Sequential Proton Resonance Assignments and Metal Cluster Topology of Lobster Metallothionein-1

Abstract

NMR studies of 111Cd6-MT 1 from lobster have been conducted to determine coordination structure of Cd-thiolate binding in the protein. Sequential proton resonance assignments were made using standard two-dimensional 1H NMR methods. Two-dimensional 1H-111Cd HMQC experiments were then carried out to determine the cadmium-cysteine connectivities in the protein. With this information, it was established that the six Cd ions exist in two different Cd3S9 clusters, each involving three bridging and six terminal thiolate ligands. Sequential cysteines in the sequence provide the sulfhydryl ligands for each cluster and do not overlap, as has been found in mammalian metallothionein. Comparison of the N-terminal, Cd3S9 B-type cluster of lobster MT 1 with the Cd3S9 cluster from rabbit MT 2 shows that while eight of the nine cysteine residues occupy homologous positions in their sequences, three of the 12 Cd-thiolate connectivities are different. Similarly, the C-terminal B-cluster of lobster MT 1 was compared with the Cd4S11 cluster of mammalian MT 2, excluding the two terminal cysteine sulfhydryl groups that convert this cluster from A- to B-type. As above, eight of nine cysteine positions are identical, yet five of 12 Cd-sulfhydryl connections are different. These differences are expanded when the role of each cysteine as bridging or terminal ligands in the clusters is considered.

A New Insight into Metallothionein (MT) Classification and Evolution

“…This is supported by the following observations: (a) a higher copper binding ability of ␤␤MTH than that of ␤␣MTH; (b) the comparable behavior of the crustacean and mammalian individual mutant domains; and (c) the capacity of MTH C-terminal domain to yield a minor Zn 4 species in vivo. The different Cd(II) connectivities reported for the C-terminal domains of Callinectes 1 and Homarus MT (11,12), despite sharing the number and position of cysteine residues, also supports the predicted flexibility of the crustacean ␤␣MT domain.…”

“…On the basis of their amino acid alignments, further application of the ␤/␣ domain terminology led to the consideration of crustacean peptides as ␤-␤ MT (10) and thus putative copper thioneins. This was corroborated by the three divalent ions contained in each domain of the crustacean Cd-MT structures solved by NMR (11,12) and thus comparable with the Cd 3 -␤MT fragment in mammalian MT.…”

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

“…However, the ability of S2ЈC receptors to coordinate Cd 2ϩ in both the native open and closed configurations of the receptor places a restriction on the maximal diameter the pore attains in both states. Using the length of the Cd-S bond (2.1-2.5 Å; (28,29,36,54)), the ionic radius of Cd 2ϩ (0.91 Å), and assuming 5-fold pseudosymmetry in the 5-HT 3A homopentameric receptor, we can estimate a maximum pore diameter of ϳ7 Å if coordination occurs across the receptor pore. If Cd 2ϩ is coordinated by sulfhydryl moieties on adjacent subunits, the pore diameter can be as wide as ϳ11 Å (using the Pythagorean theorem).…”

Structure of the 113Cd3β domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms

“…Previous structural studies carried out with lobster (Homarus americanus) and crab (Callincetes sapidus) Cd 6 -MT using 2D-NMR methods showed that the Cd-cysteine connectivities and, hence, the peptide folding, differ between their b N and b C clusters and also diverge from that found for the b N domain in mammalian MTs, despite the nearly homologous placement of cysteine residues in the crustacean and mammalian b N domains [17,18]. These three M 3 b domains display different reactivity toward reagents such as DTNB [5,5¢-dithio(2-nitrobenzoic acid)] and EDTA, leading to the hypothesis that their reactivity is related to the detailed folding of the peptide sequences around their clusters and not just to properties of the clusters themselves [19,20].…”

“…Crustacean MTs differ from the mammalian structure in that they only have 18 cysteine residues within their sequences that give rise to two M 3 S 9 clusters and peptide domains [16,17,18]. Previous structural studies carried out with lobster (Homarus americanus) and crab (Callincetes sapidus) Cd 6 -MT using 2D-NMR methods showed that the Cd-cysteine connectivities and, hence, the peptide folding, differ between their b N and b C clusters and also diverge from that found for the b N domain in mammalian MTs, despite the nearly homologous placement of cysteine residues in the crustacean and mammalian b N domains [17,18].…”