Critical Sites on Ostreolysin Are Responsible for Interaction with Cytoskeletal Proteins
Nastacia Adler Berke,
Antonella Di Pizio,
Timothy D. Vaden,
Irit Shoval,
Ofer Gover,
Daniel Waiger,
Gili Solomon,
Kristina Sepčić,
Betty Schwartz
Affiliations
Nastacia Adler Berke
The Robert H. Smith Faculty of Agriculture, Institute of Biochemistry, Food Science and Nutrition, Food and Environment, School of Nutritional Sciences, The Hebrew University of Jerusalem, Rehovot 7610001, Israel
Antonella Di Pizio
Leibniz Institute for Food Systems Biology at the Technical University of Munich, 85354 Freising, Germany
Timothy D. Vaden
Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USA
Irit Shoval
Light Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel
Ofer Gover
The Robert H. Smith Faculty of Agriculture, Institute of Biochemistry, Food Science and Nutrition, Food and Environment, School of Nutritional Sciences, The Hebrew University of Jerusalem, Rehovot 7610001, Israel
Daniel Waiger
The Robert H. Smith Faculty of Agriculture, Institute of Biochemistry, Food Science and Nutrition, Food and Environment, School of Nutritional Sciences, The Hebrew University of Jerusalem, Rehovot 7610001, Israel
Gili Solomon
The Robert H. Smith Faculty of Agriculture, Institute of Biochemistry, Food Science and Nutrition, Food and Environment, School of Nutritional Sciences, The Hebrew University of Jerusalem, Rehovot 7610001, Israel
Kristina Sepčić
Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
Betty Schwartz
The Robert H. Smith Faculty of Agriculture, Institute of Biochemistry, Food Science and Nutrition, Food and Environment, School of Nutritional Sciences, The Hebrew University of Jerusalem, Rehovot 7610001, Israel
We explored the structural features of recombinant ostreolysin A (rOlyA), a protein produced by Pleurotus ostreatus and responsible for binding to α/β-tubulin. We found that rOlyA cell internalization is essential for the induction of adipocyte-associated activity, which is mediated by the interaction of rOlyA and microtubule proteins. We created different point mutations at conserved tryptophan (W) sites in rOlyA and analyzed their biological activity in HIB-1B preadipocytes. We demonstrated that the protein’s cell-internalization ability and the differentiated phenotype induced, such as small lipid-droplet formation and gene expression of mitogenesis activity, were impaired in point-mutated proteins W96A and W28A, where W was converted to alanine (A). We also showed that an rOlyA homologue, OlyA6 complexed with mCherry, cannot bind to β-tubulin and does not induce mitochondrial biosynthesis-associated markers, suggesting that the OlyA6 region masked by mCherry is involved in β-tubulin binding. Protein–protein docking simulations were carried out to investigate the binding mode of rOlyA with β-tubulin. Taken together, we identified functional sites in rOlyA that are essential for its binding to β-tubulin and its adipocyte-associated biological activity.
