title: Unravelling the mechanism of dual-specificity GAPs.
abstract: The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1(IP4BP) and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-alpha6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.
authors.affiliation: Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
authors.forename: Begoña
authors.initials: B
authors.lastname: Sot
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.forename: Delia
authors.initials: D
authors.lastname: Deaconescu
authors.forename: Yan
authors.initials: Y
authors.lastname: Suveyzdis
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.forename: Alfred
authors.initials: A
authors.lastname: Wittinghofer
mesh_terms: D000595:Amino Acid Sequence; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D020668:rap GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020668:rap GTP-Binding Proteins; D018631:ras Proteins