Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites

摘要

A number of biological functions have been ascribed to mammalian metallothioneins (MTs) including zinc and copper homeostatic regulation, redox activity and detoxification of heavy metals like cadmium and mercury. It is unclear how these small, fluxional, cysteine rich proteins manage to play each of these vital roles. Using a combination of cadmium and pH titrations of the isolated domains of human MT isoform 1a monitored by electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report the pH dependencies that control metal binding mechanisms of these domains. We report that the alpha-domain mechanism is driven by the cooperative formation of the Cd4MT cluster at slightly acidic pH (<= 6.9) switching binding mechanisms over a physiologically relevant pH range, whereas the beta-domain metalation mechanism is dominated by terminal coordination of cadmium in a non-cooperative manner above pH 5.5. These results suggest that, in some acidic sub-cellular compartments, cadmium could be sequestered in the alpha-domain, leaving zinc or copper bound in the beta-domain and available for donation to other metalloproteins. We propose that these results can be explained by the intrinsic nature of the two domains, the four-metal alpha-cluster being more resistant to proton attack due to its lower charge-to-metal ratio, compared with the three-metal beta-domain. (c) 2016 Elsevier Inc. All rights reserved.