Alternative zinc‐binding sites explain the redox sensitivity of zinc‐containing anti‐sigma factors

Abstract

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.

The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core

“…Heo et al . 31 have suggested that the differences in redox sensitivity of different ZAS proteins are due to the differences in electronegative residues and binding of alternative zinc ions. However, as our data above illustrate, additional zinc ions play little or no role in the redox-sensing ability of RsrA.…”

“… 26 have suggested that zinc has little role to play in σ R binding and redox sensing, respectively, while Heo et al . 31 suggest that multiple zinc ions bind to RsrA to modulate its redox reactivity 31 . We re-assessed the stoichiometry of zinc binding before dissecting the redox-sensing mechanism.…”

SigR, a hub of multilayered regulation of redox and antibiotic stress responses

“…Among these residues, those that flank the two cysteines of the HCC motif (E39, E40, L45 and E46) contribute significantly to redox sensitivity (Jung et al , ). Protein modeling and zinc docking studies on redox‐sensitive vs. ‐insensitive ZAS factors suggests that a zinc ion can probabilistically occupy one of multiple sites in redox‐sensitive ZAS, thereby increasing the susceptibility of zinc‐coordinating cysteine residues to oxidation (Heo et al , ). This implies that the flexibility in zinc binding could contribute to redox‐sensitivity.…”

Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

“…The well-studied mechanism of redox-induced conformational change involves metal-derived redox reactions that occur frequently with changes in cellular thiol balance ( Ilbert et al, 2006 ; Fan et al, 2009 ). Conformational changes resulting from Zn release in proteins such as Hsp33 ( Ilbert et al, 2006 ), metallothionein ( Oteiza, 2012 ), thioredoxin 2 from Escherichia coli ( El Hajjaji et al, 2009 ), PKC ( Zhao et al, 2011 ), and antisigma factor RsrA ( Heo et al, 2013 ) routinely occur following oxidative stress. The coordinating cysteine residues in the zinc finger cluster form intramolecular disulfides releasing Zn which leads to unfolding of the redox-responsive region in Hsp33.…”