CONFORMATIONAL STABILITY STUDIES OF THE PLECKSTRIN DEP DOMAIN - DEFINITION OF THE DOMAIN BOUNDARIES

Pleckstrin is the major substrate of protein kinase C in platelets. It contains at its N- and C-termini two pleckstrin homology (PH) domains which have been proposed to mediate protein-protein and protein-lipid interactions. A new module, called DEP, has recently been identified by sequence analysis in the central region of pleckstrin. In order to study this module, several recombinant polypeptides corresponding to t he DEP module and N- and C-termini extended forms have been expressed. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) te chniques, the domain boundaries have been determined that yield a solu ble and folded pleckstrin DEP domain. This comprises 93 amino acids wi th an alpha/beta fold in agreement with secondary structure prediction s. Stability studies indicate that the regions surrounding the DEP dom ain do not contribute to its stability suggesting that the phosphoryla tion sites at S113, T114 and S117 are in an unstructured region. Ident ification of the regions of pleckstrin that are folded shall facilitat e determination of its structure and function. (C) 1998 Elsevier Scien ce B.V. All rights reserved.