Abstract
Human metallothionein (MT) is a small-size yet efficient metal-binding protein, playing an essential role in metal homeostasis and heavy metal detoxification. MT contains two domains, each forming a polynuclear metal cluster with an exquisite hexatomic ring structure. The apoprotein is intrinsically disordered, which may strongly influence the clusters and the metal-thiolate (M-S) bonds, leading to a highly dynamic structure. However, these features are challenging to identify due to the transient nature of these species. The individual signal from dynamic conformations with different states of the cluster and M-S bond will be averaged and blurred in classic ensemble measurement. To circumvent these problems, we combined a single-molecule approach and multiscale molecular simulations to investigate the rupture mechanism and chemical stability of the metal cluster by a single MT molecule, focusing on the Zn4S11 cluster in the α domain upon unfolding. Unusual multiple unfolding pathways and intermediates are observed for both domains, corresponding to different combinations of M-S bond rupture. None of the pathways is clearly preferred suggesting that unfolding proceeds from the distribution of protein conformational substates with similar M-S bond strengths. Simulations indicate that the metal cluster may rearrange, forming and breaking metal-thiolate bonds even when MT is folded independently of large protein backbone reconfiguration. Thus, a highly dynamic polynuclear metal cluster with multiple conformational states is revealed in MT, responsible for the binding promiscuity and diverse cellular functions of this metal-carrier protein.
Highlights
Transition metals are essential ingredients for life and in a large fraction are covalently bound to proteins [1]
Recent studies on nitrogenase showed that metal-coordinating sulfur atoms in the metal cluster could move during the enzymatic reaction, as a dynamic metallocofactor critical for catalysis, indicating how dynamic a metal cluster could be in a protein [4]
Metallothionein (MT) is another remarkable metalloprotein. It is an efficient metal-chelator protein with twenty metal-coordinating cysteines, capable of promiscuous binding more than ten different types of metals, forming two polynuclear metal clusters, M4S11 and M3S9, in its two domains, respectively [5, 6]
Summary
Transition metals are essential ingredients for life and in a large fraction are covalently bound to proteins [1]. It is well known that Apo-MT is intrinsically disordered and highly flexible, which may strongly influence the clusters and lead to a dynamic structure with unique metal-thiolate (M-S) bonds These features are challenging to identify due to the transient nature of the involved species. The individual signal from dynamic conformations of a protein with various populated states of the cluster and arrangements of M-S bonds will be averaged and blurred in classic ensemble characterization. To circumvent these problems, we combined a singlemolecule approach and multiscale molecular simulations to investigate the dissociation mechanism and chemical. The signal for a specific M-S bond and cluster configuration is blurred in classic ensemble methods that average upon multiple cluster connectivities, protein conformations, and a possibly heterogeneous MT sample as mentioned [35]
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