On the unfolding of alpha-lytic protease and the role of the PRO region

Molecular dynamics simulations of alpha-lytic protease (alpha LP) alone and complexed with its pro region (PRO) are performed to understand the origin of its high unfolding (and folding) barrier when it is alone and how the p ro region lowers this barrier. At room temperature, alpha LP exhibits lower dynamic fluctuations than alpha-chymotrypsin, Simulation of PRO alone led to reorientation of its N terminal helix and collapse to a more compact sta te. A model for the uncleaved proenzyme was built and found to be stable in the time scale of the simulations. Energetic analysis suggests that the or igin of strain in the uncleaved proenzyme compared with the cleaved complex is in the intramolecular backbone electrostatic interactions of the cleave d strand. In high temperature simulations, the interaction of the long beta hairpin of the enzyme with the C terminal beta sheet of PRO is among the m ost stable in the complex and a likely "nucleation site" for folding. In th e course of unfolding, the C terminal tail of PRO is sometimes observed to intervene between the long hairpin and the aspartate loop of the enzyme, pe rhaps thereby lowering the energy barrier for separation of the two hairpin s. Tighter interactions at the interface between the enzyme and its pro reg ion are also occasionally observed, providing an additional mechanism for u nfolding catalysis. Simulations of a mutant enzyme where the buried ion pai r residues R102 and D142 were replaced by W and L, respectively, did not di splay any distinguishable behavior compared with the wild type. Proteins 20 00;41:21-32. (C) 2000 Wiley-Liss, Inc.