Mechanism of heme binding by CP motifs in the BACH1 DNA-binding region
- 1Institute for Structural and Chemical Biology, Division of Molecular and Cell Biology, University of Leicester
Abstract
BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.
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BibTeX
@misc{huang2026mechanism,
title = {{Mechanism of heme binding by CP motifs in the BACH1 DNA-binding region}},
author = {Y. Huang and L. Fairall and F. W. Muskett and C. Dominguez and A. Hudson and J. W. Schwabe},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.28.747782},
url = {https://www.biorxiv.org/content/10.64898/2026.08.28.747782v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Mechanism of heme binding by CP motifs in the BACH1 DNA-binding region
AU - Y. Huang
AU - L. Fairall
AU - F. W. Muskett
AU - C. Dominguez
AU - A. Hudson
AU - J. W. Schwabe
PY - 2026
DA - 2026/08/28
PB - bioRxiv
DO - 10.64898/2026.08.28.747782
UR - https://www.biorxiv.org/content/10.64898/2026.08.28.747782v1
AB - BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.
ER -