title: Quantitative analyses of cryptochrome-mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock.
abstract: The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537(mBMAL1) acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.
authors.affiliation: Max-Planck-Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Czarna
authors.forename: Helena
authors.initials: H
authors.lastname: Breitkreuz
authors.forename: Carsten C
authors.initials: CC
authors.lastname: Mahrenholz
authors.forename: Julia
authors.initials: J
authors.lastname: Arens
authors.forename: Holger M
authors.initials: HM
authors.lastname: Strauss
authors.forename: Eva
authors.initials: E
authors.lastname: Wolf
mesh_terms: D056930:ARNTL Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D056926:CLOCK Proteins / Q000378:metabolism; D057906:Circadian Clocks / Q000235:genetics*; D056931:Cryptochromes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D055672:Static Electricity; D014158:Transcription, Genetic
chemical_list: D056930:ARNTL Transcription Factors; C538964:Cry1 protein, mouse; C538967:Cry2 protein, mouse; D056931:Cryptochromes; D008024:Ligands; D010446:Peptide Fragments; D056926:CLOCK Proteins