title: Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.
abstract: The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
authors.affiliation: DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Hořejší
authors.affiliation: MRC National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Lasse
authors.initials: L
authors.lastname: Stach
authors.affiliation: MRC National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Flower
authors.affiliation: Peptide Chemistry, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Dhira
authors.initials: D
authors.lastname: Joshi
authors.affiliation: DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Flynn
authors.affiliation: DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK; Biological Mass Spectrometry and Proteomics Group, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.
authors.forename: J Mark
authors.initials: JM
authors.lastname: Skehel
authors.affiliation: DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: O'Reilly
authors.affiliation: MRC National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Roksana W
authors.initials: RW
authors.lastname: Ogrodowicz
authors.affiliation: MRC National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, UK. Electronic address: ssmerdo@nimr.mrc.ac.uk.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Smerdon
authors.affiliation: DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK. Electronic address: simon.boulton@cancer.org.uk.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Boulton
mesh_terms: D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray / Q000379:methods*; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D050783:Proto-Oncogene Proteins c-ets / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D051017:Apoptosis Regulatory Proteins; C409823:ETV7 protein, human; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; C555143:PIH1D1 protein, human; D050783:Proto-Oncogene Proteins c-ets