Abstract:

<p>In enterobacteria such as Escherichia coli, the general stress response is mediated by σ, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σ is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the inter-domain segmented helical linker. These are accompanied by masking of the α4-β5-α5 (4-5-5) &quot;signaling&quot; face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry we identify σ binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σ engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.</p>