title: Evidence against the "Y-T coupling" mechanism of activation in the response regulator NtrC.
abstract: The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the "Y-T coupling" mechanism, wherein the χ1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC(R)), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ1 rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC(R) in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted "Y-T coupling" mechanism of activation in NtrC(R).
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Waltham, MA 02452, USA.
authors.forename: Janice
authors.initials: J
authors.lastname: Villali
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Waltham, MA 02452, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pontiggia
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Waltham, MA 02452, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Clarkson
authors.affiliation: Department of Physics, Brandeis University, Waltham, MA 02452, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Hagan
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Waltham, MA 02452, USA. Electronic address: dkern@brandeis.edu.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Kern
mesh_terms: D000495:Allosteric Site; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism
chemical_list: D051922:PII Nitrogen Regulatory Proteins
keywords: CheY; NMR spectroscopy; allostery; protein dynamics; two-component systems