Structure of actinidin: Details of the polypeptide chain conformation and active site from an electron density map at 2·8 Å resolution

Abstract

A 2·8 Å resolution electron density map of the sulphydryl protease, actinidin, has been calculated. Two isomorphous heavy-atom derivatives, prepared with uranyl acetate and dichloroethylenediamineplatinum(II), were used to calculate phases by the method of isomorphous replacement, giving an overall figure of merit of 0·81. The polypeptide chain is well-defined in the present map and many side-chains can be identified from their appearance. The molecule consists of a single chain of 220 residues, the last two of which appear disordered in the map and contains at least two, and probably three, disulphide bridges. The conformation of the polypeptide chain is remarkably similar to that of papain. It is folded into two domains, domain I consisting of residues 19–115 and 214–218, and domain II residues 1–18 and 116–213. There are three significant stretches of α-helix, involving residues 25–42, 69–81 and 120–129, together with several shorter pieces, while much of domain II consists of a twisted β-sheet structure. When compared with papain, actinidin has two additional residues inserted between 59 and 60, one inserted between 78 and 79, and four between 168 and 169 (papain numbering) while one residue (194) has been deleted from the papain structure. All these changes are in external parts of the molecule and have little effect on the conformation. The positions and orientations of the catalytically-important side-chains in the active site are virtually identical with those in papain, but some of the side-chains lining the non-polar binding pocket are clearly different.