Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1(IP4BP)

GAP1(IP4BP) is, Ras GTPase-activating protein (GAP) that in vitro is regula ted by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [I ns(1,3,4,5)P-4]. We have studied Ins(1,3,4,5)P-4 binding to GAP1(IP4BP), an d shown that the inositol phosphate specificity and binding affinity are si milar to Ins(1,3,4,5)P-4 binding to Bruton's tyrosine kinase (Btk), evidenc e which suggests a similar mechanism for Ins(1,3,4,5)P-4 binding. The cryst al structure of the Btk pleckstrin homology (PH) domain in complex with Ins (1,3,4,5)P-4 has shown that the binding site is located in a partially buri ed pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the r esidues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4 ,5)P-4 based on the Btk-Ins(1,3,4,5)P-4 complex crystal structure. This mod el 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 mu tagenesis of various residues in and around the proposed binding site. Thes e mutations have markedly reduced affinity for Ins(1,3,4,5)P-4, indicating a specific and tight fit for the substrate. The model can also be used to e xplain the specificity of inositol phosphate binding.