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• W2023590125 abstract "The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the μs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism. The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the μs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism. NMR dynamics data reveal CheY does not undergo concerted, two-state switching Asynchronous, local switching of a network of residues facilitates CheY activation Mg2+ enhances the dynamics associated with the inactive-to-active transition Allosteric conformational change is critical for the function of many proteins. Currently, it is not generally understood how allosteric conformational changes are executed or how many different execution strategies exist. As a simpler alternative to defining precise conformational change trajectories, there has been intense focus directed at the “selected fit” versus “induced fit” paradigms (Okazaki and Takada, 2008Okazaki K. Takada S. Dynamic energy landscape view of coupled binding and protein conformational change: induced-fit versus population-shift mechanisms.Proc. Natl. Acad. Sci. USA. 2008; 105: 11182-11187Crossref PubMed Scopus (267) Google Scholar, Hammes et al., 2009Hammes G.G. Chang Y.C. Oas T.G. Conformational selection or induced fit: a flux description of reaction mechanism.Proc. Natl. Acad. Sci. USA. 2009; 106: 13737-13741Crossref PubMed Scopus (410) Google Scholar, Wlodarski and Zagrovic, 2009Wlodarski T. Zagrovic B. Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin.Proc. Natl. Acad. Sci. USA. 2009; 106: 19346-19351Crossref PubMed Scopus (157) Google Scholar, Zhou, 2010Zhou H.X. From induced fit to conformational selection: a continuum of binding mechanism controlled by the timescale of conformational transitions.Biophys. J. 2010; 98: L15-L17Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, Csermely et al., 2010Csermely P. Palotai R. Nussinov R. Induced fit, conformational selection and independent dynamic segments: an extended view of binding events.Trends Biochem. Sci. 2010; 35: 539-546Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar), as well as at concerted versus propagated conformational changes (Cui and Karplus, 2008Cui Q. Karplus M. Allostery and cooperativity revisited.Protein Sci. 2008; 17: 1295-1307Crossref PubMed Scopus (517) Google Scholar, Tsai et al., 2009Tsai C.J. Del Sol A. Nussinov R. Protein allostery, signal transmission and dynamics: a classification scheme of allosteric mechanisms.Mol. Biosyst. 2009; 5: 207-216Crossref PubMed Scopus (279) Google Scholar). The pairing of selected fit with a concerted conformational change—that is, a simple “switch”—has been particularly popular. It is widely recognized that dynamics are central to these processes, yet few experiments have directly assessed the basic assumptions of allostery. Hence there is a need for experimental dynamics data on the timescale of conformational equilibria in allosteric proteins. Receiver domains from response regulator (RR) proteins have been studied extensively and, because of their small size, have become favored as models for understanding conformational allostery. RRs, along with sensor kinases, comprise the two-component system ubiquitous in prokaryotes. RRs usually consist of an input receiver domain that is activated by phosphorylation, and an output domain that transmits the signal into various activities such as DNA binding (Bourret, 2010Bourret R.B. Receiver domain structure and function in response regulator proteins.Curr. Opin. Microbiol. 2010; 13: 142-149Crossref PubMed Scopus (173) Google Scholar, Galperin, 2010Galperin M.Y. Diversity of structure and function of response regulator output domains.Curr. Opin. Microbiol. 2010; 13: 150-159Crossref PubMed Scopus (246) Google Scholar). Accordingly, the ability of the receiver domain to undergo a well-defined conformational change is a vital component of RR function (Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (140) Google Scholar, Gao et al., 2007Gao R. Mack T.R. Stock A.M. Bacterial response regulators: versatile regulatory strategies from common domains.Trends Biochem. Sci. 2007; 32: 225-234Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The chemotaxis protein Y (CheY) from Escherichia coli is an RR that regulates chemotactic flagellar rotation (Clegg and Koshland, 1984Clegg D.O. Koshland D.E. The role of a signaling protein in bacterial sensing: behavioral effects of increased gene expression.Proc. Natl. Acad. Sci. USA. 1984; 81: 5056-5060Crossref PubMed Scopus (55) Google Scholar, Matsumura et al., 1984Matsumura P. Rydel J.J. Linzmeier R. Vacante D. Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein.J. Bacteriol. 1984; 160: 36-41Crossref PubMed Google Scholar). Because it lacks an output domain, the CheY receiver domain must both accept a phosphoryl group and directly activate its downstream effector; in response to phosphorylation at D57 (Sanders et al., 1989Sanders D.A. Gillece-Castro B.L. Stock A.M. Burlingame A.L. Koshland Jr., D.E. Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY.J. Biol. Chem. 1989; 264: 21770-21778Abstract Full Text PDF PubMed Google Scholar), which requires the presence of Mg2+ (Lukat et al., 1990Lukat G.S. Stock A.M. Stock J.B. Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis.Biochemistry. 1990; 29: 5436-5442Crossref PubMed Scopus (156) Google Scholar), CheY undergoes a conformational change that enables it to directly bind the flagellar motor switch protein FliM at a surface distal to D57. CheY binding to FliM promotes a change in the flagellar rotation from counterclockwise to clockwise (Baker et al., 2006Baker M.D. Wolanin P.M. Stock J.B. Signal transduction in bacterial chemotaxis.Bioessays. 2006; 28: 9-22Crossref PubMed Scopus (239) Google Scholar). Unphosphorylated CheY also interacts with FliM, though with considerably reduced affinity (Barak and Eisenbach, 1992Barak R. Eisenbach M. Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor.Biochemistry. 1992; 31: 1821-1826Crossref PubMed Scopus (125) Google Scholar, Welch et al., 1993Welch M. Oosawa K. Aizawa S. Eisenbach M. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria.Proc. Natl. Acad. Sci. USA. 1993; 90: 8787-8791Crossref PubMed Scopus (351) Google Scholar). As is common in allosteric signaling molecules, CheY samples an active conformation (A, FliM binding competent) and an inactive conformation (I, FliM binding incompetent). In the I conformation, FliM binding is sterically hindered by the location of Y106 in a solvent-exposed (or “out”) position (Figure 1A ). Upon activation by phosphorylation, Y106 (∼11 Å away from the phosphoaspartate) rotates to a buried (or “in”) position, relieving the hindrance. T87 hydrogen bonds with the phosphoryl group and has been shown to be an important link for Y106 rotation (Appleby and Bourret, 1998Appleby J.L. Bourret R.B. Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet.J. Bacteriol. 1998; 180: 3563-3569Crossref PubMed Google Scholar, Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (140) Google Scholar). Motion of the β4-α4 loop, consisting of residues 88–91, facilitates burial of Y106 (Ma and Cui, 2007Ma L. Cui Q. Activation mechanism of a signaling protein at atomic resolution from advanced computations.J. Am. Chem. Soc. 2007; 129: 10261-10268Crossref PubMed Scopus (42) Google Scholar, Simonovic and Volz, 2001Simonovic M. Volz K. A distinct meta-active conformation in the 1.1-A resolution structure of wild-type ApoCheY.J. Biol. Chem. 2001; 276: 28637-28640Crossref PubMed Scopus (63) Google Scholar), leading to the possibility that residues in the loop (in addition to T87) may be involved in the signaling. Together with small changes in the β5-α5 loop and side-chain motions of K109 and F14, the I and A conformations differ mainly in the Y106 rotation and the location of the β4-α4 loop (Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (140) Google Scholar). The prevailing conceptual framework for receiver domain activation/allostery has been that the protein exists in a dynamic equilibrium that accesses both the I and A conformations, in line with the Monod-Wyman-Changeux (MWC) model of allostery. The following findings provide evidence of an I-to-A state conformational equilibrium: (1) in the absence of phosphorylation, CheY still has the ability to stimulate clockwise flagellar rotation (Barak and Eisenbach, 1992Barak R. Eisenbach M. Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor.Biochemistry. 1992; 31: 1821-1826Crossref PubMed Scopus (125) Google Scholar); (2) in the crystal structure of unphosphorylated, Mg2+-free CheY, both “out” and “in” conformations of the Y106 side chain were observed (Volz and Matsumura, 1991Volz K. Matsumura P. Crystal structure of Escherichia coli CheY refined at 1.7-A resolution.J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar); (3) binding of FliM, CheA, and CheZ peptides to CheY affect its ability to phosphorylate and can be explained by a ligand-induced shift of the I-to-A equilibrium (Schuster et al., 2001Schuster M. Silversmith R.E. Bourret R.B. Conformational coupling in the chemotaxis response regulator CheY.Proc. Natl. Acad. Sci. USA. 2001; 98: 6003-6008Crossref PubMed Scopus (69) Google Scholar); (4) NMR studies of the receiver domains of Spo0F and NtrC showed that in regions where structural changes upon phosphorylation were observed, there was enhanced transverse relaxation due to conformational exchange on the μs-ms timescale (Volkman et al., 2001Volkman B.F. Lipson D. Wemmer D.E. Kern D. Two-state allosteric behavior in a single-domain signaling protein.Science. 2001; 291: 2429-2433Crossref PubMed Scopus (534) Google Scholar, Feher and Cavanagh, 1999Feher V.A. Cavanagh J. Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F.Nature. 1999; 400: 289-293Crossref PubMed Scopus (203) Google Scholar); and (5) mutant NtrC proteins revealed a correlation between activity/inactivity and a two-state equilibrium between active and inactive conformations (Volkman et al., 2001Volkman B.F. Lipson D. Wemmer D.E. Kern D. Two-state allosteric behavior in a single-domain signaling protein.Science. 2001; 291: 2429-2433Crossref PubMed Scopus (534) Google Scholar, Gardino et al., 2009Gardino A.K. Villali J. Kivenson A. Lei M. Liu C.F. Steindel P. Eisenmesser E.Z. Labeikovsky W. Wolf-Watz M. Clarkson M.W. Kern D. Transient non-native hydrogen bonds promote activation of a signaling protein.Cell. 2009; 139: 1109-1118Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). While these data are consistent with a two-state switch of RRs, direct detection of a two-state dynamic process between active and inactive conformations has been elusive. Complicating the simple idea of a preexisting equilibrium between I and A conformations are several crystal structures of CheY that show intermediate conformations (Simonovic and Volz, 2001Simonovic M. Volz K. A distinct meta-active conformation in the 1.1-A resolution structure of wild-type ApoCheY.J. Biol. Chem. 2001; 276: 28637-28640Crossref PubMed Scopus (63) Google Scholar, Dyer and Dahlquist, 2006Dyer C.M. Dahlquist F.W. Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM.J. Bacteriol. 2006; 188: 7354-7363Crossref PubMed Scopus (81) Google Scholar, Guhaniyogi et al., 2006Guhaniyogi J. Robinson V.L. Stock A.M. Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation.J. Mol. Biol. 2006; 359: 624-645Crossref PubMed Scopus (40) Google Scholar). Additionally, molecular dynamics simulations indicated that Y106 rotation and the formation of a hydrogen bond between T87 and the phosphoryl group are independent of one another (Ma and Cui, 2007Ma L. Cui Q. Activation mechanism of a signaling protein at atomic resolution from advanced computations.J. Am. Chem. Soc. 2007; 129: 10261-10268Crossref PubMed Scopus (42) Google Scholar). These studies showed that CheY is not restricted to the two end states, and taken together, they suggest that it can be trapped in metastable states that presumably are sampled along the allosteric conformational change trajectory. It is unknown, however, whether such trapped states are functionally relevant or are merely artifacts from crystallization; it also remains unknown what the relevant timescales are for conformational switching in CheY. In order to further understand the allosteric switch CheY undergoes upon phosphorylation, we investigated the conformational equilibrium that occurs in the unphosphorylated protein. NMR relaxation dispersion was used to measure the dynamics of CheY switching to test for consistency with a two-state model. We found that a physiological level of Mg2+ likely plays a critical role in promoting allosteric conformational changes. Nevertheless, whether Mg2+ is present or absent, unphosphorylated CheY appears not to undergo two-state concerted switching between I and A conformations. Rather, the data are more suggestive of a model in which CheY switches in a nonconcerted, segmental fashion. Local sites may occupy their active conformations at different times using a previously undescribed signaling network consisting of T87, A88, and the quartet of W58, M85, E89, and Y106. Large-scale conformational changes in proteins frequently occur on the slow, or μs-ms, timescale (Henzler-Wildman and Kern, 2007Henzler-Wildman K. Kern D. Dynamic personalities of proteins.Nature. 2007; 450: 964-972Crossref PubMed Scopus (1710) Google Scholar, Kleckner and Foster, 2011Kleckner I.R. Foster M.P. An introduction to NMR-based approaches for measuring protein dynamics.Biochim. Biophys. Acta. 2011; 1814: 942-968Crossref PubMed Scopus (359) Google Scholar). In an attempt to measure the dynamic switching between the I and A conformations of unphosphorylated CheY, we used 15N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments to measure motions on this timescale (Palmer et al., 2001Palmer 3rd, A.G. Kroenke C.D. Loria J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules.Methods Enzymol. 2001; 339: 204-238Crossref PubMed Scopus (837) Google Scholar). These experiments elucidate processes on the μs-ms timescale that contribute to broadening (or “width”) of NMR peaks. Specifically, the NMR linewidth is proportional to the transverse relaxation rate, R2, which is comprised of an “intrinsic” rate, R2°, and a rate that arises from conformational exchange processes on the μs-ms timescale, Rex:R2=R2o+Rex.(1) The CPMG relaxation dispersion experiment measures the suppression of Rex contributions to R2 as a function of spacing between 180° pulses in the CPMG train, τcp (Loria et al., 1999Loria J.P. Rance M. Palmer A.G. A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy.J. Am. Chem. Soc. 1999; 121: 2331-2332Crossref Scopus (564) Google Scholar). For a two-state exchange process in the limit of fast exchange on the NMR timescale,R2,eff(1τcp)=R2o+(pIpAΔω2kex)[1−2tanh(kexτcp2)kexτcp],(2) where pI and pA are populations of the major and minor states, Δω is the difference in chemical shift between the two states, and kex is the rate of exchange between I and A (= k1 + k-1). A longer expression (the Carver-Richards equation) exists for the general case (Palmer et al., 2001Palmer 3rd, A.G. Kroenke C.D. Loria J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules.Methods Enzymol. 2001; 339: 204-238Crossref PubMed Scopus (837) Google Scholar). A particular residue's Rex value is considered to be non-zero (i.e., affected by conformational exchange) if Rex > 2 s−1, as described in the Experimental Procedures. Pilot relaxation dispersion experiments indicated that the dispersion curves were more pronounced at lower temperatures, and 15°C was determined to be a good compromise between pronounced dispersion curves and signal-to-noise ratio. Under the standard conditions of 10 mM Mg2+, 15N CPMG relaxation dispersion data were collected at 500, 600, and 700 MHz for CheY. Residues with non-zero Rex values localized to the active site and FliM binding interface, which includes residues T87, A88, and E89 (in the β4-α4 loop), as well as Y106 and V107 (Figure 1A). This was not surprising, since all substantial conformational differences between the I and A states are limited to this region (Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (140) Google Scholar). There were no non-zero Rex values elsewhere in the protein, detected either by CPMG relaxation dispersion or by model-free analysis of T1, T2, and {1H}-15N NOE data. Therefore, at a qualitative level, these data are consistent with motion corresponding to the I-to-A transition. The relaxation dispersion curves (Figure 1B) were quantitatively analyzed using the general Carver-Richards equation (Palmer et al., 2001Palmer 3rd, A.G. Kroenke C.D. Loria J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules.Methods Enzymol. 2001; 339: 204-238Crossref PubMed Scopus (837) Google Scholar) to fit each residue individually (i.e., “local fits”; see Experimental Procedures). A total of 11 residues could be fit with reasonable precision. If CheY switches concertedly in a simple two-state manner between I and A conformations, we would expect to measure the same exchange rate and populations for all residues. However, the fits yielded a range of kinetic and thermodynamic parameters (Table 1). Exchange rates varied from slow (∼1,300 s−1 for residues 12 and 38) to fast (∼3,000 s−1 for residues 106 and 107), and populations varied from 90% to 99% of I. The different exchange rates were also evident in the raw dispersion curves (compare rows of Figure 1B). In addition, two-state or three-state concerted switching could not be rationalized based on attempts to group fit the data (see Experimental Procedures for details).Table 1Local Fits of 15N CPMG Relaxation Dispersion for CheY in the Presence of 10 mM Mg2+Residuekex (s−1)Δω (ppm)pIR2° (s−1)χ2500 MHz600 MHz700 MHz121,220 ± 501.5 ± 0.10.94 ± 0.0113.3 ± 0.313.9 ± 0.214.6 ± 0.14.0361,910 ± 3000.52 ± 0.040.91 ± 0.0213.1 ± 0.213.2 ± 0.114.1 ± 0.12.6381,370 ± 701.7 ± 0.10.95 ± 0.0114.0 ± 0.414.3 ± 0.215.1 ± 0.246622,060 ± 1802.7 ± 0.10.99 ± 0.0112.0 ± 0.112.2 ± 0.113.1 ± 0.10.75643,620 ± 4606.0 ± 0.60.99 ± 0.0113.7 ± 0.513.0 ± 0.613.8 ± 0.75.3862,100 ± 3303.6 ± 0.20.99 ± 0.0115.1 ± 0.315.5 ± 0.216.9 ± 0.213871,370 ± 1801.8 ± 0.20.98 ± 0.0115.3 ± 0.414.8 ± 0.316.6 ± 0.23.6881,230 ± 2801.4 ± 0.40.91 ± 0.0420.1 ± 120.2 ± 0.921.0 ± 0.81489246 ± 313.2 ± 0.50.99 ± 0.0114.5 ± 0.315.8 ± 0.217.5 ± 0.12.91063,100 ± 4102.0 ± 0.10.99 ± 0.0116.4 ± 0.217.1 ± 0.218.5 ± 0.24.51072,840 ± 3804.2 ± 0.40.99 ± 0.0117.6 ± 0.317.7 ± 0.319.3 ± 0.45.5 Open table in a new tab Yet another test for two-state behavior is to compare the dispersion-based differences in chemical shifts to known differences in chemical shifts for two defined structural states. Accordingly, we compared Δω from the local fits of relaxation dispersions to Δδ from the chemical shift perturbations between unphosphorylated and BeFx-bound (phosphoryl mimic [Yan et al., 1999Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.Proc. Natl. Acad. Sci. USA. 1999; 96: 14789-14794Crossref PubMed Scopus (122) Google Scholar]) CheY. A poor correlation between Δω and Δδ was obtained (Figure 1C), further indicating that the I-to-A transition cannot be described by a simple two-state transition. Taken together, the nonuniform values of the individual exchange parameters, the inability to group fit, and poor correlation of Δω and Δδ indicate that CheY does not undergo concerted, two-state switching in the presence of 10 mM Mg2+ at 15°C. Another possible source of non-zero Rex values in CheY and other RRs is the reversible binding of Mg2+. A divalent metal ion is necessary for all RR phosphorylation and dephosphorylation (Lukat et al., 1990Lukat G.S. Stock A.M. Stock J.B. Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis.Biochemistry. 1990; 29: 5436-5442Crossref PubMed Scopus (156) Google Scholar). In the experiments described in the previous section, near physiological concentrations of Mg2+ (10 mM) were used to characterize CheY in an environment similar to the interior of cells and to have consistency with previous biochemical and NMR work on CheY (Moy et al., 1994Moy F.J. Lowry D.F. Matsumura P. Dahlquist F.W. Krywko J.E. Domaille P.J. Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy.Biochemistry. 1994; 33: 10731-10742Crossref PubMed Scopus (74) Google Scholar, Hubbard et al., 2003Hubbard J.A. MacLachlan L.K. King G.W. Jones J.J. Fosberry A.P. Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli.Mol. Microbiol. 2003; 49: 1191-1200Crossref PubMed Scopus (51) Google Scholar). Because the binding affinity of Mg2+ to CheY is 1.5 ± 0.3 mM under our conditions (determined by NMR 1H-15N HSQC peak shifts; Figure S1, available online), at 10 mM Mg2+ and 1 mM CheY, 86% of CheY is bound by the ion. Thus, if the Mg2+ binding kinetics are on the appropriate timescale, the binding of Mg2+ and associated side-chain rearrangements could be detected by relaxation dispersion, and this could complicate the interpretation in terms of conformational exchange. To separate motions associated with Mg2+ binding and release from those intrinsic to CheY, we performed relaxation dispersion experiments without Mg2+ present (i.e., with 1 mM EDTA). In the absence of Mg2+, 13 residues displayed non-zero Rex values for CheY, compared to 11 when Mg2+ is present at 10 mM. These residues were localized to the FliM binding interface or the active site (Figure 2A ), just as when Mg2+ was present (Figure 1A). The locally fit parameters, when assuming a two-state mechanism, were diverse (Figure 2B and Table 2). In comparison with dynamics in the presence of Mg2+, many of the same residues displayed significant dispersion, including T87 and V107. The locally fit parameters of the common residues differed significantly between the Mg2+-free and Mg2+-present conditions (Tables 1 and 2). Additionally, Δω and Δδ did not correlate (Figure 2C), as was the case with Mg2+ (Figure 1C). Therefore, even without the potentially complicating effects of Mg2+, CheY appears to not undergo two-state switching.Table 2Local Fits of 15N CPMG Relaxation Dispersion for CheY in the Presence of 1 mM EDTAResiduekex (s−1)Δω (ppm)pIR2° (s−1)χ2600 MHz700 MHz18288 ± 573.9 ± 0.70.99 ± 0.0115.7 ± 0.317.0 ± 0.32.919180 ± 305.7 ± 10.96 ± 0.0116.1 ± 0.717.4 ± 0.97.6362,400 ± 5000.6 ± 0.10.86 ± 0.0413.3 ± 0.214.1 ± 0.32.6382,360 ± 6403.6 ± 0.40.99 ± 0.0114.0 ± 0.515.8 ± 0.74.3621,820 ± 5203.7 ± 0.40.99 ± 0.0113.4 ± 0.413.8 ± 0.7116496 ± 169.4 ± 10.80 ± 0.0420.0 ± 122.2 ± 213671,860 ± 9200.4 ± 0.10.73 ± 0.115.2 ± 0.416.3 ± 0.63.5681,400 ± 7903.5 ± 0.80.99 ± 0.0116.1 ± 0.415.9 ± 0.61.5692,400 ± 11002.3 ± 0.50.99 ± 0.0115.0 ± 0.515.0 ± 0.81.1872,130 ± 5400.53 ± 0.10.81 ± 0.0514.2 ± 0.315.9 ± 0.42.3941,330 ± 8000.27 ± 0.10.67 ± 0.215.4 ± 0.216.1 ± 0.33.31072,440 ± 5700.52 ± 0.10.80 ± 0.0814.8 ± 0.216.1 ± 0.42.61271,980 ± 5501.6 ± 0.20.98 ± 0.0115.8 ± 0.316.5 ± 0.52.5 Open table in a new tab It is possible that bound Mg2+ induces a conformational change in CheY. The crystal structure of Mg2+-bound E. coli CheY reveals a conformational difference from Mg2+-free CheY that localizes to α4 and the β4-α4 loop (Bellsolell et al., 1994Bellsolell L. Prieto J. Serrano L. Coll M. Magnesium binding to the bacterial chemotaxis protein CheY results in large conformational changes involving its functional surface.J. Mol. Biol. 1994; 238: 489-495Crossref PubMed Scopus (121) Google Scholar). However, chemical shift perturbations of CheY upon the addition of Mg2+ were strongly correlated with closeness to the ion and in general are small in regions of allosteric conformational change (Figure S2), suggesting no significant structural rearrangements. In addition, the crystal structure of Salmonella typhimurium CheY (Stock et al., 1993Stock A.M. Martinez-Hackert E. Rasmussen B.F. West A.H. Stock J.B. Ringe D. Petsko G.A. Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis.Biochemistry. 1993; 32: 13375-13380Crossref PubMed Scopus (197) Google Scholar) (which differs by three amino acids from E. coli) and the NMR structure of E. coli CheY (Moy et al., 1994Moy F.J. Lowry D.F. Matsumura P. Dahlquist F.W. Krywko J.E. Domaille P.J. Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy.Biochemistry. 1994; 33: 10731-10742Crossref PubMed Scopus (74) Google Scholar), both with Mg2+ present, have no indication of any large structural rearrangement. For these reasons, the different CheY dynamics observed with or without Mg2+ do not appear to be the direct result of Mg2+-induced conformational change. To gain greater insight into the effect of Mg2+ on CheY dynamics, we obtained estimates of Rex at additional Mg2+ concentrations of 1 mM and 75 mM (Figure 3). Thus, assuming a single binding site (see Supplemental Discussion), 1 mM CheY was calculated (based on the Kd of 1.5 mM) to be bound by Mg2+ at a level of 0%, 31%, 86%, and 98% for 0 mM, 1 mM, 10 mM, and 75 mM concentrations of Mg2+, respectively. For all concentrations of Mg2+, estimates of Rex were obtained as the difference of R2,eff values at the lowest and highest values of τcp used for dispersion measurements. Overall, the dependence of Rex on the concentration of Mg2+ was immediately apparent: Rex in the presence of no Mg2+ or very high concentration of Mg2+ (75 mM) was relatively low, and Rex at intermediate levels of Mg2+ (1 mM) was very high (Figure 3). Furthermore, peak broadening was evident in many residues at intermediate Mg2+ concentrations, implying increased motion. While high Rex levels at intermediate concentrations of Mg2+ were expected for residues in close proximity to the bound ion (assuming appropriate line-broadening kinetics), high Rex levels were not expected at residues distal to Mg2+ that were not struc" @default.
• W2023590125 created "2016-06-24" @default.
• W2023590125 creator A5010573181 @default.
• W2023590125 creator A5028716214 @default.
• W2023590125 creator A5037260416 @default.
• W2023590125 date "2012-08-01" @default.
• W2023590125 modified "2023-10-18" @default.
• W2023590125 title "Segmental Motions, Not a Two-State Concerted Switch, Underlie Allostery in CheY" @default.
• W2023590125 cites W1514082093 @default.
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