:Rabbits; D013816:Thermodynamics
D008670:Metals; D010446:Peptide Fragments; D002104:Cadmium; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid