Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein

• Published in: The Journal of biological chemistry Vol. 294; no. 45; pp. 16604 - 16619
• Main Authors: Garg, Archit, Orru, Roberto, Ye, Weixiang, Distler, Ute, Chojnacki, Jeremy E., Köhn, Maja, Tenzer, Stefan, Sönnichsen, Carsten, Wolf, Eva
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.11.2019; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; ARNTL Transcription Factors - chemistry; ARNTL Transcription Factors - genetics; ARNTL Transcription Factors - metabolism; Binding Sites; biophysics; BMAL1 G-region; brain and muscle ARNT-like protein-1 (BMAL1); chromatin modification; circadian clock; Circadian Clocks; circadian gene regulation; CREB-binding protein (CBP); CREB-Binding Protein - chemistry; CREB-Binding Protein - metabolism; gene regulation; Histone-Lysine N-Methyltransferase - chemistry; Histone-Lysine N-Methyltransferase - metabolism; kinase inducible domain interacting (KIX) domain of CBP; Mice; Mutagenesis, Site-Directed; Myeloid-Lymphoid Leukemia Protein - chemistry; Myeloid-Lymphoid Leukemia Protein - metabolism; Protein Binding; Protein Domains; protein interactions; Protein Structure and Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myb - chemistry; Proto-Oncogene Proteins c-myb - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; SAXS; Scattering, Small Angle; structural biology; Surface Plasmon Resonance; transactivation domain (TAD); transcription regulation; transcriptional rhythmicity; X-Ray Diffraction; transcription regulation; brain and muscle ARNT-like protein-1 (BMAL1); chromatin modification; CREB-binding protein (CBP); circadian clock; SAXS; protein interactions; kinase inducible domain interacting (KIX) domain of CBP; gene regulation; BMAL1 G-region; transcriptional rhythmicity; structural biology; transactivation domain (TAD); biophysics; circadian gene regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009845

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1’s C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537. Tethering the small compound 1–10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb–binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.