Co-evolutionary analysis reveals a conserved dual binding interface between extracytoplasmic function (ECF) σ factors and class I anti-σ factors

Abstract

AbstractExtracytoplasmic function σ factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Amongst 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 non-inducing conditions, the binding interface between ECFs and ASDIs is surprisingly variable between the published co-crystal 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 co-evolution 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 α-helix of the ASDI to the DNA binding region in the σ4 domain of the ECF, and the fourth α-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 are likely a universal feature of other ECF anti-σ factors, preventing the formation of non-functional trimeric complexes between σ/anti-σ factors and RNAP or DNA.SignificanceIn 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 co-evolved 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.