Implications of the three‐dimensional structure of astacin for the structure and function of the astacin family of zinc‐endopeptidases

Abstract

Astacin, a zinc-endopeptidase from the crayfish Astacus astucus 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-~-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 a-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 Hisl02, 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 replacemen...

The astacin family of metalloendopeptidases

“…Interestingly, flavastacin lacks the four conserved cysteine residues of the other astacin family members. The conservation of most of the residues identified by Stocker et al (1993) to be involved in internal bonds may be particularly important to the folding and stability of the bacterial enzyme. The enzyme contains an unusual 0-linked oligosaccharide, and initial experiments indicate a preference for cleavage of peptides with aspartic acid in the P1' position.…”

The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes

“…Astacin metalloproteases are structurally distinct zinc metallo-endopeptidases, characterized by 2 conserved motifs in the catalytically-active astacin domain: the catalytic active-site (HExxHxxGFxHExxRxDRD) for binding the essential Zn 2 + and the methionineturn (SxMHY) which maintains the enzyme conformation. The active-site zinc is penta-coordinated and is bound by the 3 histidine residues and the glutamic acid residue in the active-site and the tyrosine residue in the methionine-turn (Stöcker et al 1993). There are 39 Nematode AStacin (NAS) metalloproteases in the free-living nematode, C. elegans, divided into 6 subgroups (I to VI) that correlate with the functionally significant C-terminal domain structure (Möhrlen et al 2003).…”

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

“…Meprin is a membranal metalloendoproteinase found in the intestinal and renal brush-border membranes of the mouse (14,15), rat (16 -18), and man (19,20). Meprins belong to the astacin family of endopeptidases (21)(22)(23)(24) and are usually composed of two types of subunits, ␣ and ␤, that exist as homo-and heterotetramers bound to each other through disulfide bridges (25,26). In spite of a large number of studies on meprins, the substrate specificity and the physiological assignment are not established yet even though several suggestions have been set forth.…”