Phosphoinositides are phosphatidylinositol derived, well known to be second messengers in various cell signaling pathways as well as in processes such as cell differentiation, cellular stress response, gene transcription, and chromatin remodeling. The pleckstrin homology domain of phospholipase C-delta 1 is responsible for recognizing and binding to PI(4,5)P2 and for this reason has been widely used to study this phosphoinositide as a biosensor when it is conjugated to a fluorescent tag. In this work, we modified the primary structure of pleckstrin homology domain by site-specific mutagenesis to change the specificity for phosphoinositides. We obtained 3 mutants: K30A, W36F, and W36Y with different specificity to phosphoinositides. Mutant domain K30A recognized PI(4,5)P2 , PI(3,4,5)P3 , phosphatidic acid (PA), and weakly PI(3,5)P2 . Mutant domain W36F recognized all the phosphoinositides studied and the PA. Finally, mutant domain W36Y seemed to interact with PA and all the other phosphoinositides studied, except PI(3)P. The changes in recognition argue against a simple charge and nonpolar region model for these interactions and more in favor of a specific docking region with a specific recognition site. We conducted in silico modeling that explains the mechanisms behind the observed changes and showed that aromatic amino acids appear to play more important role, than previously thought, in the specificity of phospholipids' binding domains.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic v v i Prague Czech Republic

Unidad de Bioquímica y Biología molecular de plantas Centro de Investigación Científica de Yucatán Mérida Yucatan Mexico

Unidad de Biotecnología Centro de Investigación Científica de Yucatán Mérida Yucatán Mexico

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