Cellulose membrane supported peptide arrays for deciphering protein-protein interaction sites: the case of PIN, a protein with multiple natural partners.
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.
Lajoix
Anne-Dominique
AD
UMR 5160 CNRS, Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France. anne.lajoix@ibph.pharma.univ-montp1.fr
Gross
René
R
Aknin
Cindy
C
Dietz
Samuel
S
Granier
Claude
C
Laune
Daniel
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002482:Cellulose; D056445:Cytoplasmic Dyneins; D004398:Dyneins / Q000138:chemical synthesis / Q000737:chemistry*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D024701:Myosin Type V / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D019151:Peptide Library; D040081:Protein Array Analysis; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats
D007202:Indicators and Reagents; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins; D002482:Cellulose; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C486069:Dynll1 protein, rat; D024701:Myosin Type V; D056445:Cytoplasmic Dyneins; D004398:Dyneins