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

Extracytoplasmic function {sigma} factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Amongst the diverse regulators of ECF activity, class I anti-{sigma} 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-{sigma} 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 -helix of the ASDI to the DNA binding region in the {sigma}4 domain of the ECF, and the fourth -helix of the ASDI to the RNA polymerase (RNAP) binding region of the {sigma}2 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-{sigma} factors, preventing the formation of non-functional trimeric complexes between {sigma}/anti-{sigma} factors and RNAP or DNA. SignificanceIn the bacterial world, extracytoplasmic function {sigma} factors (ECFs) are the most widespread family of alternative {sigma} factors, mediating many cellular responses to environmental cues, such as stress. This work uses a computational approach to investigate how these {sigma} factors interact with class I anti-{sigma} factors - the most abundant regulators of ECF activity. By comprehensively classifying the anti-{sigma}s 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-{sigma}s in different phylogenetic families and set the basis for the rational design of anti-{sigma}s to specifically target certain ECFs. This will help to prevent the cross-talk between heterologous ECF/anti-{sigma} pairs, allowing their use as orthogonal regulators for the construction of genetic circuits in synthetic biology.