Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.
Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.
Laboratoire des BioMolécules (LBM), CNRS - UMR 7203, Ecole Normale Supérieure / Université Pierre et Marie Curie 24, rue Lhomond, 75231, Paris Cedex 05, France; Fondation Pierre-Gilles de Gennes pour la Recherche, 29, rue d'Ulm, 75005, Paris, France.
C
C
Byrne
E
E
Miclet
I
I
Broutin
D
D
Gallo
V
V
Pelekanou
M
M
Kampa
E
E
Castanas
G
G
Leclercq
Y
Y
Jacquot
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000502:physiology; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000502:physiology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology; D056918:Nuclear Receptor Coactivators / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D018909:src Homology Domains / Q000502:physiology*
C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D051380:GRB2 Adaptor Protein; D009687:Nuclear Proteins; D056918:Nuclear Receptor Coactivators; C096395:PNRC1 protein, human; C437718:PNRC2 protein, human; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D014157:Transcription Factors; C011083:polyproline; D011392:Proline
NMR; electronic circular dichroism; estrogen receptor α; molecular dynamics; polyproline II; proline-rich nuclear receptor coactivator PNRC