Probing the Role of Dynamics in Allosteric Signal Transduction in the CO‐Sensing Transcription Factor CooA Using Site‐Directed Spin Label EPR Spectroscopy

Abstract

CooA (CO oxidation activator) is a CO‐sensing transcription factor that is a member of the cyclic AMP receptor protein/fumarate and nitrate reductase regulatory protein (CRP/FNR) superfamily. The CO‐sensing function of CooA is carried out by a heme cofactor: the heme can be reduced from Fe(III) to Fe(II) under anaerobic conditions, and binding of CO to Fe(II) heme results in allosteric activation of DNA binding activity. While crystal structures of CooA in both active and inactive states exist, there is no clear consensus on how the signal of CO binding propagates from the regulatory domain to the DNA binding domain. Growing evidence supports the notion that dynamics, in addition to structure, plays a crucial role in allosteric regulation. In CRP, NMR experiments highlighted the importance of entropy‐driven dynamics in effector‐mediated allostery. To understand the role of dynamics in propagating the effector‐binding signal in CooA, we employed site‐directed spin label electron paramagnetic resonance (SDSL‐EPR) spectroscopy. Through site‐directed mutagenesis of a fully active protein missing four native Cys residues, we reintroduced Cys residues in key locations for attachment of spin labels: the surface of the heme domain (K26C), the top of the 4/5 loop (E60C), the domain‐linking hinge (F132C, D134C) and the end of the DNA‐binding helix (S175C). All variants exhibited heme characteristics, and most exhibited CO‐dependent DNA binding affinities, nearly identical to those of wild‐type CooA. The variant F132C, missing a phenyl ring believed to make a key allosteric contact with the DNA‐binding helix, exhibits weak, constitutive DNA binding. Multicomponent signals were observed in EPR spectra of all Fe(III) variants labelled with the nitroxide probes MTSL and MAL‐6. EPR spectra of these probes are sensitive to variations on the nanosecond timescale, consistent with large‐scale motions of entire protein domains, and report the same multiplicity of dynamic populations. The presence of multicomponent signals that are responsive to osmolytes implicate multiple dynamic populations that arise from different local conformational states. The relative intensities of the component signals varied with the site of label attachment, demonstrating that local dynamics vary as a function of position in the CooA protein. These observations suggest that interconversion among different dynamic populations may play a role in the allosteric mechanism of signal transduction in CooA.Support or Funding InformationNational Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)