A Slow Conformational Switch in the BMAL1 Transactivation Domain Modulates Circadian Rhythms

Abstract

The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.

Keywords: NMR spectroscopy; circadian rhythms; cyclophilins; cyclosporin A; proline isomerization; transcriptional activation.

Figure 1. Isomerization about a conserved Trp-Pro imide bond in the BMAL1 C-terminal TAD

(A) Domain schematic of mouse BMAL1 showing the chemical shifts (Δδ) in the TAD after binding CRY1 CC helix (purple) or CBP KIX domain (green). *, proline residues that lack crosspeaks in ¹⁵N-¹H HSQC NMR spectra. (B) Selected regions of ¹⁵N-¹H HSQC spectra of ¹⁵N BMAL1 TAD displaying backbone amide peaks for two isomers at W624 and L626. (C) Cross peaks for P623 and P625 C_β and C_γ atoms are shown in strips from the ¹⁵N-edited (H)C(CO)NH-TOCSY of ¹³C/¹⁵N BMAL1 TAD at ¹⁵N planes for W624 and L626 amides. Average ¹³C shifts for trans (gray) and cis (blue) isomers from (Shen and Bax, 2010). (D) The W624-P625 imide bond in cis and trans conformations. (E) Sequence alignment BMAL1, BMAL2 and CYCLE from insects with a vertebrate-like clock (iBMAL1). (F) Regions of ¹⁵N-¹H HSQC spectra of 8-mer switch peptides from mouse (FSDLPWPL, black) and dwarf honey bee (FSGLPWPLP, peach) showing cis and trans peaks for W624 indole. See also Figure S1.

Figure 2. Locked mutants of the TAD switch shorten the circadian period

(A) Representation of cis content of 8-mer TAD switch peptides for P625 and W624 mutants compared to the intact ¹⁵N BMAL1 TAD. Cis content was calculated from peak volumes of residues 624 and 626 in ¹⁵N-¹H HSQC and ¹H-¹H TOCSY NMR spectra. (B) ¹H NMR spectra from FSDLPWPL (black), FSDLPWAL (red) and FSDLPWdmPL (blue) 8-mer TAD switch peptides highlighting the W624 indole region. (C) Synchronized circadian bioluminescence records from Bmal1^−/−; Per2^Luc mouse fibroblasts complemented with WT (gray), W624A (pink), P625A (red), or Δswitch (619X, green) Bmal1. Black line, mean luminescence ± s.d. from n = 6–8 replicates from 2 independent clonal lines in indicated colors. D) Circadian period of complemented fibroblast lines from panel (C). Individual period measurements with mean ± s.d. ***P<0.01 and ****P<0.0001 compared to WT Bmal1 by two-tailed t test. See also Figure S2.

Figure 3. Both isomers of the TAD switch interact with transcriptional activators and repressors

(A) Regions of the ¹⁵N-¹H HSQC spectra showing 100 μM WT (left panels) or P625A (right panels) ¹⁵N BMAL1 TAD upon titration of CRY1 CC (purple) or CBP KIX (green), with increasing concentrations from 25–200 μM indicated by darker colors. (B) Fluorescence anisotropy data for CRY1 PHR (left) and CBP KIX (right) with wild type (black), P625A (red), P625dmP (blue) and Δswitch (green) 5,6-TAMRA-labeled short BMAL1 TAD (residues 594–626). Mean polarization data from one representative experiment (n = 4 replicates) of 3 independent assays. See also Figure S3.

Figure 4. Slow isomerization of the TAD switch occurs on the timescale of minutes

(A) Highlighted region of the 2D spectrum from a ¹⁵N-¹H ZZ-exchange assay performed at 25°C of ¹⁵N BMAL1 TAD displaying cis (dark green), trans (dark blue), cis to trans (light green) and trans to cis (light blue) cross peaks for L626 at a delay = 1 s. Dashed circles, location of exchange cross peaks. (B) Overlay of ¹⁵N-¹H HSQC spectra showing the cis and trans peaks of L626 at increasing temperatures. (C) Snapshot of ZZ-exchange assays at delay = 1 s at indicated temperatures. (D) Eyring analysis of exchange rates vs. temperature from ZZ-exchange assays with errors displayed in s.d. (E) Free energy plot showing the calculated activation energy of isomerization for the BMAL1 TAD. See also Figure S4.

Figure 5. Cyclophilins accelerate isomerization of the TAD switch to regulate circadian period

(A) Domain schematics for cyclophilins tested against the BMAL1 TAD and relative rate enhancement compared to uncatalyzed isomerization at room temperature. (B) Highlighted region of ¹⁵N-¹H ZZ-exchange spectra displaying the W624 indole of the ¹⁵N TAD with indicated cyclophilins. Spectra from a ZZ-exchange time delay series (delay = 0–1 s) are overlaid in sequentially darker colors. (C) Synchronized circadian bioluminescence records from U2OS Bmal1-dLuc fibroblasts in the presence of DMSO, Cyclosporin A (CsA), or Deltamethrin. One individual trace shown for clarity from 2 independent assays with n = 3 replicates. (D) Mean circadian period of cultures upon treatment with DMSO (black), CsA (green) or Deltamethrin (purple) ± s.d. from n = 3 replicates with 2 independent assays. (E) Comparison of mean period changes ± s.d. as a function of CsA concentration in Bmal1^−/−; Per2^Luc fibroblasts complemented with WT (black), P625A (red), or W624A/P625A (gray) Bmal1. ***P<0.01 compared to WT Bmal1 by two-tailed t test. See also Figure S5.