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 Phe¹³² 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.

Keywords: allostery; carbon monoxide; electron paramagnetic resonance spectroscopy; heme; nitroxide spin label; protein dynamics; transcription factor.

Figure 1

The three functional states of CooA and corresponding structural data for the Fe(II) “ready‐off” state in Rr CooA (PDB 1FT9)15 and Fe(II)–CO “on” state of Ch CooA (PDB 2HKX).24 For each structure, the effector binding domain is shown in magenta, DNA binding domain in dark purple, and heme cofactors in red. The DNA‐binding F‐helices are highlighted in orange. Images were created using PyMOL Molecular Graphics System (Version 1.3 Schrödinger, LLC.). The unique heme ligation/oxidation state associated with each functional state is depicted below the structural data.

Figure 2

(Left) Crystal structure of WT Rr CooA (PDB 1FT9) in the inactive, Fe(II) “ready‐off” state15 highlighting the positions of Cys substitution sites. For clarity, one monomer is opaque and the other is translucent. The Cys substitution sites in the opaque monomer are labeled and shown in green. The effector binding domain is shown in magenta, DNA binding domain in dark purple, and heme cofactors in red. Images were created using PyMOL Molecular Graphics System (Version 1.3 Schrödinger, LLC.). (Right) Nitroxide spin labels used in this study.

Figure 3

EPR spectra of MTSL‐labeled and MAL6‐labeled Fe(III) ΔCys₄CooA variants in 100 mM MOPS, 500 mM NaCl, pH 7.4 (grey) and 100 mM MOPS, 500 mM NaCl, pH 7.4 with 25% (w/w) Ficoll‐70 (black). The spectra are scaled to equivalent central line amplitude. More mobile (m) and less mobile (l) components of the m _I = +1 line are labeled for clarity.

Figure 4

EPR spectra of MTSL‐labeled and MAL‐6‐labeled Fe(III) ΔCys₄CooA variants in 100 mM MOPS, 500 mM NaCl, pH 7.4 with 30% (w/w) sucrose (red) and 100 mM MOPS, 500 mM NaCl, pH 7.4 with 25% (w/w) Ficoll‐70 (black). The spectra are scaled to equivalent central line amplitude. More mobile (m) and less mobile (l) components of the m _I = +1 line are labeled for clarity.