The structural basis for regulated assembly and function of the transcriptional activator NtrC

Abstract

In two-component signal transduction, an input triggers phosphorylation of receiver domains that regulate the status of output modules. One such module is the AAA+ ATPase domain in bacterial enhancer-binding proteins that remodel the 54 form of RNA polymerase. We report X-ray solution scattering and electron microscopy structures of the activated, full-length nitrogen-regulatory protein C (NtrC) showing a novel mechanism for regulation of AAA+ ATPase assembly via the juxtaposition of the receiver domains and ATPase ring. Accompanying the hydrolysis cycle that is required for transcriptional activation, we observed major order-disorder changes in the GAFTGA loops involved in 54 binding, as well as in the DNA-binding domains. The 54 holoenzyme form of RNA polymerase forms closed DNA complexes that open only with the help of enhancer-binding proteins (EBPs). For the nitrogen-regulatory protein C of enteric bacteria (NtrC), such activity starts a cascade of events that may ultimately lead to the activation of transcription for as much as 2% of the genome (Zimmer et al. 2000). Sequence analysis of >160054 -dependent transcriptional activators shows that in addition to their oligomerization/AAA+ ATPase domain, they also have a DNA-binding domain and a regulatory domain, which in 50% of cases is a twocomponent receiver domain (Bateman et al. 2004). The helix-turn-helix DNA-binding domain recognizes enhancer-like sequences between 100 and 150 bp upstream of the promoter. In their inactive states these proteins are usually dimers that bind to pairs of tandem sites in the enhancer elements. Upon activation via the regulatory domains, they oligomerize into ATPase-active rings that use the energy from ATP hydrolysis to physically remodel closed complexes of 54 holoenzyme and promoter DNA (Rombel et al. 1998;Neuwald et al. 1999;Chaney et al. 2001;Lee et al. 2003).Crystal or NMR structures are available for several isolated domains and truncation constructs of EBPs including NtrC, DctD, PspF, ZraR, and NtrC1 (Volkman et al. 1995;Kern et al. 1999;Pelton et al. 1999;Meyer et al. 2001;Park et al. 2002;Hastings et al. 2003;Lee et al. 2003;Doucleff et al. 2005;Rappas et al. 2005;Sallai and Tucker 2005). Existing structural information on DctD and NtrC1 has been used to provide a model of how two-component signal transduction can regulate assembly of their AAA+ ATPase domains ), but this model fails to explain regulation in the closely related protein NtrC (40% sequence identity, 60% sequence similarity) Hastings et al. 2003). Here we report both SAXS/WAXS (small-and wide-angle X-ray scattering) and EM (electron microscopy) structures of the full-length, activated form of NtrC from Salmonella typhimurium. Docking of atomic models for the individual domains into the structures reveals their organization within the activated ring and uncovers a novel mechanism for the use of two-compo-

Sequence-, structure-, and dynamics-based comparisons of structurally homologous CheY-like proteins

“…S5 (blue/red highlights). The flexibility of these regions is closely related to their biological functions, and our results are in good agreement with previous experimental and computational studies (19,(39)(40)(41). Fig.…”

Analysis of the Campylobacter jejuni FlgR Response Regulator Suggests Integration of Diverse Mechanisms To Activate an NtrC-Like Protein

“…The phosphorylation of the receiver domain removes this inhibition, allowing for oligomerization and ATPase activity, requirements for the initiation of target gene transcription. In contrast, the receiver domain of NtrC of S. enterica serovar Typhimurium positively regulates protein activation upon phosphorylation, as this modification induces conformational changes necessary for the function of the response regulator (15). In NtrC, the receiver domain is essential for activation and its removal results in an inactive protein.…”

VasH Is a Transcriptional Regulator of the Type VI Secretion System Functional in Endemic and Pandemic Vibrio cholerae

“…In the case of VasH V52 , deletion of the HTH motif from the C terminus resulted in a nonfunctional protein that was unable to rescue T6SS gene expression and Hcp synthesis, compete with wild-type VasH V52 , or restore virulence of V52 ⌬vasH toward D. discoideum. A well-studied 54 activator, NtrC from Salmonella enterica serovar Typhimurium, requires binding to the enhancer sequence through the HTH motif for efficient formation of ATPase-active oligomers (6). It is possible that VasH follows a pattern similar to that of NtrC in that DNA binding is required for efficient activation of the protein and/or for its activity, which cannot be compensated by increased protein-protein interaction due to overexpression.…”