1. In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.
- Author
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Šakanović A, Kranjc N, Omersa N, Aden S, Kežar A, Kisovec M, Zavec AB, Caserman S, Gilbert RJC, Podobnik M, Crnković A, and Anderluh G
- Subjects
- Epistasis, Genetic, Protein Binding, Amino Acid Motifs, Mutation, Hemolysin Proteins metabolism, Hemolysin Proteins chemistry, Hemolysin Proteins genetics, Cholesterol metabolism, Cholesterol genetics, Bacterial Toxins metabolism, Bacterial Toxins chemistry, Bacterial Toxins genetics, Clostridium perfringens genetics, Clostridium perfringens metabolism
- Abstract
The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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