Separation and characterization of the metal-thiolate-cluster domains of recombinant sea urchin metallothionein.

Abstract

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.