Determination of the solution three dimensional structure of chemically-synthesized native ¹¹³Cd₃S₉ domain of Lobster Metallothionein by NMR Spectroscopy

Amalia Muņoz^*, F. Holger Forsterling, C. Frank Shaw III⁺ and David H. Petering

INTRODUCTION

Metallothionein (MT) was discovered by Margoshes and Vallee in 1957. It is a small molecular weight protein that lacks aromatic amino acids but is rich in cysteine residues (in reduced form) that confer a high ability to bind metal ions. Some of the multiple biological functions in which MT appears to be involved are heavy metal detoxification (Hg²⁺, Cd⁺²), metabolism of essential ions such as Zn⁺² or Cu, detoxification of reactive oxygen species and metabolism of metallodrugs and alkylating agents^1-3.

(Note: Click here to view this structure in interactive 3D using the Chime plug-in)

Human and mammalian MTs bind 7 Zn⁺² or Cd⁺² ions via 20 cysteine residues, which are distributed in two independent and very dynamic clusters Cd₄S₁₁ and Cd₃S₉ located at the alpha- and beta- domains of the protein, respectively^3,4.

Crustacean MTs contain only 18 cysteines and 6 Cd⁺², which generate two Cd₃S₉ clusters^8,9. Previous structural studies carried out in both species by 2D-NMR techniques show that the Cd-cysteine coordination and hence the folding differs between these Cd₃S₉ clusters in the beta-domains of crustacean and mammalian MTs.

Based on the biphasic kinetic behaviour of the reaction between lobster MT and thiol reagents such as DTNB (5,5'-dithio-2,2'dinitro benzoic acid) or DTP¹¹ (dithiopyridine) and the different thermodynamic and kinetic reactivity exhibited by the isolated domains of mammalian MTs^6,7,12, also biphasic behaviour, but are more rapid than the mammalian MT reactions. It was proposed that the kinetic and thermodynamic reactivity of the MT domains is related to the detailed folding of the peptides around their clusters.

To analyze structure-function relationships among these domains, in this poster we describe the 3D-structure of the chemically synthesized native lobster beta_C as described by 2D-NMR spectroscopy and analyzed its reactivity toward EDTA and DTNB.

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Amalia Muņoz - April, 2000