Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

摘要

pGAP1supIP4BP/sup is a Ras GTPase-activating protein (GAP) that iin vitro/i is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)Psub4/sub]. We have studied Ins(1,3,4,5)Psub4/sub binding to GAP1supIP4BP/sup, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)Psub4/sub binding to Bruton9s tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)Psub4/sub binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)Psub4/sub has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1supIP4BP/sup. Therefore we generated a model of the PH domain of GAP1supIP4BP/sup in complex with Ins(1,3,4,5)Psub4/sub based on the Btk-Ins(1,3,4,5)Psub4/sub complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)Psub4/sub, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding./p