title: Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.
abstract: The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kast
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Changsong
authors.initials: C
authors.lastname: Yang
authors.affiliation: FIRC Institute of Molecular Oncology, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Disanza
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Boczkowska
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Yadaiah
authors.initials: Y
authors.lastname: Madasu
authors.affiliation: 1] FIRC Institute of Molecular Oncology, Milan, Italy. [2] Department of Health Sciences, University of Milan School of Medicine, Milan, Italy.
authors.forename: Giorgio
authors.identifier: 0000000179841889
authors.initials: G
authors.lastname: Scita
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Svitkina
authors.affiliation: Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Dominguez
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains
chemical_list: C119539:BAIAP2 protein, human; D009419:Nerve Tissue Proteins; D020764:cdc42 GTP-Binding Protein