.J. Mol. Wlol. (1987) l%, 877-W) Structure and Refinement at 143 A Resolution of the Aspartic Proteinase from Rhizopus chinensis K. Suguna, Richard R. Bottt, Eduardo A. Padlan, E. Subramanianl Steven Sheriff, Gerson H. Cohen and David R. Davies Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda MD 20892, U.S.A. ( Rewived 6 November 1986, and in revised form 14 April 1987) The structure of rhizopuspepsin (EC 3.4.23.6), the aspartic proteinase from Rhizop,us chinensis, has been refined to a crystallographic R-factor of 0.143 at 1.8 A resolution. The positions of 2417 protein atoms have been determined with a root-mean-square (r.m.s.) error of 0.12 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.010 A, and for angle distances it is O-034 A. During the course of the refinement, a calcium ion and 373 water molecules, of which 17 are internal, have been located. The ache aspartate residues, Asp35 and Asp218, are involved in similar hydrogen-bonding interactions with neighboring residues and with several water molecules. One water molecule is located between the two carboxyl groups of the catalytic aspartate residues in a t)ightly hydrogen-bonded position. The refinement resulted in an unambiguous interpretation of the highly mobile “flap”, a /?-hairpin loop region that projects over the binding pocket. Large solvent channels are formed when the molecules pack in the crystal, exposing the binding pocket and making it easily accessible. Intermolecular contacts involve mainly solvent molecules and a few protein atoms. The three-dimensional st’ructure of rhizopuspepsin closely resembles other aspartic proteinase structures. A detailed comparison wit’h the structure of penicillopepsin showed striking similarities as well as subtle differences in the active site geometry and molecular packing. 1. Introduction The aspartic proteinases form one of the major classes of proteolytic enzymes, together with the serine, t’hiol and metallo proteinases. Enzymes of this class are widely distributed, having been found in micro-organisms, plants and mammals. They have been isolated from stomach (pepsin, chymosin and gastricsin), lysozome (cathepsin D) and kidney and submaxillary gland (renin). They are charac- terized by having t’wo aspartyl residues in their catalytic apparatus, and are inhibited by diazonor- leucinr met,hyl ester, epoxy(p-nitrophenoxy)- propane and by pepstatin, a hexapeptide from Streptomyces. All of these enzymes (with the exception of renin) operate optimally at acidic pH, have molecular t Present address: Genentech, 460 San Bruno Blvd., Ho. San Francisco, (‘A 94080, U.S.A. $ Present address: Department of Crystallography and Biophysics. University of Madras, Madras-600 0%. India. weights between 35,000 and 42,000, and show significant sequence homology. Three-dimensional structures have been determined for porcine pepsin (Andreeva et al., 1977. 1985), pepsinogen (James & Sielecki, 1986), and for three fungal proteinases: penicillopepsin (Hsu et al., 1977; James & Sielecki. 1983), endothiapepsin (Jenkins et al., 1977; Sub- ramanian et al., 19770; Hlundell of al., 1985) and rhizopuspepsin (Subramanian et al., 1977a.6). In this paper, we describe the refined structure of rhizopuspepsin (EC 3.4.23.6), the aspartic proteinase from Rhizopua chinensis, based on X-ray diffraction dat)a to a resolution of’ approximately 1.75 A. 2. Materials and Methods (a) (‘rystallizatiovi Rhizopuspepsin with p/ = 5.15 was provided k)y Robert Delaney of the IJnirersity of Oklahoma. The crystals were initially grown from 20 mw-oalrium acetate solution with a protein concentration of 50 mg/ml at pH 6.0