title: Activation of leukemia-associated RhoGEF by Galpha13 with significant conformational rearrangements in the interface.
abstract: The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).
authors.affiliation: Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
authors.forename: Nobuchika
authors.initials: N
authors.lastname: Suzuki
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Nicole
authors.initials: N
authors.lastname: Hajicek
authors.forename: Kenji
authors.initials: K
authors.lastname: Daigo
authors.forename: Reiko
authors.initials: R
authors.lastname: Tokita
authors.forename: Shiro
authors.initials: S
authors.lastname: Minami
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Tohru
authors.initials: T
authors.lastname: Kozasa
mesh_terms: D019943:Amino Acid Substitution; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D064067:Rho Guanine Nucleotide Exchange Factors; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: C577222:ARHGEF12 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D044365:GTP-Binding Protein alpha Subunits, G12-G13