Abstract
Pleckstrin is a major substrate of protein kinase C in platelets and leukocytes and appears to play an important role in exocytosis through a currently unknown mechanism. Pleckstrin function is regulated by phosphorylation, which is thought to cause dissociation of pleckstrin dimers, thereby facilitating phosphoinositide interactions and membrane localization. Evidence also exists suggesting that phosphorylation causes a subtle conformational change in pleckstrin. Structural studies of pleckstrin have been initiated in order to characterize these structural changes and ultimately advance understanding of pleckstrin function. Here, the crystallization and preliminary X-ray diffraction analysis of a truncated version of pleckstrin consisting of the N-terminal PH domain, the protein kinase C phosphorylation sites and the DEP domain (NPHDEP) are reported. In addition, the oligomeric state and phospholipid-binding properties of NPHDEP were analyzed. This work demonstrates that NPHDEP behaves as a monomer in solution and suggests that all three pleckstrin domains contribute to the dimerization interface. Furthermore, based on the binding properties of NPHDEP, the C-terminal PH domain appears to increase the specificity of pleckstrin for phosphoinositides. This work represents a significant step towards determining the structure of pleckstrin.