Mehlenbacher Matthew R, Elsiesy Rahma, Lakha Rabina, Villones Rhiza Lyne E, Orman Marina, Vizcarra Christina L, Meloni Gabriele, Wilcox Dean E, Austin Rachel N
Department of Chemistry, Dartmouth College Hanover NH 03755 USA.
Department of Chemistry, Barnard College of Columbia University New York NY 10027 USA
Chem Sci. 2022 Apr 4;13(18):5289-5304. doi: 10.1039/d2sc00676f. eCollection 2022 May 11.
Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn and Cu binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn binding was measured by chelation titrations of ZnMT-3, while Cu binding was measured by Zn displacement from ZnMT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant () and the change in enthalpy (Δ) and entropy (Δ) for these metal ions binding to MT-3. Zn was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of ZnMT-2 with Cu revealed that both MT isoforms have similar Cu affinities and binding thermodynamics, indicating that Δ and Δ are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu -thiolate clusters when Cu displaces Zn under physiological conditions. Comparison of the Zn and Cu binding thermodynamics reveal that enthalpically-favoured Cu, which forms Cu -thiolate clusters, displaces the entropically-favoured Zn. These results provide a detailed thermodynamic analysis of d metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.
金属硫蛋白（MTs）是一类广泛存在的小金属结合蛋白，参与金属稳态和解毒过程。尽管它们对d金属离子具有高亲和力，但关于金属与MTs结合的热力学数据却惊人地匮乏。在本研究中，通过等温滴定量热法（ITC）在pH 7.4条件下对锌和铜与哺乳动物金属硫蛋白-3（MT-3）的结合进行了定量分析。通过对ZnMT-3进行螯合滴定来测量锌的结合，而通过谷胱甘肽（GSH）竞争从ZnMT-3中置换锌来测量铜的结合。在多种缓冲液中进行滴定能够进行详细分析，得出这些金属离子与MT-3结合的条件无关的缔合常数（）以及焓变（Δ）和熵变（Δ）值。还从MT-3的单个α和β结构域中螯合锌，以量化金属结合中结构域间相互作用的热力学。用铜对ZnMT-2进行比较滴定表明，两种MT同工型具有相似的铜亲和力和结合热力学，表明Δ和Δ主要由保守的半胱氨酸残基决定。对富含铜的样品进行电感耦合等离子体质谱（ICP-MS）分析和低温发光测量表明，在生理条件下当铜置换锌时，两种蛋白质都会形成两个铜硫醇盐簇。锌和铜结合热力学的比较表明，形成铜硫醇盐簇的焓有利的铜会取代熵有利的锌。这些结果提供了对d金属与这些富含硫醇盐的蛋白质结合的详细热力学分析，并为MT-3在铜的神经元化学中所起的作用提供了定量支持以及分子层面的见解。
