Metal-dependent protein folding mechanisms of the alpha domain of human metallothionein

Over 30% of all proteins require metal ions for functional or structural purposes. The effect of metal-protein interactions on the stability of the folded protein structure and the role that the metal ion(s) play in the folding mechanism has been largely overlooked. Metallothionein (MT) is a unique metalloprotein that folds in the presence of divalent metal ions (M) to form two distinct domains, each containing a metal-thiolate cluster with stoichiometries of M3(Scys)9 (beta domain) and M4(S cys)11 (alpha domain), respectively, making it an ideal model to study metal-induced folding mechanisms. The present study investigates the metal binding reactivity of the isolated alpha domain of human MT-1a with Cd2+ using the techniques of CD, UV absorption, NMR and stopped-flow spectroscopy, ESI mass spectrometry and molecular dynamics/mechanics computational techniques.;From the results of these studies, it is proposed that the metal-free protein can adopt two different structural motifs: (1) a well-defined H-bonded structure that is induced by the template effect of the previously coordinated metal-thiolate cluster, or (2) a random conformation that is associated with the de nova synthesized protein. Furthermore, the metallation mechanism is predicted to he dependent on the initial structure of the apo protein.;Keywords: Metallothionein, metal-dependent folding, cooperativity, cadmium, CD spectroscopy, ESI mass spectrometry, stopped-flow kinetics, 113 Cd NMR spectroscopy, molecular mechanics, molecular dynamics.;The results of the metallation studies were as follows: (1) the rate of the metallation reaction of Cd2+ was shown to be very fast (kobs = 106 108 M-1 s-1) when the demetallated protein was the reacting species. (2) a reduction in the metallation rate was observed in the presence of a chemical denaturant. (3) the MM3/MD calculations of the demetallated protein domains showed significant retention of the backbone structural features imposed by the metal-thiolate cluster. (4) the MM3/MD calculations of the in silico-synthesized metal-free protein as a linear strand showed random behaviour of the polypeptide backbone. (5) partially-metallated domain intermediates were detected in the presence of sub-stoichiometric Cd2+ concentrations confirming that metal binding proceeds by four sequential, non-cooperative metallation reactions, and (6) excess Cd2+ was shown to induce the formation of a novel Cd5alpha species in which the fifth Cd2+ ion inserted into the cluster by coordination to two cysteinyl sulfur ligands.