Structural studies of the activation of the two component receiver domain NTRC by multidimensional heteronuclear NMR

Multidimensional heteronuclear NMR spectroscopy was used to investigate the N-terminal domain of the transcriptional enhancer NTRC (NiTrogen Regulatory protein C). This domain belongs to the family of receiver domains of two-component regulatory systems involved in signal transduction. Phosphorylation of NTRC at D54 leads to an activated form of the molecule which stimulates transcription of genes involved in nitrogen regulation. Three and four dimensional NMR techniques were used to determine an intermediate resolution structure of the unphosphorylated, inactive form of the N-terminal domain of NTRC. The structure is comprised of five {alpha}-helices and a five-stranded {beta}-sheet in a ({beta}/{alpha}){sub 5} topology. Analysis of the backbone dynamics of NTRC indicate that helix 4 and strand 5 are significantly more flexible than the rest of the secondary structure of the protein and that the loops making up the active site are flexible. The short lifetime of phospho-NTRC hampers the study of this form. However, conditions for determining the resonance assignments and, possibly, the three dimensional structure of phosphorylated NTRC have been obtained. Tentative assignments of the phosphorylated form indicate that the majority of the changes that NTRC experiences upon phosphorylation occur in helix 3, strand 4, helix 4, strand 5, and the loop between strand 5 and helix 5 (the 3445 face of NTRC) as well as near the site of phosphorylation. In order to examine a stable, activated form of the protein, constitutively active mutants of NTRC were investigated

Probing Microsecond Time Scale Dynamics in Proteins by Methyl 1H Carr−Purcell−Meiboom−Gill Relaxation Dispersion NMR Measurements. Application to Activation of the Signaling Protein NtrCr

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ H-1 Carr-Purcell-Meiboom-Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [H-1, C-13]-D-glucose in similar to 100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using C-13 TOCSY NMR spectroscopy, as was recently demonstrated (Often, R.; et al. J. Am. Chem. Soc. 2010, 132, 2952-2960). In this Article, NMR pulse schemes are presented to measure H-1 CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-delta 1 and Thr-gamma 2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong C-13 scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrC(r), for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone N-15 CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the H-1 line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 +/- 0.5) x 10(3) per second (i.e., tau(ex) = 64.7 +/- 1.9 mu s). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by H-1 CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone N-15 relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins

Altering dimerization specificity by changes in surface electrostatics

Arc repressor forms a homodimer in which the subunits intertwine to create a single globular domain. To obtain Arc sequences that fold preferentially as heterodimers, variants with surface patches of excess positive or negative charge were designed. Several but not all oppositely charged sequence pairs showed preferential heterodimer formation. In the most successful design pair, α helix B of one subunit contained glutamic acids at positions 43, 46, 47, 48, and 50, whereas the other subunit contained lysines or arginines at these positions. A continuum electrostatic model captures many features of the experimental results and suggests that the most successful designs include elements of both positive and negative design

Conformational coupling in the chemotaxis response regulator CheY

CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation