Structural Analysis of Bacillus subtilis Sigma Factors

Abstract

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.

Keywords: Alphafold; Bacillus subtilis; SigE; X-ray crystallography; sigma factor; sporulation; structure.

Figure 1

NMR spectra of B. subtilis SigE. (A) Overlaid ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled SigE^17–239 (blue) and SigE^17–133 (orange) constructs. The broad peaks indicate that the former had likely aggregated and/or degraded. The substantial overlap with the peaks of the latter construct, and the lack of many additional peaks, imply that the SigE^17–239 sample now resembles SigE^17–133. (B) ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled SigE^17-133 alone (turquoise) and in the presence of a two-fold excess CsfB^A48E (red). Chemical shift perturbation clearly indicates interaction between the two proteins.

Figure 2

Crystal structure of SigE. (A) View of the crystallographic asymmetric unit showing six copies of SigE residues 52–133, each representing a classic helix-turn-helix domain. (B) Alignment of the six units of SigE 52–133 from the asymmetric unit. This overlay shows some minor differences between the different biological units (in the same colours as shown in (A), with slight structural deviations in the flexible loop regions).

Figure 3

Structural alignments of SigE. (A) Structural overlay of SigE^52–133 crystal structure (cream) with Thermus aquaticus RNAP sigma factor A (light blue; PDB: 3UGO [53]) bound to a −10 promoter element ssDNA oligo (TACAAT). The structures align with RMSD: 0.99 over 72 residues, indicating how SigE^52–133 likely interacts with the −10 promoter in B. subtilis. (B) SigE^52–133 crystal structure (cream) overlaid with the AlphaFold model of full-length SigE (light blue) from B. subtilis (UniProt ID: P06222). Regions of helix overprediction (residues 77–80, 104–106) by AlphaFold are indicated in red.

Figure 4

Structural alignments of experimentally solved (partial) B. subtilis sigma factors (cream) with the equivalent AlphaFold models (light blue): (A) Overlay of the NMR structure of σ1.1 domain of SigA (5MWW) [12] with the AlphaFold model of SigA. (B) SigA structure excised from the cryo-EM (7CKQ [23]) structure of the BmrR transcription activation complex [23] overlaid with the full-length AlphaFold model of SigA. (C) Overlay of the NMR structure of SigE σ2 domain (5OR5; unpublished) with the equivalent AlphaFold model of SigE. (D) 2.6 Å crystal structure of SigW (5WUR [11]) excised from the co-crystal complex with the anti-sigma factor RsiW [11] overlaid with the AlphaFold model of SigW; structured regions are a near perfect match. (E) Overlay of the 3.1 Å crystal structure (6JHE [39]) domain bound to the −35 element DNA [39] (hidden) with the AlphaFold model of full-length SigW.