Refined 1·8 Å X-ray Crystal Structure of Astacin, a Zinc-endopeptidase from the Crayfish Astacus astacus L. : Structure Determination, Refinement, Molecular Structure and Comparison with Thermolysin

Abstract

Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1·8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0·158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long α-helices, one centrally located and one more peripheral, and of a five-stranded pleated β-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-sit distinguish their structures from astacin. The five-stranded β-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central α-helix and zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and HIs142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue). The site of the third zinc-liganding histidine, His102, is, however, occupied by the sequentially more distant Glu1665TL, while Tyr149 has no direct counterpart in thermolysin. The astacins share an even longer consensus sequence His-Glu-Uaa-Xaa-His-Xaa-Uaa-Gly-Uaa-Xaa-His (where Uaa is a bulky, apolar residue) with several other metalloendo-peptidases of hitherto unknown tertiary structure, namely the vertebrate collagenases, snake-venom proteins and some "large" bacterial proteinases. It is therefore likely that in these otherwise sequentially unrelated proteinases the three adjacent histidine residues (corresponding to His92, His96 and His102) represent three of their zinc ligands. In Hg-astacin, the mercury is displaced by 0·45 Å relative to the site occupied by zinc in the native enzyme. The mercury ion exhibits a strongly distored tetrahedral co-ordination and is liganded by three histidine and one tyrosine residues. In apoastacin, the zinc site appears to be completely empty, whereas the rest of the crystal structure is virtually identical with the zinc enzyme.