title: 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.
abstract: GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal 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 buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues 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 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)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: G
authors.initials: G
authors.lastname: Cozier
authors.forename: R
authors.initials: R
authors.lastname: Sessions
authors.forename: J R
authors.initials: JR
authors.lastname: Bottomley
authors.forename: J S
authors.initials: JS
authors.lastname: Reynolds
authors.forename: P J
authors.initials: PJ
authors.lastname: Cullen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D007295:Inositol Phosphates / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001798:Blood Proteins; D007295:Inositol Phosphates; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; C039077:platelet protein P47; C081655:inositol-1,3,4,5-tetrakisphosphate; D014364:Tryptophan; D008715:Methionine; D008239:Lysine; D002118:Calcium