Nonhydrolyzable analogs of phosphatidylinositol as ligands of phospholipases C

Abstract

Phosphatidylinositol-specific phospholipases C (PI-PLCs) are important enzymes participating in transmembrane signal transduction. The structures of the two major species of these enzymes: bacterial Ca²⁺-nondependent enzyme from B. cereus and mammalian Ca²⁺-dependent PLCδ₁ from rat brain in the complexes with the polar head groups of their substrates have been previously solved. Although these structures show few differences as compared to free enzymes, there is a compelling evidence that full catalytic activity of PI-PLC necessitates interaction of the enzyme with the entire substrate, including the hydrophobic fatty acid chains. In this work we develop new tightly binding and cleavage-resistant analogs of phosphatidylinositol, using relatively minor modifications of the structure. Two synthesized analogs, 2-amino-2-deoxy-PI (8) and the conformationally constrained analog (10) had binding affinities (K_i) in 10 μM range. ¹⁵N-¹H HSQC NMR spectra of uniformly ¹⁵N-labeled bacterial Ca²⁺-nondependent and Ca²⁺-dependent phospholipases C, btPLC and saPLC1, respectively, displayed changes upon ligand binding that suggest an occurrence of a conformational change.