Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Abstract

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ EcfG , was identified. σ EcfG is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ EcfG sigma factor-like output domain (PhyR SL ). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR SL –NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR SL binding, NepR forms two helices that extend over the surface of the PhyR SL subdomains. Homology modeling and comparative analysis of NepR, PhyR SL , and σ EcfG mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ EcfG –NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ EcfG . Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

Single-domain response regulator involved in the general stress response of Methylobacterium extorquens

“…In response to stress, the PhyR receiver domain becomes phosphorylated and its N-terminal sigma-factorlike domain mimics s EcfG to sequester NepR, thereby releasing s EcfG and allowing it to initiate the transcription of stress-related genes . In agreement with the model, recent studies have shown that differences in the affinity of the anti-sigma factor NepR for its two partners, s EcfG and phosphorylated PhyR, are sufficient to trigger the partner switch (Campagne et al, 2012).…”

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

“…Regulated PhyR Proteolysis Is a GSR Control Feature in B. abortus-Previous biochemical analyses of the interaction between PhyR and the anti-factor, NepR, support a model in which high affinity association between these proteins requires aspartyl phosphorylation of the PhyR receiver domain (10,12,13,15,16,33). The equilibrium affinity of PhyRϳP for NepR has been measured in the tens (16) to hundreds (33) nanomolar range, using purified proteins from Sphingomonas sp.…”

“…Thus, the in vitro half-lifetime of the PhyRϳP⅐NepR complex is 129 days, so, as in other species, phosphorylation of B. abortus PhyR increases its affinity for NepR. However, the absolute affinity of PhyR for NepR is substantially higher both in its phosphorylated and unphosphorylated states than PhyR-NepR pairs from other ␣-proteobacteria (16,33). Moreover, the measured association and dissociation rate constants for these B. abortus proteins are orders of magnitude slower than what our group has measured previously on orthologous proteins from C. crescentus (33).…”

“…Studies in related ␣-proteobacteria have shown that stress-induced phosphorylation of the PhyR receiver domain promotes binding of its -like domain to the anti-factor, NepR (10,(12)(13)(14)(15). PhyR binding titrates the NepR anti-factor away from a bona fide ECF factor (16), thereby initiating transcription of genes that mediate bacterial cell survival during stress (Fig. 1A).…”

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

“…As is the case for other response regulators, the activity of PhyR is controlled by phosphorylation of a conserved Asp residue in its REC domain. Despite a rather advanced understanding of the partner switch itself (15)(16)(17), little is known about the decisive step of GSR activation, that is, regulation of PhyR phosphorylation. As a response regulator, PhyR is expected to be part of a two-component system, or phosphorelay, working together with one or several histidine kinases that control PhyR phosphorylation upon sensing a particular stimulus.…”