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 clust...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 theαdomain 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.展开更多
基金was funded by the Fundamental Research Funds for the Central Universities(Grant No.14380259)the Natural Science Foundation of Jiangsu Province(Nos.BK20200058 and BK20202004)+1 种基金the National Natural Science Foundation of China(Grant Nos.21771103 and 21977047)the Fundação para Amparo a Pesquisa do Estado de São Paulo(FAPESP)(scholarship 2017/26109-0 to FC and Grants 2018/08311-9 and 2019/21856-7 to GMA)。
文摘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 theαdomain 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.
