8J3Q

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

(2023) Biochemistry 62: 2952-2969

• PubMed: 37796763 Search on PubMed
• DOI: https://doi.org/10.1021/acs.biochem.3c00252
• Primary Citation of Related Structures:  
  8HWU, 8J3Q


• PubMed Abstract: 
  

  Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis , is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated 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 an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile ₃₁ as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

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Organizational Affiliation: 

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.