Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pH

Biochem Biophys Res Commun. 2017 Nov 4;493(1):650-653. doi: 10.1016/j.bbrc.2017.08.137. Epub 2017 Sep 1.

Abstract

Maintenance of the homeostasis of zinc is very important in regulating bodily functions. There are over 300 Zn-dependent enzymes identified where Zn(II) plays a structural or catalytic role. However, an excess of Zn(II) in a cell is toxic and free Zn(II) is tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins that can bind up to seven Zn(II) and act as a Zn(II) reservoir. The MT2a isoform is predominantly found in the liver. This study focused on designing an MT2a construct of recombinant human MT2a to determine the Zn(II) binding profile of MT2a in vitro. We analyzed the pH dependence of Zn-MT2a speciation from electrospray ionization mass spectral data. At physiological pH, Zn(II) is terminally bound to the cysteine thiols of MT2a, making bead-like structures (non-cooperative metal binding), while at low pH, Zn(II) formed Zn4S11-MT2a clusters involving bridged cysteinyl thiols to the Zn(II) (cooperative metal binding). The Zn(II) binding profile of MT2a was compared to Zn(II) binding profile of human kidney MT1a, which was reported in literature, and found that the Zn(II) binding profile of MT2a is similar to that of MT1a. The facility of forming bead-like structures at physiological pH for Zn5-MT2a means that Zn7-MT2a can donate up to two Zn(II) to Zn-dependent enzymes.

Keywords: Cooperative metalation; ESI-MS; Metallothionein 2a (MT2a); Non-cooperative metalation; Zinc binding to proteins; Zinc homeostasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Humans
  • Hydrogen-Ion Concentration
  • Liver / chemistry*
  • Liver / enzymology*
  • Metallothionein / chemistry*
  • Metallothionein / metabolism*
  • Protein Binding
  • Protein Interaction Mapping
  • Zinc / chemistry*
  • Zinc / metabolism*

Substances

  • MT2A protein, human
  • Metallothionein
  • Zinc