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., re presents a structurally distinct group of metalloproteinases termed th e 'astacin family'. This protein family includes oligomeric membrane-b ound proteins with zinc proteinase domains found in rodent kidneys (me prins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminob enzoate hydrolase). Another branch of this family comprises morphogene tically active proteins, which induce bone formation (human bone morph ogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ra y crystal structure of astacin has recently been solved to a resolutio n of 0.18 nm [Bode et al. (1992) Nature 358,164-1671. This structure i s different from hitherto known metalloendopeptidase structures and ha s been used in the present study to analyze the structures of the othe r members of the astacin protein family. Computer-assisted modelling o f the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue d eletions and three single residue insertions are implied. The proteina se domains of the other astacins can be included in the model-based se quence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop s egments connecting regular secondary structure elements and should lea ve the overall structure unaltered. The topology of residues forming t he zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewis e metalloproteinases. Based on this similarity, it is proposed that th e active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigo nal bipyramidal geometry. Other remarkable common features are a hydro phobic cluster in the N-terminal domain and a conserved, solvent-fille d cavity buried in the C-terminal domain. Most interestingly, the amin o-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the termi nal alanine residue forms a water-linked salt bridge to Glu103, direct ly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like pr oteinases 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 t he astacins. A striking example are variations in the presumed S', sub strate-binding site, which binds the amino acid side chain on the C-te rminal side of the scissile bond of the substrate. In this subsite the crayfish proteinase astacin prefers short, uncharged residues. By con trast, meprin A accepts bulky, charged side chains in this position. T his difference presumably can be explained by both the replacement of Pro176 (astacin) by Gly176 (all other astacins) and the concomitant de letion of Tyr177 (all other astacins). Interestingly, the three imidaz ole-zinc ligands are included in a consensus sequence (HEXXHX-XGXXH) w hich the astacins share with otherwise sequentially unrelated enzymes like vertebrate matrix metalloproteinases (matrixins), snake venom hae morrhagic toxins and certain large bacterial enzymes. Hence, a zinc li gation similar to that seen in astacin is probable also for these prot einases.