Site‐directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) “locked‐off” state of the CO‐sensing transcription factor CooA

Abstract

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs-ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild-type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) "locked-off" CooA variants labeled with either MTSL or MAL-6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs-ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) "locked-off" CooA. This observation suggests that, in direct contrast to effector-free CRP, Fe(III) "locked-off" CooA undergoes conformational exchange on the μs-ms timescale. Importantly, we establish MAL-6 as a spin label with a redox-stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.

Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands

“…(Values between 6 and 16 nM have been reported. 18,37,38 ) It is in agreement with a high functional affinity, similar to what has been observed for the binding of RcoM-1 to the a + b + c region 14 and an efficient CO-specific recruitment of RcoM-2.…”

PgRsp Is a Novel Redox-Sensing Transcription Regulator Essential for Porphyromonas gingivalis Virulence

“…We demonstrated that PgRsp protein belongs to the Crp/Fnr superfamily of transcription factors and could be classified as a relatively close homolog to the CooA family. Proteins homologous to CooA can sense CO [31,53], employing interaction of CO with heme bound to the protein [51,53], thus regulating genes important for utilization of CO as an energy source [72,73]. The molecular mechanism of the best-characterized homolog of CooA protein from R. rubrum, RrCooA, implies binding of Fe(III)heme by Pro2 and Cys75, when the protein is not active.…”

“…For protein activation, the CO molecule is bound to heme by a Pro2 residue, resulting in conformational changes of the protein, leading to interaction with DNA. This kind of protein activation is unique for RrCooA protein [51][52][53], since in the CooA homolog from Carboxydothermus hydrogenoformans, the heme molecule is coordinated by Ala2 and His82 residues and no transfer of the heme group occurs during the change of iron redox state [52,73].…”

“…The modeled structure showed fold typical for Crp proteins, comprising Nand C-terminal (DNA-binding domain) domains, both connected by an alpha helix ( Figure 4). Besides Cys and His, CooA homologs use N-terminal Pro or Ala as the second heme-binding ligand in the non-active state [52,53]. Neither PgRsp nor its closest homologs have such an amino acid residue at the N terminus ( Figure 2).…”

How to assess the structural dynamics of transcription factors by integrating sparse NMR and EPR constraints with molecular dynamics simulations

“…When a transcription factor or its target DNA carries a paramagnetic moiety, e.g., a spin-label or a metal center as used in PRE-NMR, direct detection of the electron spin by electron paramagnetic resonance (EPR) spectroscopy can provide valuable information complementary to NMR data [103] , [104] .…”