Effector‐induced self‐association and conformational changes in the enhancer‐binding protein NTRC

Abstract

The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM. In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM). The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

“…Data are not shown for the other mutant forms of DctD (⌬1-142) , all of which gave similar patterns of protection from DNase I digestion. subunit dissociation constants of 6 M and 0.5 M respectively (Farez-Vidal et al, 1996). One of the mutant proteins, DctD (⌬1-142, T223I) , tended to aggregate, and the absorbance distribution (data not shown) suggested that higher molecular weight species were present.…”

“…induce self-association of the protein to higher order states (Farez-Vidal et al, 1996). We plan to determine whether ATP␥S will similarly induce self-association of DctD (⌬1-142) , and if any of the mutant proteins that activate transcription poorly in the absence of the UAS are deficient in selfassociation.…”

Mutant Forms of theAzotobacter vinelandiiTranscriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

“…Members of the AAAϩ family function as oligomers, often as hexamers arranged in a ring structure (23,34). The binding of a nucleotide promotes the oligomerization of 54 -dependent activators (10,27,35), and it is likely that NifL perturbs nucleotide-dependent interactions required for protomer association and/or conformational changes necessary for the interaction of NifA with 54 -RNA polymerase. Mutations in the central AAAϩ domain of NifA which result in constitutive insensitivity to NifL may affect residues which are either required for direct interaction with NifL or involved in intramolecular repression mediated by the GAF domain when NifL is present.…”

The Central Domain of Escherichia coli TyrR Is Responsible for Hexamerization Associated with Tyrosine-mediated Repression of Gene Expression

“…NtrC is believed to be in a dimer-octamer or -hexamer equilibrium in the presence of DNA (42,47). ATP␥S causes a mutant NtrC that activates transcription in the absence of phosphorylation to oligomerize to a hexamer or octamer in the absence of DNA (48). The major dimerization determinants of NtrC are located in its C-terminal domain, and indirect evidence points to oligomerization determinants being located in the central domain (46,48).…”

“…ATP␥S causes a mutant NtrC that activates transcription in the absence of phosphorylation to oligomerize to a hexamer or octamer in the absence of DNA (48). The major dimerization determinants of NtrC are located in its C-terminal domain, and indirect evidence points to oligomerization determinants being located in the central domain (46,48). In contrast to NtrC, the ligand binding sites that promote the higher order oligomerization of TyrR are located solely within the central domain.…”