Coevolutionary Analysis Reveals a Conserved Dual Binding Interface between Extracytoplasmic Function σ Factors and Class I Anti-σ Factors

Abstract

Extracytoplasmic function σ factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Among the diverse regulators of ECF activity, class I anti-σ factors are the most important signal transducers in response to internal and external stress conditions. Despite the conserved secondary structure of the class I anti-σ factor domain (ASDI) that binds and inhibits the ECF under noninducing conditions, the binding interface between ECFs and ASDIs is surprisingly variable between the published cocrystal structures. In this work, we provide a comprehensive computational analysis of the ASDI protein family and study the different contact themes between ECFs and ASDIs. To this end, we harness the coevolution of these diverse protein families and predict covarying amino acid residues as likely candidates of an interaction interface. As a result, we find two common binding interfaces linking the first alpha-helix of the ASDI to the DNA-binding region in the σ₄ domain of the ECF, and the fourth alpha-helix of the ASDI to the RNA polymerase (RNAP)-binding region of the σ₂ domain. The conservation of these two binding interfaces contrasts with the apparent quaternary structure diversity of the ECF/ASDI complexes, partially explaining the high specificity between cognate ECF and ASDI pairs. Furthermore, we suggest that the dual inhibition of RNAP- and DNA-binding interfaces is likely a universal feature of other ECF anti-σ factors, preventing the formation of nonfunctional trimeric complexes between σ/anti-σ factors and RNAP or DNA.IMPORTANCE In the bacterial world, extracytoplasmic function σ factors (ECFs) are the most widespread family of alternative σ factors, mediating many cellular responses to environmental cues, such as stress. This work uses a computational approach to investigate how these σ factors interact with class I anti-σ factors-the most abundant regulators of ECF activity. By comprehensively classifying the anti-σs into phylogenetic groups and by comparing this phylogeny to the one of the cognate ECFs, the study shows how these protein families have coevolved to maintain their interaction over evolutionary time. These results shed light on the common contact residues that link ECFs and anti-σs in different phylogenetic families and set the basis for the rational design of anti-σs to specifically target certain ECFs. This will help to prevent the cross talk between heterologous ECF/anti-σ pairs, allowing their use as orthogonal regulators for the construction of genetic circuits in synthetic biology.

Keywords: RNA polymerase; coevolutionary analysis; comparative genomics; computational biology; direct coupling analysis; gene regulation; transcription factors.

FIG 1

Structures of ECF σ factors in complex with class I anti-σ factors. ECFs are shown in shades of pink, whereas anti-σ factors appear in shades of blue. Different areas of the protein are differentially colored (see legend). Different anti-σ factors show different binding conformations. (A) SigE-ChrR from R. sphaeroides (PDB accession no. 2Q1Z [13]). (B) SigW-RsiW from B. subtilis (PDB accession no. 5WUQ [14]). (C) RpoE-RseA from E. coli (PDB accession no. 1OR7 [16]). (D) SigK-RskA from M. tuberculosis (PDB accession no. 4NQW [15]).

FIG 2

ASDI phylogenetic tree. Phylogenetic tree of the consensus sequences of subgroups of class I anti-σ factor domains. The tree is rooted at the sequence of the class II anti-σ factor CnrY, from Cupriavidus metallidurans, used as an outlier. Branch length indicates evolutionary distance. Internal branch colors indicate bootstrap values, where 0% is red and 100% is green. Rings are explained as follows: 1, ASDI group defined in this work; 2, ECF group of the cognate ECFs encoded in the same genetic neighborhoods; 3, presence of Zn-binding motif; and 4, average domain composition of the anti-σ factors associated with each subgroup. The most important domains are explained in the legend.

FIG 3

DCA results on the contact between ECFs and ASDIs. (A) DCA contact map. Each axis represents the concatenated protein sequences of RpoE and RseA, from E. coli, used as reference for the amino acid labeling. High DCA scores, indicated by darker colors, correspond to residues with a high likelihood to bind in vivo. The 14 highest scores (DCA score ≥0.255) are marked in the heatmap and labeled according to their rank. (B) Table of the 14 highest-scoring DCA predictions, mapped to the amino acid coordinates of RpoE and RseA from E. coli. The common contact (CC) column indicates the DCA predictions that are also common contacts observed in the four crystal structures of ECFs/ASDIs, as derived by Voronoi tessellation (Table 2). (C) Scatterplot of the top 21 DCA predictions against the distance between the alpha carbons of the predicted contacts, as derived from the four structures of ECF/ASDI complexes (Fig. 1). The top 14 predictions are in close proximity in most of the three-dimensional structures. Complexes are labeled after their anti-σ factor, where RseA corresponds to RpoE/RseA complex from E. coli (PDB accession no. 1OR7 [16]), ChrR to SigE/ChrR from R. sphaeroides (PDB accession no. 2Q1Z [13]), RsiW to SigW/RsiW from B. subtilis (PDB accession no. 5WUQ [14]), and RskA to SigK/RskA from M. tuberculosis (PDB accession no. 4NQW [15]). (D) Multiple-sequence alignment of two selected ECF/ASDI pairs, RpoE/RseA from E. coli and SigK/RskA from M. tuberculosis. Labels of the top 14 contacts indicate their position. The presence of alpha-helices and their names are depicted on top of the alignment. The sequence logo depicts the amino acid composition of the full ECF and ASDI alignments derived from 10,930 sequences, respectively. (E) Three-dimensional depiction of the top 14 predictions in the structure of RpoE/RseA complex (PDB accession no. 1OR7 [16]). ECF is colored in beige, and anti-σ factor is in gray. Predicted contacts are labeled according to their rank. N and C termini from ECF and anti-σ factor are labeled.

FIG 4

Sequence logos of the top 14 DCA predictions, computed for the 12 ASDI groups with more than 100 sequences. The sequence logos show the amino acid composition for the DCA-predicted contact points for both the ECF and anti-σ factor in each ECF/ASDI group. The contacts are ordered from left to right according to their DCA rank, as indicated on top. The sequence logos are manually arranged based on their similarity.

FIG 5

Description of the specificity determining positions (SDPs) that distinguish different ASDI groups. (A) Multiple-sequence alignment of the anti-σ factors RseA from E. coli and RskA from M. tuberculosis showing the position of the SDPs, labeled with numbers according to sequence position. Alpha-helices and their names are indicated with red boxes on the ASDI sequences. The sequence logo shows the amino acid composition of the full ASDI alignment. (B) Logo of SDPs in every ASDI group with more than 100 proteins. Positions are labeled as in panel A. (C) ASDI specificity determining positions plotted in the structure of ECF/ASDI complexes. ECFs are colored in beige, and anti-σ factors are in gray; SDPs are colored in green and labeled with their identifier as in panel A. The RpoE/RseA complex is present in E. coli (PDB accession no. 1OR7 [16]), the SigK/RskA complex is in M. tuberculosis (Mtu, PDB accession no. 4NQW [15]), SigW/RsiW is in B. subtilis (Bsu, PDB accession no. 5WUQ [14]), and SigE/ChrR is in R. sphaeroides (Rsp, PDB accession no. 2Q1Z [13]). Contacts with the ECF are represented by connector lines.