title: Self-recognition behavior of a helix-loop-helix domain by a fragment scan.
abstract: The inhibitors of DNA binding Id1-4 are helix-loop-helix (HLH) proteins that exert their biological function by interacting with members of the basic-HLH (bHLH) transcription-factor family. The HLH domains of the Id and bHLH proteins allow both self- and hetero-association. Due to their abnormal expression in cancer cells, the Id proteins are potential protein targets for cancer treatment. Suitable Id-protein inactivators should promote self-association and/or prevent hetero-association. In this work we evaluated the ability of the Id-protein HLH domain to recognize itself in form of short sequences extracted from the helical and loop regions. We performed a peptide scan of the Id1 HLH domain 64-106 based on three-residue overlapping octapeptides. Interaction of each octapeptide with the natively folded Id1 HLH domain was investigated by CD and fluorescence spectroscopy. The results from both techniques showed that the helix-based but not the loop-based octapeptides interacted with the Id1 HLH domain in the low-micromolar range. In contrast, a nitrotyrosine-containing analog of the Id1 HLH region, which was unable to reproduce the native-like conformation, quenched only the 2-amino-benzoyl-(Abz)-labeled loop-based octapeptides. This opposite self-recognition pattern suggests that the short helix-based and loop-based sequences should be able to distinguish different folding states of the Id1 HLH domain. This feature may be biologically relevant, as the Id proteins are predicted to behave as intrinsically disordered proteins, being in equilibrium between rapidly exchanging monomeric conformations and structurally better-defined homo-/heterodimers displaying the parallel four-helix bundle.
authors: Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany.
authors: Michael
authors: M
authors: Beisswenger
authors: Faculty of Chemistry and Biochemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany; Department of Molecular Biology, Division of Chemistry and Bioanalytics, Paris-Lodron University Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria. Electronic address: chiara.cabrele@sbg.ac.at.
authors: Chiara
authors: C
authors: Cabrele
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018257:Helix-Loop-Helix Motifs; D057166:High-Throughput Screening Assays; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D051798:Inhibitor of Differentiation Protein 1; D008024:Ligands; D009842:Oligopeptides; D062367:ortho-Aminobenzoates; C002744:3-nitrotyrosine; D014443:Tyrosine
keywords: Circular dichroism; Helix–loop–helix; Id proteins; Protein–ligand interaction