共查询到20条相似文献,搜索用时 31 毫秒
1.
Mingshan Li Gerald L. Hazelbauer 《Protein science : a publication of the Protein Society》2020,29(2):443-454
Methylation of specific chemoreceptor glutamyl residues by methyltransferase CheR mediates sensory adaptation and gradient sensing in bacterial chemotaxis. Enzyme action is a function of chemoreceptor signaling conformation: kinase‐off receptors are more readily methylated than kinase‐on, a feature central to adaptational and gradient‐sensing mechanisms. Differential enzyme action could reflect differential binding, catalysis or both. We investigated by measuring CheR binding to kinase‐off and kinase‐on forms of Escherichia coli aspartate receptor Tar deleted of its CheR‐tethering, carboxyl terminus pentapeptide. This allowed characterization of the low‐affinity binding of enzyme to the substrate receptor body, otherwise masked by high‐affinity interaction with pentapeptide. We quantified the low‐affinity protein–protein interactions by determining kinetic rate constants of association and dissociation using bio‐layer interferometry and from those values calculating equilibrium constants. Whether Tar signaling conformations were shifted by ligand occupancy or adaptational modification, there was little or no difference between the two signaling conformations in kinetic or equilibrium parameters of enzyme‐receptor binding. Thus, differential methyltransferase action does not reflect differential binding. Instead, the predominant determinants of binding must be common to different signaling conformations. Characterization of the dependence of association rate constants on Deybe length, a measure of the influence of electrostatics, implicated electrostatic interactions as a common binding determinant. Taken together, our observations indicate that differential action of methyltransferase on kinase‐off and kinase‐on chemoreceptors is not the result of differential binding and suggest it reflects differential catalytic propensity. Differential catalysis rather than binding could well be central to other enzymes distinguishing alternative conformations of protein substrates. 相似文献
2.
The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties. 相似文献
3.
The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar. 相似文献
4.
The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (Δ tar ). However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled Δ tar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the 'opposite' orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation. When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit. In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar + cells. 相似文献
5.
Sensory adaptation of low-abundance chemoreceptors in Escherichia coli requires assistance from high-abundance receptors, because only high-abundance receptors carry the carboxyl-terminal pentapeptide sequence NWETF that enhances adaptational covalent modification. Using membrane vesicles containing both high-abundance receptor Tar and low-abundance receptor Trg, we observed effective assistance in vitro for all three adaptational modifications: methylation, demethylation and deamidation. These results demonstrated that adaptational assistance involves not only the previously documented assistance for methylation but also assistance for the two CheB-catalysed reactions. We determined rates of assisted methylation and demethylation at many ratios of assisting to assisted receptor. Analysis by a model of assistance indicated one Tar dimer could assist seven Trg dimers in methylation or five in demethylation, defining assistance neighbourhoods. These neighbourhoods were larger than a trimer of homodimers, required only receptors and were minimally affected by formation of signalling complexes. Time courses of assisted Trg methylation in membranes with low amounts of Tar showed that assisting receptors did not diffuse beyond initial neighbourhoods for at least two hours. Taken together, these observations indicate that chemoreceptors can form stable neighbourhoods larger than trimers in the absence of other chemotaxis proteins. Such interactions are likely to occur in natural receptor clusters in vivo. 相似文献
6.
Mutations in the aspartate receptor of Escherichia coli which affect aspartate binding 总被引:4,自引:0,他引:4
The effects of five mutations at arginines 64, 69, and 73 of the Tar protein were analyzed using swarm assays, aspartate binding in purified membranes, and methylation both in vitro and in vivo. The defects in the responses of these mutant receptors to aspartate were shown to be directly attributable to reduced binding of aspartate to the receptor rather than to defects in their signaling characteristics. Mutations at residues 64, 69, and 73 reduced aspartate binding by factors of greater than 10(-4), 10(-3), and 10(-2), respectively. Once aspartate was bound, the mutants exhibited normal signaling properties. No cooperativity was observed in the coupling of aspartate binding to methylation, indicating that the monomers of the receptor dimer act independently. The in vitro methylation system was thus shown to be an effective way of measuring aspartate binding constants and examining the functional integrity of the proteins. The maltose responses of the receptor proteins were affected slightly, or not at all, in an in vivo methylation assay. Two models for the roles of these arginine residues in receptor function are discussed. 相似文献
7.
Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli. 总被引:5,自引:0,他引:5
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The Tar protein of Escherichia coli is a chemotactic signal transducer that spans the cytoplasmic membrane and mediates responses to the attractants aspartate and maltose. Aspartate binds directly to Tar, whereas maltose binds to the periplasmic maltose-binding protein, which then interacts with Tar. The Arg-64, Arg-69, and Arg-73 residues of Tar have previously been shown to be involved in aspartate sensing. When lysine residues are introduced at these positions by site-directed mutagenesis, aspartate taxis is disrupted most by substitution at position 64, and maltose taxis is disrupted most by substitution at position 73. To explore the spatial distribution of ligand recognition sites on Tar further, we performed doped-primer mutagenesis in selected regions of the tar gene. A number of mutations that interfere specifically with aspartate taxis (Asp-), maltose taxis (Mal-), or both were identified. Mutations affecting residues 64 to 73 or 149 to 154 in the periplasmic domain of Tar are associated with an Asp- phenotype, whereas mutations affecting residues 73 to 83 or 141 to 150 are associated with a Mal- phenotype. We conclude that aspartate and maltose-binding protein interact with adjacent and partially overlapping regions in the periplasmic domain of Tar to initiate attractant signalling. 相似文献
8.
Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers. 相似文献
9.
The Tar protein of Escherichia coli belongs to a family of methyl-accepting inner membrane proteins that mediate chemotactic responses to a variety of compounds. These transmembrane signalers monitor the chemical environment by means of specific ligand-binding sites arrayed on the periplasmic side of the membrane, and in turn control cytoplasmic signals that modulate the flagellar rotational machinery. The periplasmic receptor domain of Tar senses two quite different chemoeffectors, aspartate and maltose. Aspartate is detected through direct binding to Tar molecules, whereas maltose is detected indirectly when complexed with the periplasmic maltose-binding protein. Saturating levels of either aspartate or maltose do not block behavioral responses to the other compound, indicating that the detection sites for these two attractants are not identical. We initiated structure-function studies of these chemoreceptor sites by isolating tar mutants which eliminate aspartate or maltose taxis, while retaining the ability to respond to the other chemoeffector. Mutants with greatly reduced aspartate taxis are described and characterized in this report. When present in single copy in the chromosome, these tar mutations generally eliminated chemotactic responses to aspartate and structurally related compounds, such as glutamate and methionine. Residual responses to these compounds were shifted to higher concentrations, indicating a reduced affinity of the aspartate-binding site in the mutant receptors. Maltose responses in the mutants ranged from 10 to 80% of normal, but had no detectable threshold shifts, indicating that these receptor alterations may have little effect on maltose detection sensitivity. The mutational changes in 17 mutants were determined by DNA sequence analysis. Each mutant exhibited a single amino acid replacement at residue 64, 69, or 73 in the Tar molecule. The wild-type Tar transducer contains arginines at all three of these positions, implying that electrostatic forces may play an important role in aspartate detection. 相似文献
10.
Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions 总被引:1,自引:0,他引:1
Ichiro Tatsuno Lan Lee Ikuro Kawagishi Michio Homma Yasuo Imae 《Molecular microbiology》1994,14(4):755-762
The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming. 相似文献
11.
Chemotactic Adaptation Is Altered by Changes in the Carboxy-Terminal Sequence Conserved among the Major Methyl-Accepting Chemoreceptors 总被引:12,自引:6,他引:6
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Hisashi Okumura So-ichiro Nishiyama Akie Sasaki Michio Homma Ikuro Kawagishi 《Journal of bacteriology》1998,180(7):1862-1868
In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR. 相似文献
12.
CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli
Boukhvalova MS Dahlquist FW Stewart RC 《The Journal of biological chemistry》2002,277(25):22251-22259
The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation. 相似文献
13.
The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediates signaling and adaptation. These two domains communicate through the second transmembrane helix (TM2) that connects them. In the high-abundance receptors Tar and Tsr, TM2 is flanked by tryptophan residues, which should localize preferentially to the interfacial zone between the polar and hydrophobic layers of the phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 of Tar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192A protein retains full activity in vivo and in vitro, but it increases the K(i) for aspartate in the in vitro assay 3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leads to an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changes seen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrude farther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similar displacement. Moving Trp to each position from residue 206 to residue 212 generated a wide variety of Tar signaling states that are generally consistent with the predictions of the piston model of transmembrane signaling. None of these receptors was completely locked in one signaling mode, although most showed pronounced signaling biases. Our findings suggest that the Trp residues flanking TM2, especially Trp-209, are important in setting the baseline activity and ligand sensitivity of the Tar receptor. We also conclude that the Tyr-210 residue plays at least an auxiliary role in this control. 相似文献
14.
The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology. 相似文献
15.
Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar. 相似文献
16.
《Journal of molecular biology》2021,433(15):167036
Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand–protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues. 相似文献
17.
Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form. 相似文献
18.
The remarkably wide dynamic range of the chemotactic pathway of Escherichia coli, a model signal transduction system, is achieved by methylation/amidation of the transmembrane chemoreceptors that regulate the histidine kinase CheA in response to extracellular stimuli. The chemoreceptors cluster at a cell pole together with CheA and the adaptor CheW. Several lines of evidence have led to models that assume high cooperativity and sensitivity via collaboration of receptor dimers within a cluster. Here, using in vivo disulfide cross-linking assays, we have demonstrated a well defined arrangement of the aspartate chemoreceptor (Tar). The differential effects of amidation on cross-linking at different positions indicate that amidation alters the relative orientation of Tar dimers to each other (presumably inducing rotational displacements) without much affecting the conformation of the periplasmic domains. Interestingly, the effect of aspartate on cross-linking at any position tested was roughly opposite to that of receptor amidation. Furthermore, amidation attenuated the effects of aspartate by several orders of magnitude. These results suggest that receptor covalent modification controls signal gain by altering the arrangement or packing of receptor dimers in a pre-formed cluster. 相似文献
19.
Tajima H Imada K Sakuma M Hattori F Nara T Kamo N Homma M Kawagishi I 《The Journal of biological chemistry》2011,286(49):42200-42210
Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical. 相似文献
20.
Matthias Habjan Philipp Hubel Livia Lacerda Christian Benda Cathleen Holze Christian H. Eberl Angelika Mann Eveline Kindler Cristina Gil-Cruz John Ziebuhr Volker Thiel Andreas Pichlmair 《PLoS pathogens》2013,9(10)
Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells. 相似文献