Abstract
Rev-erbβ is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes. Heme binding to Rev-erbβ indirectly facilitates its interaction with the nuclear receptor co-repressor (NCoR1), resulting in repression of Rev-erbβ target genes. Fe3+-heme binds in a 6-coordinate complex with axial His and Cys ligands, the latter provided by a heme-regulatory motif (HRM). Rev-erbβ was thought to be a heme sensor based on a weak Kd value for the Rev-erbβ·heme complex of 2 μm determined with isothermal titration calorimetry. However, our group demonstrated with UV-visible difference titrations that the Kd value is in the low nanomolar range, and the Fe3+-heme off-rate is on the order of 10-6 s-1 making Rev-erbβ ineffective as a sensor of Fe3+-heme. In this study, we dissected the kinetics of heme binding to Rev-erbβ and provided a Kd for Fe3+-heme of ∼0.1 nm Loss of the HRM axial thiolate via redox processes, including oxidation to a disulfide with a neighboring cysteine or dissociation upon reduction of Fe3+- to Fe2+-heme, decreased binding affinity by >20-fold. Furthermore, as measured in a co-immunoprecipitation assay, substitution of the His or Cys heme ligands in Rev-erbβ was accompanied by a significant loss of NCoR1 binding. These results demonstrate the importance of the Rev-erbβ HRM in regulating interactions with heme and NCoR1 and advance our understanding of how signaling through HRMs affects the major cellular processes of circadian rhythm maintenance and metabolism.
Highlights
Rev-erb is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes
Previous equilibrium titrations yielded Kd values for Fe3ϩheme complexes with wild-type and C384A ligand-binding domain (LBD) of 23 Ϯ 3 and 46 Ϯ 14 nM (Table 1), respectively, suggesting the axial thiolate has a minor impact on heme affinity (37)
Assessing the effect of redox changes on the affinity of Rev-erb for heme required the development of a kinetic system to establish accurate Kd values
Summary
Previous equilibrium titrations yielded Kd values for Fe3ϩheme complexes with wild-type and C384A LBDs of 23 Ϯ 3 and 46 Ϯ 14 nM (Table 1), respectively, suggesting the axial thiolate has a minor impact on heme affinity (37). Previous biophysical and crystallographic analyses as well as the transient kinetics studies described using truncated Rev-erb LBD constructs have demonstrated Cys-384 is a Fe3ϩ-heme axial ligand that dissociates upon heme reduction (36, 38) To ensure these properties are maintained in the context of the full-length protein, UV-visible spectra were acquired of wild-type and C384A FLRev-erb (Fig. 8). Comparison of that value with the koff for the predominant kinetic phase of wild-type FLRev-erb (see Table 1) (35), koff-slow ϭ (3.9 Ϯ 0.8) ϫ 10Ϫ6 sϪ1 (82 Ϯ 3% of the total ⌬absorbance) indicates that loss of the axial thiolate leads to a 55-fold decrease in heme affinity, assuming a direct correlation between off-rate and Kd. Cys-384 does provide a strong axial ligand to Fe3ϩ-heme, suggesting that any processes, e.g. redox or gas-binding, that cause Cys-384 dissociation significantly decreases the affinity of Rev-erb for heme
Sign-in/Register to view highlights & summary 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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
