RGD Reference Report - Cellulose membrane supported peptide arrays for deciphering protein-protein interaction sites: the case of PIN, a protein with multiple natural partners. - Rat Genome Database

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Cellulose membrane supported peptide arrays for deciphering protein-protein interaction sites: the case of PIN, a protein with multiple natural partners.

Authors: Lajoix, Anne-Dominique  Gross, RenĂ©  Aknin, Cindy  Dietz, Samuel  Granier, Claude  Laune, Daniel 
Citation: Lajoix AD, etal., Mol Divers. 2004;8(3):281-90.
RGD ID: 13207431
Pubmed: PMID:15384421   (View Abstract at PubMed)

Cellulose membrane supported peptide arrays, prepared according to the Spot method, allow the rapid identification and characterization of protein-protein interaction sites. Here, the method was used to screen reactive peptides from different proteins that bind to a single molecule, the PIN protein. PIN possesses two binding grooves, that have been shown to interact with several targets, including neuronal NO synthase, dynein intermediate chain, myosin V, the proapoptotic protein Bim, the scaffolding proteins DAP1alpha and gephyrin, and the transcription factor NRF-1. Arrays of peptides representing sequences of these targets were probed for reactivity with GST-tagged PIN, enabling the precise identification of binding motifs. Binding motifs were then minimized to seven or eight amino acid long peptides: YSKETQT for dynein IC, CDKSTQT for Bim, KDTGIQVD for nNOS, QSVGVQV for DAP1alpha and EDKNTMTD for myosin V. Alascan and substitution analysis provided proof that the Gln residue is critical for the interaction and cannot be easily replaced. Positions -1 and +1, just flanking the pivotal Gln, are also important; they consist of hydrophobic residues (Thr, Val) that could only be replaced by hydrophobic or aromatic amino acids. Position -4 is also critical for binding, with its Asp or Ser being replaceable to some extent. Alignment of sequences of proteins known to bind PIN shows that the most frequent amino acids in the motif are DKGTQT, consistent with the Spot results. We postulate that the degenerate character of binding to PIN is based on the propensity of several sequences to adopt a beta-strand conformation that allows the Gln residue to position itself in the PIN channel and on the conformational breathing of the PIN binding groove.



Gene Ontology Annotations    Click to see Annotation Detail View

Molecular Function

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Dynll1Ratprotein binding  IPINos1 (Rattus norvegicus) RGD 
Nos1Ratprotein binding  IPIDynll1 (Rattus norvegicus) RGD 

Objects Annotated

Genes (Rattus norvegicus)
Dynll1  (dynein light chain LC8-type 1)
Nos1  (nitric oxide synthase 1)


Additional Information