Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions [Gene Regulation]

April 14th, 2014 by Lin, W., Wang, Y., Han, X., Zhang, Z., Wang, C., Wang, J., Yang, H., Lu, Y., Jiang, W., Zhao, G.-P., Zhang, P.

The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator (RR) that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp50 residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical RR while the so-called phosphorylation pocket is not conserved with its space being occupied by an Arg52 from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms functional homodimer via its receiver domain and suggest that the charge interactions of Asp50 with the highly conserved Arg52 and Thr9 in the receiver domain may be crucial in maintaining the proper conformation for homodimerization as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR, i.e. D50N, D50E, R52A and T9A, which have only minor effects upon both dimerization and physiological function of GlnR in vivo albeit their DNA binding ability is weakened comparing to that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical R/D/T charge interactions, which is essential for maintaining the biologically active homodimer conformation.
  • Posted in Journal of Biological Chemistry, Publications
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