Correlation between sites of limited proteolysis and segmental mobility in thermolysin.

Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors ( B values) determined crystallographically [Holmes, M. A., & Matthews, B. W. (1982) J . Mol. Biol. 160, 623-6391. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes. A native globular protein is not a static entity but rather one that undergoes conformational fluctuations about its most stable conformation, as clearly emphasized in recent years by numerous studies using a number of varied physical methods (Gurd & Rothgeb, 1979; McCammon & Karplus, 1983). Crystallographic results have provided direct evidence of intramolecular motions in proteins, as indicated by the fact that certain parts of the protein molecule are systematically poorly resolved, e.g., the terminal atoms of long, flexible, external side chains such as that of lysine (Takano, 1977), the ends of polypeptide chains (Thornton & Sibanda, 1983), or some segments within the chains such as segment 18-23 in ribonuclease S (Richards & Wyckoff, 1971, 1973). More clear-cut evidence for motion in proteins comes directly from highly refined analysis of the diffraction data giving information about mean atom displacements, as usually expressed by the crystallographic temperature factors ( B values). The B value represents the mean-square displacement of each atom and, when plotted against residue number, provides a graphic image of the degree of mobility existing along the polypeptide chain. In particular, it has been found that B values are, in general, low in helical regions and high at bends and chain termini (Frauenfelder et al., 1979; Artymiuk et al., 1979; Sternberg et al., 1979). We will show in this paper that the sites of the polypeptide chain characterized by highest mobility are the more susceptible ones to proteolytic attack. This study was carried out with thermolysin, the highly thermostable metalloendoprotease from Bacillus thermoproteolyticits (Endo, 1962), for which the amino acid sequence of its chain of 3 16 amino acid residues (Titani et al., 1972) and three-dimensional structure (Matthews et al., 1972, 1974; Colman et al., 1972) are known. More recently, a highly refined structure of thermolysin at 1.6-A resolution has been reported, together with the average thermal motion (segmental mobility) of its polypeptide chain (Holmes & Matthews, 1982). To perform limited proteolysis of thermolysin, subtilisin was chosen as the attacking protease, or autolytic degradation was used. Because of the broad specificity of both subtilisin (Harris & Roos, 1959) and thermolysin (Morihara & Tzusuki, 1970; Keil, 1982), it was anticipated that peptide bond fission would occur at sites dictated by the stereochemistry and flexibility of the polypeptide substrate, and not by the specificity of the protease. MATERIALS AND METHODS -. 'This study was supported by the Consiglio Nazionale delle Ricerche, Thermolysin from Bacillus thermoproteolyticus (Rokko) was obtained from Sigma Chemical Co. (St. Louis, MO) as special program on Biotechnology. 0006-2960/86/04251847$01.50/0