Abstract
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.
Highlights
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
We propose that the somewhat lower ϳ1 M binding affinity of BMAL1(530 – 625)P624A (BMAL530) (WT, P624A, K537Q) to WT KIX determined in this nano-surface plasmon resonance (NanoSPR) experiment (Fig. 4) compared with the experiment shown in Fig. 3 is correlated with the fact that N-terminally His6-tagged BMAL1 is used as surface-bound receptor, consistent with the overall lower KIX-binding affinities of N-terminally labeled and His6-tagged BMAL1 observed in microscale thermophoresis (MST) and fluorescence polarization (FP) (Fig. 2)
To further our mechanistic understanding of the roles of the BMAL1-transactivation domain (TAD)-CBP-KIX interaction and its interplay with other KIX ligands and with the BMAL1-CRY interaction in circadian regulation, we investigated the contributions of the mixed lineage leukemia (MLL)- and cMyb/phosphorylated kinase-inducible domain (pKID)-binding pockets of CBPKIX, of BMAL1 folding, and of BMAL1 Lys[537] acetylation to the BMAL1-KIX interactions
Summary
We initially purified the complex of the mouse CBPKIX domain (residues 586 – 672) with the previously described ϳ14-kDa C-terminal mouse BMAL1(490 – 625) fragment (termed BMAL490) that includes the BMAL1-TAD, Lys[537], and parts of the BMAL1 G-region (14) (Fig. 1). Modeling of N- and C-terminal residues (Gly[586], Val[587], Glu666–Leu672) that were not included in the 1–10-KIX crystal structure, due to conformational disorder, resulted in a much better fit of the experimental SAXS data (2 ϭ 1.51 (CRYSOL) and 2 ϭ 1.49 (FoXS); Fig. S5 (E and F)) As these residues are included in the NMR structure of the MLL1:KIX complex (PDB entry 2LXS) and, the MLL pocket binds the 1–10 compound (27) and is important for BMAL1 interactions (this study), we used the MLL1: KIX complex structure to position the KIX domain in the 1–10KIX SAXS envelope (Fig. 7D). The fitting of the KIX NMR structure (model obtained after SREFLEX) and of the 1–10-KIX crystal structure to the SAXS envelope of 1–10tethered KIX and its further superposition onto the SAXS envelope of the BMAL530:KIX complex (Fig. 7D) suggest that the N-terminal BMAL1 region emerges from the KIX domain near the MLL-binding site. They would result in additional extensions of the SAXS envelopes emerging from the KIX:BMAL1-TAD core complex on the opposite side of the N-terminal BMAL1 regions, which is less consistent with the fact that the most C-terminal BMAL1 residues are engaged in KIX binding (9)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
