Solution NMR studies of a potent subtilisin inhibitor from Budgett’s frog and the catalytic subunit of AHAS

Abstract

This thesis involves a detailed structural study of two molecular systems, a small disulfide-rich peptide LL-TIL and a large catalytic subunit of AHAS. Subtilisin inhibitors play an important role in fighting against these harmful microorganisms. LL-TIL, found in skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays established that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided a detailed view of the structural basis of inhibition. The energy calculation by MM-PBSA analysis for the LL-TIL-subtilisin complex predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL was generated, which broadened the inhibitory profile and attenuated subtilisin inhibition by two orders of magnitude. These results provide a valuable template to engineer more specific and potent TIL-type subtilisin inhibitors. AHAS catalyzes the first step in the biosynthetic pathway of branched-chain amino acids. The active site resides within the interface of the independently folded catalytic domains α and γ. The α domain of the catalytic subunit of E. coli AHAS I (ilvBα) aggregated at high concentrations required for NMR. A new approach was designed involving the study of the α and γ domains from T. maritima (TmCSUα and TmCSUγ). The domains were found to be tethered by a disulfide bond. Domain-specific isotopically labelled samples were successfully prepared for the NMR study. However, the high-resolution multidimensional NMR experiments suffered from low sensitivity, which hindered the sequence-specific assignments.