A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

Abstract

SummaryPhyR is a hybrid stress regulator conserved in a-proteobacteria that contains an N-terminal s-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-s factor. PhyR thus functions as an anti-anti-s factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as s T and its anti-s factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti-anti-s regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions s2 and s4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of a4-b5-a5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions s 2 and s4 in the SL domain to open about a flexible connector loop and bind anti-s factor.

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

“…Superimposition of the individual subdomains σ 2 and σ 4 of PhyR SL with the corresponding regions of a prototypical ECF sigma factor, σ E [Protein Data Bank (PDB) ID code 1OR7] (28) showed that the PhyR SL subdomains essentially retain the typical fold of ECF sigma factors, with rmsds of 3.12 Å and 0.80 Å, respectively (Fig. S2), except for helix α4 of bona fide ECF sigma factors, which is absent in PhyR orthologs (16) (Fig. S3).…”

“…3A). Whereas the σ 2 -σ 4 linker was described as a dynamic hinge in unphosphorylated PhyR (16), it undergoes an outward movement and positions helix α4 to allow its participation in NepR helix α1′ recognition in the complex. In the PhyR SL -NepR complex, the position of helix α4 is not compatible with the position of the receiver domain in unphosphorylated PhyR and generates a steric clash with α12 (Fig.…”

“…PhyR is a member of the response regulator family and is unique in that its output domain shows homology to ECF sigma factors (14,15). While the PhyR receiver domain (PhyR REC ) has a classic receiver fold with the conserved phosphorylation site and catalytical motifs, the sigma factor-like output domain (PhyR SL ) essentially retains an ECF sigma factor fold but lacks the σ 2.4 region and is degenerate in the σ 4.2 region, which are normally involved in promoter binding in bona fide ECF sigma factors (12,16). According to the proposed model (12), under unstressed conditions, the sigma factor σ EcfG is sequestered by its anti-sigma factor, NepR, and the anti-sigma factor antagonist, PhyR, remains in an unphosphorylated inactive state.…”

“…The crystal structure of the unphosphorylated form of PhyR from Caulobacter crescentus shows that the two domains of PhyR, PhyR REC and PhyR SL , interact through a polar interface that buries one face of the PhyR SL domain and the α4-β5-α5 face of the receiver domain (16), a region known to undergo conformational changes on phosphorylation of the receiver domain in other response regulators (17)(18)(19). It was proposed that the activation of PhyR releases the PhyR SL domain to bind the antisigma factor NepR (12); however, the mode of binding of NepR to PhyR SL remains unknown.…”

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.…”