共查询到20条相似文献,搜索用时 109 毫秒
1.
Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Roche P Mouawad L Perahia D Samama JP Kahn D 《Protein science : a publication of the Protein Society》2002,11(11):2622-2630
FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed. 相似文献
2.
CheY is the best characterized member of the response regulator superfamily, and as such it has become the principal model for understanding the initial molecular mechanisms of signaling in two-component systems. Normal signaling by response regulators requires phosphorylation, in combination with an activation mechanism whose conformational effects are not completely understood. CheY activation involves three events, phosphorylation, a conformational change in the beta(4)--alpha(4) loop, and a rotational restriction of the side chain of tyrosine 106. An outstanding question concerns the nature of an active conformation in the apoCheY population. The details of this 1.08-A resolution crystal structure of wild-type apoCheY shows the beta(4)--alpha(4) loop in two distinctly different conformations that sterically correlate with the two rotameric positions of the tyrosine 106 side chain. One of these conformational states of CheY is the inactive form, and we propose that the other is a meta-active form, responsible for the active properties seen in apoCheY. 相似文献
3.
Ozkirimli E Yadav SS Miller WT Post CB 《Protein science : a publication of the Protein Society》2008,17(11):1871-1880
The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain-domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. 相似文献
4.
Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state. 相似文献
5.
The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation. Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli. In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti. The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti. 相似文献
6.
7.
The N-terminal receiver domain of NtrC is the molecular switch in the two-component signal transduction. It is the first protein where structures of both the active (phosphyroylated) and inactive (unphosphyroylated) states are determined experimentally. Phosphorylation of the NtrC at the active site induces large structural change. NMR experiments suggested that the wild type unphosphorylated NtrC adopts both the active and the inactive conformations and the phosphorylation stabilizes the active conformations. We applied free (unconstrained) molecular dynamic (MD) simulation to examine the intrinsic flexibilities and stabilities of the NtrC receiver domain in both the active and inactive conformations. Molecular dynamic simulations showed that the inactive state of NtrC receiver domain is more flexible than the active state. There were large movements in helix 4 and loop beta3-alpha3 which coincide with major structural differences between the inactive and active states. We observed large root-mean-square deviations from the initial starting structure and the large root-mean-square fluctuations during MD simulation for the inactive state. We then investigated the activation pathway with Targeted MD simulation. We show that the intrinsic flexibility in the loop beta3-alpha3 plays an important role in triggering the conformational change. Phosphorylation at the active site may serve to stabilize the conformational change. These results together suggest that the unphosphorylated NtrC receiver domain could be involved in a conformational equilibrium between two different states. 相似文献
8.
beta helix proteins are characterized by a repetitive fold, in which the repeating unit is a beta-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed beta helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into beta helices. Left-handed beta helices attain their equilateral prism fold by incorporating "corners" with the conformational sequence P(II)-P(II)-alpha(L)-P(II), which imposes sequence restrictions, resulting in the first and third P(II) residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed beta helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an alpha-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (phi,psi) that permit the formation of continuous, loopless beta helices (Perutz nanotubes) have also been investigated. 相似文献
9.
Wynn RM Kato M Machius M Chuang JL Li J Tomchick DR Chuang DT 《Structure (London, England : 1993)》2004,12(12):2185-2196
The human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a 4 MDa macromolecular machine comprising three catalytic components (E1b, E2b, and E3), a kinase, and a phosphatase. The BCKDC overall activity is tightly regulated by phosphorylation in response to hormonal and dietary stimuli. We report that phosphorylation of Ser292-alpha in the E1b active site channel results in an order-to-disorder transition of the conserved phosphorylation loop carrying the phosphoryl serine. The conformational change is triggered by steric clashes of the phosphoryl group with invariant His291-alpha that serves as an indispensable anchor for the phosphorylation loop through bound thiamin diphosphate. Phosphorylation of Ser292-alpha does not severely impede the E1b-dependent decarboxylation of alpha-ketoacids. However, the disordered loop conformation prevents phosphorylated E1b from binding the E2b lipoyl-bearing domain, which effectively shuts off the E1b-catalyzed reductive acylation reaction and therefore completely inactivates BCKDC. This mechanism provides a paradigm for regulation of mitochondrial alpha-ketoacid dehydrogenase complexes by phosphorylation. 相似文献
10.
Src kinase activity is implicated in the regulation of downstream signal transduction pathways involved in cell growth processes. Crystallographic studies indicate that activation of Hematopoietic cell kinase (Hck), a member of the Src kinase family, is accompanied structurally by a large conformational change in two specific parts of its catalytic domain: the alpha-C helix and the activation loop. In the present study, molecular dynamics (MD) simulations are used to characterize the transformation pathway from the inactive to the active state. Four different conditions are considered: the presence or absence of Tyr416 phosphorylation in the activation loop, and the presence or absence of substrate ATP-2Mg(+2) in the active site. Effective free energy landscapes for local residues are determined using a combination of restrained MD simulations with a Root Mean Square Distance (RMSD) biasing potential to enforce the change followed by free MD simulations to allow relaxation from artificially enforced intermediates. A conceptual subdivision of the kinase catalytic domain into four moving parts: the flexible activation loop segment, the buried activation loop segment, the alpha-C helix, and the N-terminal end linker, leads to a concise hypothesis in which each of the moving parts are only required to be coupled to their nearest neighbor to ensure bidirectional allostery in the regulation of protein tyrosine kinases. Both Tyr416 phosphorylation and ATP-2Mg(+2) affect the local backbone torsional free energy landscapes accompanying the structural transition. When these two factors are present together, a metastable coordinated state of ATP-2Mg(+2) and the phosphorylated Tyr416 is observed that offers a possible explanation for the inhibition of protein kinase activity due to increase in Mg(+2) ion concentration. 相似文献
11.
Porter SL Wadhams GH Martin AC Byles ED Lancaster DE Armitage JP 《The Journal of biological chemistry》2006,281(43):32694-32704
The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues. The roles and cellular localization of the CheYs in Rhodobacter sphaeroides were determined. Only two CheYs were required for chemotaxis, CheY(6) and either CheY(3) or CheY(4). These CheYs were partially localized to either of the two chemotaxis signaling clusters, with the remaining protein delocalized. Interestingly, mutation of the CheY(6) phosphorylatable aspartate to asparagine produced a stopped motor, caused by phosphorylation on alternative site Ser-83 by CheA. Extensive mutagenesis of E. coli CheY has identified a number of activating mutations, which have been extrapolated to other response regulators (D13K, Y106W, and I95V). Analogous mutations in R. sphaeroides CheYs did not cause activation. These results suggest that although the R. sphaeroides and E. coli CheYs are similar in that they require phosphorylation for activation, they may differ in both the nature of the phosphorylation-induced conformational change and their subsequent interactions with the flagellar motor. Caution should therefore be used when projecting from E. coli CheY onto novel response regulators. 相似文献
12.
Lee SY Cho HS Pelton JG Yan D Henderson RK King DS Huang L Kustu S Berry EA Wemmer DE 《Nature structural biology》2001,8(1):52-56
The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation. 相似文献
13.
CheY's acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli
下载免费PDF全文
![点击此处可从《Molecular microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Milana Fraiberg Oshri Afanzar C. Keith Cassidy Alexandra Gabashvili Klaus Schulten Yishai Levin Michael Eisenbach 《Molecular microbiology》2015,95(2):231-244
Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high‐resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate‐dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the β4α4 loop, shifted toward an active state upon mutation. Removal of β4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation. 相似文献
14.
Crystal structure of Escherichia coli CheY refined at 1.7-A resolution 总被引:19,自引:0,他引:19
The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57. 相似文献
15.
Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles. 相似文献
16.
Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis 总被引:34,自引:0,他引:34
G S Lukat B H Lee J M Mottonen A M Stock J B Stock 《The Journal of biological chemistry》1991,266(13):8348-8354
The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY. 相似文献
17.
Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Da Re S Tolstykh T Wolanin PM Stock JB 《Protein science : a publication of the Protein Society》2002,11(11):2644-2654
Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY-P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by alpha4, beta5, alpha5, and alpha1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets. 相似文献
18.
VanSchouwen B Selvaratnam R Fogolari F Melacini G 《The Journal of biological chemistry》2011,286(49):42655-42669
The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation. 相似文献
19.
Cho HS Lee SY Yan D Pan X Parkinson JS Kustu S Wemmer DE Pelton JG 《Journal of molecular biology》2000,297(3):543-551
The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains. 相似文献
20.
Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY 总被引:30,自引:0,他引:30
D A Sanders B L Gillece-Castro A M Stock A L Burlingame D E Koshland 《The Journal of biological chemistry》1989,264(36):21770-21778
The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein. 相似文献