In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.
Aleksandra ��akanovi��, Nace Kranjc, Ne��a Omersa, Sa��a Aden, Andreja Ke��ar, Matic Kisovec, Apolonija Bedina Zavec, Simon Caserman, Robert J C Gilbert, Marjetka Podobnik, Ana Crnkovi��, Gregor Anderluh
Author Information
Aleksandra ��akanovi��: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Nace Kranjc: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Ne��a Omersa: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Sa��a Aden: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Andreja Ke��ar: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Matic Kisovec: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Apolonija Bedina Zavec: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Simon Caserman: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Robert J C Gilbert: Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
Marjetka Podobnik: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Ana Crnkovi��: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia. Electronic address: ana.crnkovic@ki.si.
Gregor Anderluh: Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia. Electronic address: gregor.anderluh@ki.si.
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.
