Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases

Abstract

ASTACIN, a digestive zinc-endopeptidase from the crayfish Astacus astacus L.^1,2, is the prototype for the 'astacin family'^3–5, which includes mammalian metallo-endopeptidases⁵ and develop-mentally regulated proteins of man⁶, fruitfly⁷, frog⁸ and sea urchin^9,10. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases¹¹. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.