IMPLICATIONS OF THE 3-DIMENSIONAL STRUCTURE OF ASTACIN FOR THE STRUCTURE AND FUNCTION OF THE ASTACIN FAMILY OF ZINC-ENDOPEPTIDASES

Astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358,164-1671. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family. Computer-assisted modelling of the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered. The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins. Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins. A striking example are variations in the presumed S', substrate-binding site, which binds the amino acid side chain on the C-terminal side of the scissile bond of the substrate. In this subsite the crayfish proteinase astacin prefers short, uncharged residues. By contrast, meprin A accepts bulky, charged side chains in this position. This difference presumably can be explained by both the replacement of Pro176 (astacin) by Gly176 (all other astacins) and the concomitant deletion of Tyr177 (all other astacins). Interestingly, the three imidazole-zinc ligands are included in a consensus sequence (HEXXHX-XGXXH) which the astacins share with otherwise sequentially unrelated enzymes like vertebrate matrix metalloproteinases (matrixins), snake venom haemorrhagic toxins and certain large bacterial enzymes. Hence, a zinc ligation similar to that seen in astacin is probable also for these proteinases.