IngentaConnect Effector-induced self-association and conformational changes in t...

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 σ^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 ATPγS concentration suggested an apparent dissociation constant (K_d) for ATPγS of 150 μM. 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 K_d of 6 μM. In the presence of ATPγS the equilibrium was shifted towards the dimer form (K_d = 0.8 μM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATPγS (K_d = 0.5 μM). The addition of ATPγS 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.

Document Type: Research article

DOI: 10.1046/j.1365-2958.1996.01530.x

Affiliations: 1: Nitrogen Fixation Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. 2: Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.

The full text electronic article is available for purchase. You will be able to download the full text electronic article after payment.

$50.39 plus tax Refund Policy

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