Structural features of neutral protease from Bacillus subtilis deduced from model‐building and limited proteolysis experiments

Abstract

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20: 1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1 -214 and 21 5 -300 are separated under protein-denaturing conditions.Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins. subtilis [lo] are known. These sequence data, obtained by direct protein sequencing or by analyzing the corresponding cDNA, clearly established that these proteases show high degrees of sequence similarities, this being higher among thennostable proteases derived from thermophilic microorganisms, as well as among thermolabile ones from mesophilic sources. Moreover, the crystal structure of thermolysin [I 1 -141 and of the mesophilic neutral protease from B. cereus [15] have been reported. Thus, this ample set of well characterized (thermophilic and mesophilic) neutral proteases constitute an excellent model system for addressing problems of structure/ functionlstability relationships. As a matter of fact, initial studies of protein engineering experiments on neutral proteases by site-directed mutagenesis have already appeared [16, 171. Correspondence to A. Fontana, Department of Organic Chemistry, Via Marzolo 1, 1-35131 Padua, Italy.Abbreviations. Gdn In the ambit of a research program aimed to prepare and study mutants of B. subtilis neutral protease (NP) [17], it was of importance to develop a model of this protease in order to design interesting mutants and to interpret the possible effects of site-specific mutagenesis in terms of conformational details and interactions within the protein molecule. In this paper we report a proposed model of NP predicted on the basis of the known three-dimensional structure of TLN [14] with the aid of a graphics display. As expected from the 50% seq...

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

“…Finally, the results reported here provide some insights into the partly folded TFE-state of proteins utilizing a proteolytic probe, and complement those obtained by other investigators using spectroscopic techniques (Buck et al, 1993(Buck et al, , 1995Alexandrescu et al, 1994;Shiraki et al, 1994;Schonbrunner et al, 1996). This study represents a continuation of our efforts to demonstrate that proteolytic enzymes can be used as reliable probes of protein structure and dynamics (Vita et al, 1985;Fontana et al, 1989Fontana et al, , 1993Signor et al, 1990;Polverino de Laureto et al, 1994a, 1994b, 1995a, 1995bVindigni et al, 1994).…”

“…The aim of the present work was to probe the conformational state of RNase when dissolved in aqueous TFE by using proteolytic enzymes as probes of protein structure and dynamics (Mihalyi, 1978;Neurath, 1980;Fontana et al, 1989Fontana et al, , 1993Price & Johnson, 1990;Signor et al, 1990;Wilson, 1991). This experimental approach relies on the fact that proteolysis of a polypeptide chain requires the binding of a chain segment of 6-8 amino acid residues of the polypeptide substrate at the active site cleft of the protease (Berger & Schechter, 1970).…”

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

“…Indeed, the use of proteolytic enzymes as probes of protein structure and dynamics is a well recognized and often employed approach (Mihalyi, 1978 ;Neurath, 1980;Fontana et al, 1989Fontana et al, , 1993Signor et al, 1990). In analogy to all enzymic reactions, a proteolytic enzyme can cleave a polypeptide chain only if the site(s) of chain fission can bind and adapt to the specific stereochemistry of the enzyme active site.…”

Limited proteolysis of cytochrome c in trifluoroethanol

“…In previous studies, we have shown that the specific or preferential fission of peptide bond(s) by limited proteolysis [11,12] is a useful procedure to probe structural and dynamic aspects of globular proteins [13][14][15][16][17]. A detailed analysis of the structural characteristics of the sites of specific cleavages in proteins allowed us to establish that native globular proteins are cleaved at exposed and flexible loops only and never at chain segments of regular secondary structure (helices) [13,16].…”