Metallobiological Necklaces: Mass Spectrometric and Molecular Modeling Study of Metallation in Concatenated Domains of Metallothionein

Abstract

The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of Cu(I) and Zn(II), but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys-cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal-thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd(4)-containing domain of alpha-rhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 Cd(II) ions to the 11, 22, and 33 cysteinyl sulfur atoms in the alpha-rhMT1a, alphaalpha-rhMT1a, and alphaalphaalpha-rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal-sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.

Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

“…We can only postulate that each a domain is also somewhat discrete from the other two-domains when binding 3 As 3+ but upon coordinating the 10th and 11th As 3+ the individuality of each domain structure collapses, leaving a single globular structure, which may resemble that calculated for the apo-aaahMT [56]. A similar proposal was suggested to account for the very strong CD spectrum of Hg 18 -MT [57,58].…”

“…show that each a domain is discrete for Cd 12 -aaahMT [56]. We can only postulate that each a domain is also somewhat discrete from the other two-domains when binding 3 As 3+ but upon coordinating the 10th and 11th As 3+ the individuality of each domain structure collapses, leaving a single globular structure, which may resemble that calculated for the apo-aaahMT [56].…”

“…7) show the dependence of the rate constant values on the number of available binding sites. [56]. Our As 3+ -binding studies of the aaa human metallothionein show that by the end of the reaction, 11 As 3+ ions are bound to the 33 cysteines in the protein.…”

The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A

“…rhMT1A was expressed and purified following previously reported methods . The MT sequence used in this study is based on the recombinant human MT1A sequence that consists of 72 residues: MGKAAAACSC ATGGSCTCTG SCKCKECKCN SCKKAAAACC SCCPMSCAKC AQGCVCKGAS EKCSCCK KAA AA.…”

Metalation of metallothioneins

“…Over the next 50 years these optical properties have proven to provide excellent probes of the metalation status of the protein because of the atypical amino acid composition of mammalian MTs (2) (see Fig. 1 in ref 3…”