SEPARATION AND CHARACTERIZATION OF THE METAL-THIOLATE-CLUSTER DOMAINSOF RECOMBINANT SEA-URCHIN METALLOTHIONEIN

Partial metal depletion and Cd-113-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylo centrotus purpuratus (Cd-7-MTA) binds its metal ions in a four-metal ( Cd(4)Cys(11)) and a three-metal (Cd(3)Cys(9)) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wan g, Y., Mackay, E. A., Zerbe, O., Hess, D., Hunziker, P. E., Vasak, M. & Kagi, J. H. R. (1995) Biochemistry 34, 7460-7467]. This partitioning has now been confirmed by bisecting native Cd-7-MTA with subtilisin i nto products bearing only a single metal-thiolate cluster Their separa tion by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of t he protein into a set of N-terminal polypeptides containing 37-39 resi dues 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 do mains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two meta l-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 conse quences of this repartition were reflected in massive chiroptical chan ges 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 f eatures attributable to the intact three-metal cluster were largely lo st on cleavage. The vanished features bear strong resemblance to the l arge biphasic ellipticity signal at 250 nm which dominates the CD spec trum of native Cd-7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the th ree-metal cluster.