Structural insights into the regulation of Bacillus subtilis SigW activity by anti-sigma RsiW

Abstract

Bacillus subtilis SigW is localized to the cell membrane and is inactivated by the tight interaction with anti-sigma RsiW under normal growth conditions. Whereas SigW is discharged from RsiW binding and thus initiates the transcription of its regulon under diverse stress conditions such as antibiotics and alkaline shock. The release and activation of SigW in response to extracytoplasmic signals is induced by the regulated intramembrane proteolysis of RsiW. As a ZAS (Zinc-containing anti-sigma) family protein, RsiW has a CHCC zinc binding motif, which implies that its anti-sigma activity may be regulated by the state of zinc coordination in addition to the proteolytic cleavage of RsiW. To understand the regulation mode of SigW activity by RsiW, we determined the crystal structures of SigW in complex with the cytoplasmic domain of RsiW, and compared the conformation of the CHCC motif in the reduced/zinc binding and the oxidized states. The structures revealed that RsiW inhibits the promoter binding of SigW by interacting with the surface groove of SigW. The interaction between SigW and RsiW is not disrupted by the oxidation of the CHCC motif in RsiW, suggesting that SigW activity might not be regulated by the zinc coordination states of the CHCC motif.

Themes and variations in gene regulation by extracytoplasmic function (ECF) sigma factors

“…Co-crystal structures have been solved for four ECF sigma factors from different families bound to their cognate anti-sigma factors, E. coli σ E -RseA, R. sphaeroides σ E -ChrR, B.subtilis σ W -RsiW, and M. tuberculosis σ K -RskA [26,27,29,30 • ]. RseA, RskA, and RsiW are all inner membrane proteins and the cytoplasmic domain was used for structural studies, while ChrR is a soluble cytoplasmic protein and a proteolytically stable fragment lacking the C-terminal 10 amino acids was used [26,27,29,30 • ].…”

“…RseA, RskA, and RsiW are all inner membrane proteins and the cytoplasmic domain was used for structural studies, while ChrR is a soluble cytoplasmic protein and a proteolytically stable fragment lacking the C-terminal 10 amino acids was used [26,27,29,30 • ]. The ASD fold formed by the first three helices is highly conserved, although the fourth helix assumes a different position in each structure (Figure 2).…”

“…The ASD fold formed by the first three helices is highly conserved, although the fourth helix assumes a different position in each structure (Figure 2). Interestingly, the way in which each anti-sigma factor binds to the ECF sigma factor varies, despite conservation of the ASD fold (Figure 3) [26,27,29,30 • ]. RseA, ChrR, and RskA all bind between the σ2 and σ4 domains, but they dock on the cognate sigma factor in different manners (Figure 3) [26,27,30 • ].…”

“…RseA and ChrR block RNAP binding surfaces in a similar fashion on σ2, but interact differently with σ4 [26,27,30 • ]. Remarkably, the structure of RsiW bound to σ W reveals a completely different mode of inhibition (Figure 3) [29]. Instead of the two sigma factor domains sandwiching the anti-sigma factor, σ W forms a compact structure in which σ W 2 and σ W 4 interact with each other in a way that masks the DNA binding surface of σ W 2 [29].…”

“…Remarkably, the structure of RsiW bound to σ W reveals a completely different mode of inhibition (Figure 3) [29]. Instead of the two sigma factor domains sandwiching the anti-sigma factor, σ W forms a compact structure in which σ W 2 and σ W 4 interact with each other in a way that masks the DNA binding surface of σ W 2 [29]. RsiW wraps around the outside of σ W with the ASD occluding the promoter-binding surface of σ W 4 and the fourth helix reaching around σ W 2 [29].…”