title: Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).
abstract: CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.
authors: From the Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, United Kingdom.
authors: Evgenia
authors: E
authors: Rouka
authors: From the Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, United Kingdom, philip.simister@imm.ox.ac.uk.
authors: Philip C
authors: PC
authors: Simister
authors: From the Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, United Kingdom.
authors: Melanie
authors: M
authors: Janning
authors: the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors: Joerg
authors: J
authors: Kumbrink
authors: From the Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, United Kingdom.
authors: Tassos
authors: T
authors: Konstantinou
authors: the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.
authors: João R C
authors: JR
authors: Muniz
authors: the Peptide Chemistry Laboratory, London Research Institute Cancer Research UK, London WC2A 3LY, United Kingdom.
authors: Dhira
authors: D
authors: Joshi
authors: the Peptide Chemistry Laboratory, London Research Institute Cancer Research UK, London WC2A 3LY, United Kingdom.
authors: Nicola
authors: N
authors: O'Reilly
authors: the Institute of Medical Immunology, Charité-Universitätsmedizin Berlin, 10115 Berlin, Germany.
authors: Rudolf
authors: R
authors: Volkmer
authors: the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors: Brigitte
authors: B
authors: Ritter
authors: the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.
authors: Stefan
authors: S
authors: Knapp
authors: the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, the Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0QX, United Kingdom, and the Department of Biochemistry, University of Johannesburg, Auckland Park 2006, South Africa.
authors: Frank
authors: F
authors: von Delft
authors: the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
authors: Kathrin H
authors: KH
authors: Kirsch
authors: From the Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, United Kingdom, the Institute of Molecular Medicine, Martin Luther University Halle-Wittenberg, D-06120 Halle, Germany, stephan.feller@uk-halle.de.
authors: Stephan M
authors: SM
authors: Feller
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D019295:Computational Biology; D018360:Crystallography, X-Ray; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; C487967:RIN3 protein, human; D018631:ras Proteins
keywords: Alzheimer disease; CD2AP (CD2-associated protein); RIN3 (Ras and Rab interactor 3); T-cell biology; breast cancer; domain plasticity; focal segmental glomerulosclerosis (FSGS); nephrology; polyproline type II (PP-II) helix; x-ray crystallography