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The crystal structure of purine-specific ribonuclease (RNase) U2 from Ustilago sphaerogena has been solved by the molecular replacement methods using RNase T1 as a search model. The structure, with 114 amino acid residues, 141 water molecules, and a sulfate ion, is refined to an R factor of 0.143 at 1.8 A resolution. As evidenced by the electron densities, residues 49 and 50 are revised to Glu 49 and Asp 50, respectively, and also Asp 45 is identified as a beta-isomerized form to L-isoaspartate with a beta-peptide linkage. RNase U2 consists of a beta-hairpin at residues from 7 to 14, a 4.4-turn alpha-helix from 16 to 32, a central beta-sheet with five strands, and a protruding beta-turn from 74 to 77. As for the catalytic site residues, His 41, Glu 62, and Arg 85 are located as constituents of the central beta-sheet, and Tyr 39 and His 101 are situated at either end of the beta-sheet. The side chains of Tyr 39, Glu 62, Arg 85, and His 101 are hydrogen-bonded to the sulfate ion which marks the RNA phosphate position. Though the side chain of His 41 is pointing away from the sulfate, small conformational adjustments of His 41 enable the side chain to interact with either the phosphate or the ribose group of RNA. The loop region from Tyr 44 to Asp 50 is ascribed to the base recognition site where Glu 49 is involved in adenine recognition. beta-Isomerized Asp 45 suggests that this region is conformationally flexible and alterable.

Heat shock protein (Hsp) 40s play essential roles in cellular processes by cooperating with Hsp70 proteins. Hsp40 proteins recognize non-native polypeptides, deliver these peptides to Hsp70 proteins, and stimulate the ATPase activity of Hsp70 proteins to facilitate the correct folding of the polypeptides. We have determined the crystal structures of the C-terminal peptide-binding domain of human Hsp40 Hdj1 (CTD) and of its complex with the C-terminal octapeptide of human Hsp70, (634')GPTIEEVD(641'). CTD exists as a twisted, horseshoe-shaped homodimer. The protomer consists of two domains, I and II, with similar topologies. The octapeptides are located in two sites, 1 and 2, of domain I. In site 1, the octapeptide forms an antiparallel β-sheet with CTD. The negatively charged residues of the EEVD motif in the octapeptide form electrostatic interactions with the positively charged Lys residues of CTD. The Ile side chain of the octapeptide fits into the narrow concave formed by the hydrophobic residues of CTD. In site 2, the octapeptide also forms an antiparallel β-sheet with CTD, and the EEVD motif forms electrostatic interactions. The side chains of Pro and Ile of the octapeptide interact with the hydrophobic surface region of CTD site 2, which is broader and shallower than the concave binding region of site 1. This region seems to be capable of binding hydrophobic side chains that are bulkier than the Ile side chain. The roles of these two peptide-binding sites of Hdj1 are discussed.

Unlike trypsin-like serine proteases having only one conspicuous binding pocket in the active site, subtilisin BPN' has two such pockets, the S1 and S4 pockets, which accommodate the P1 and P4 residues of ligands (after Schechter and Berger notation) respectively. Using computer graphics, the geometrical nature of the two pockets was carefully examined and strategies for site-directed mutagenesis studies were set up against a protein SSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceous inhibitor (or a substrate analogue) of subtilisin BPN'. It was decided to convert the P1 residue, methionine 73, into lysine (M73K) with or without additional conversion of the P4 residue, methionine 70, into glycine (M70G). The crystal structures of the two complexes of subtilisin BPN', one with the single mutant SSI (M73K) and the other with the double mutant SSI (M73K, M70G) were solved showing that (i) small 'electrostatic induced-fit movement' occurs in the S1 pocket upon introducing the terminal plus charge of the lysine side chain, and (ii) large 'mechanical induced-fit movement' occurs in the S4 pocket upon reducing the size of the P4 side chain from methionine to glycine. In both (i) and (ii), the induced-fit movement occurred in a concerted fashion involving both the enzyme and 'substrate' amino acid residues. The term 'substrate-assisted stabilization' was coined to stress the cooperative nature of the induced-fit movements.

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