Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

Abstract

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new techniques have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

“…For molecular modeling and database searches, the program WHAT IF was used [24]. The modeling procedures have been described in detail elsewhere [6]. A threedimensional model of TLP-ste was built on the basis of the crystal structure of thermolysin [4].…”

“…TLP consist of an N-terminal domain (residues 1 -154; thermolysin numbering) characterized by a predominance of P-pleated sheet and a C-terminal domain (residues 155-31 6) that is mainly a-helical [4, 51. TLP differ in thermal stability [6] and the structural determinants of these differences have been analyzed by several sitedirected mutagenesis studies [7-111. At elevated temperatures TLP are irreversibly inactivated as a result of autolysis.…”

“…It has been shown that local unfolding processes that render the protein susceptible towards autolysis are the rate-limiting steps in thermal inactivation [6,[15][16]. It has been proposed that these thermally induced local unfolding processes mainly involve surface located regions of the protein 16, 9, 141.…”

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

“…The modeling procedures have been described in detail elsewhere (32). Because of the high sequence similarity, the model was expected to be sufficiently reliable for prediction and analysis of the effects of most amino acid substitutions (32,33).…”

“…quantified by T 50 , being the temperature giving 50% residual activity after a 30-min period of incubation (32,40).…”