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1.
Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes. 相似文献
2.
Manson MD 《Molecular microbiology》2011,80(3):573-576
HAMP domains communicate between input and output signalling modules in a wide variety of bacterial sensor proteins. In the Tsr chemoreceptor, they convert a signal initiated by binding of serine to the periplasmic domain of the protein into regulation of receptor control of the CheA kinase, and ultimately of the direction of flagellar rotation. In this issue, Zhou et al. report an extensive mutational analysis of the Tsr HAMP domain that shows that it can assume a number of different signalling states, which presumably correspond to a variety of different conformations. The two conformational extremes of a tightly packed and a loosely packed HAMP four‐helix bundle support only low levels of CheA activity. Thus, Tsr HAMP does not function as a simple on‐off, two‐state device but rather as a dynamic structure with biphasic control. The normal physiological operating range of Tsr is proposed to be at intermediate degrees of packing of the HAMP four‐helix bundle, but HAMP domains in other proteins could occupy different portions of the conformational spectrum. 相似文献
3.
Cysteine-scanning analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA
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The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes. 相似文献
4.
HAMP domains mediate input–output transactions in many bacterial signalling proteins. To clarify the mechanistic logic of HAMP signalling, we constructed Tsr‐HAMP deletion derivatives and characterized their steady‐state signal outputs and sensory adaptation properties with flagellar rotation and receptor methylation assays. Tsr molecules lacking the entire HAMP domain or just the HAMP‐AS2 helix generated clockwise output signals, confirming that kinase activation is the default output state of the chemoreceptor signalling domain and that attractant stimuli shift HAMP to an overriding kinase‐off signalling state to elicit counter‐clockwise flagellar responses. Receptors with deletions of the AS1 helices, which free the AS2 helices from bundle‐packing constraints, exhibited kinase‐off signalling behaviour that depended on three C‐terminal hydrophobic residues of AS2. We conclude that AS2/AS2′ packing interactions alone can play an important role in controlling output kinase activity. Neither kinase‐on nor kinase‐off HAMP deletion outputs responded to sensory adaptation control, implying that out‐of‐range conformations or bundle‐packing stabilities of their methylation helices prevent substrate recognition by the adaptation enzymes. These observations support the previously proposed biphasic, dynamic‐bundle mechanism of HAMP signalling and additionally show that the structural interplay of helix‐packing interactions between HAMP and the adjoining methylation helices is critical for sensory adaptation control of receptor output. 相似文献
5.
SAXS study of the PIR domain from the Grb14 molecular adaptor: a natively unfolded protein with a transient structure primer?
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Moncoq K Broutin I Craescu CT Vachette P Ducruix A Durand D 《Biophysical journal》2004,87(6):4056-4064
Grb14 belongs to the Grb7 family of adapters and was identified as a negative regulator of insulin signal transduction. Between the PH (pleckstrin homology) and SH2 (Src homology 2) domains is a new binding domain implicated in the interaction with receptor tyrosine kinases called PIR (phosphorylated insulin receptor interaction region). Both PIR and SH2 domains interact with the insulin receptor, but their relative role varies considering the member of the Grb7 family and the tyrosine kinase receptor. In the case of Grb14, PIR is the main binding domain and is sufficient to inhibit the insulin receptor kinase activity. We have proposed, on the basis of NMR measurements, that PIR lacks ordered structure and presents a high flexibility, although remaining fully active. To complement this first study, we have used small-angle x-ray scattering in solution together with a modeling approach representing the PIR domain as a chain of pseudo residues. Circular dichroism experiments were also performed in the presence of variable amounts of trifluoroethanol. These observations, together with an ensemble of sequence analyses and previous NMR results, all support the view of PIR as essentially unstructured but with a potentially structured short stretch encompassing residues 399-407. This stretch, which may be only structured transiently in the isolated molecule, could play a major role in Grb14 PIR binding to a biological partner by undergoing a structural transition. 相似文献
6.
Poly-HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer-2, has an unusual domain architecture consisting of a PAS-sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains, followed by a conserved kinase control module. The structure of the N-terminal HAMP domains was recently solved, making Aer-2 the first protein with resolved poly-HAMP structure. The role of Aer-2 in P. aeruginosa is unclear, but here we show that Aer-2 can interact with the chemotaxis system of Escherichia coli to mediate repellent responses to oxygen, carbon monoxide and nitric oxide. Using this model system to investigate signalling and poly-HAMP function, we determined that the Aer-2 PAS domain binds penta-co-ordinated b-type haem and that reversible signalling requires four of the five HAMP domains. Deleting HAMP 2 and/or 3 resulted in a kinase-off phenotype, whereas deleting HAMP 4 and/or 5 resulted in a kinase-on phenotype. Overall, these data support a model in which ligand-bound Aer-2 PAS and HAMP 2 and 3 act together to relieve inhibition of the kinase control module by HAMP 4 and 5, resulting in the kinase-on state of the Aer-2 receptor. 相似文献
7.
The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the β-strand 8 of domain 1 and the β-strands 1 and 3 of domain 2. The receptor-CheW complex shares a similar binding interface to that found in the "trimer-of-dimers" oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor-CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW. 相似文献
8.
Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors. 总被引:12,自引:4,他引:8
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The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing. 相似文献
9.
Membrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligand-binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX-Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand-dependent activation of a sensor kinase and repellent-initiated activation of receptor-coupled CheA kinase involve a similar transmembrane signal. 相似文献
10.
Src homology 2 (SH2) domains are protein modules (of approximately 100 amino acids) found in many proteins involved in tyrosine kinase signalling cascades. Their function is to bind tyrosine-phosphorylated sequences in specific protein targets. Binding of an SH2 domain to its cognate tyrosine-phosphorylated target links receptor activation to downstream signalling, both to the nucleus to regulate gene expression and throughout the cytoplasm of the cell. This review recapitulates the roles that SH2 domains play in normal and diseased states, describes the successes of SH2 domain research in deciphering their mechanism of action, and provides an overview of the use of SH2 domains as structural templates for the design of inhibitor drugs. 相似文献
11.
Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems. 相似文献
12.
Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. 总被引:6,自引:1,他引:5
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J W Baumgartner C Kim R E Brissette M Inouye C Park G L Hazelbauer 《Journal of bacteriology》1994,176(4):1157-1163
Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition. 相似文献
13.
Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA
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During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA. 相似文献
14.
HAMP domains mediate input-output communication in many bacterial signalling proteins. To explore the dynamic bundle model of HAMP signalling (Zhou et al., Mol. Microbiol. 73: 801, 2009), we characterized the signal outputs of 118 HAMP missense mutants of the serine chemoreceptor, Tsr, by flagellar rotation patterns. Receptors with proline or charged amino acid replacements at critical hydrophobic packing residues in the AS1 and AS2 HAMP helices had locked kinase-off outputs, indicating that drastic destabilization of the Tsr-HAMP bundle prevents kinase activation, both in the absence and presence of the sensory adaptation enzymes, CheB and CheR. Attractant-mimic lesions that enhance the structural stability of the HAMP bundle also suppressed kinase activity, demonstrating that Tsr-HAMP has two kinase-off output states at opposite extremes of its stability range. HAMP mutants with locked-on kinase outputs appeared to have intermediate bundle stabilities, implying a biphasic relationship between HAMP stability and kinase activity. Some Tsr-HAMP mutant receptors exhibited reversed output responses to CheB and CheR action that are readily explained by a biphasic control logic. The findings of this study provide strong support for a three-state dynamic bundle model of HAMP signalling in Tsr, and possibly in other bacterial transducers as well. 相似文献
15.
Madeline Hayes Sebastian Guettler Ian Clarke Frank Sicheri Peter Dirks Brian Ciruna Daniela Rotin 《The EMBO journal》2011,30(16):3259-3273
Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4‐1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4‐1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non‐canonical sequence (non‐PY motif) on FGFR1. Deletion of this recognition motif (FGFR1‐Δ6) abolishes Nedd4‐1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4‐1 knockdown. Accordingly, FGFR1‐Δ6, or Nedd4‐1 knockdown, exhibits sustained FGF‐dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1‐Δ6 in human embryonic neural stem cells strongly promotes FGF2‐dependent neuronal differentiation. Furthermore, expression of this FGFR1‐Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4‐1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development. 相似文献
16.
George D. Glekas Richard M. Foster Joseph R. Cates Jeffrey A. Estrella Michael J. Wawrzyniak Christopher V. Rao George W. Ordal 《The Journal of biological chemistry》2010,285(3):1870-1878
During chemotaxis toward asparagine by Bacillus subtilis, the ligand is thought to bind to the chemoreceptor McpB on the exterior of the cell and induce a conformational change. This change affects the degree of phosphorylation of the CheA kinase bound to the cytoplasmic region of the receptor. Until recently, the sensing domains of the B. subtilis receptors were thought to be structurally similar to the well studied Escherichia coli four-helical bundle. However, sequence analysis has shown the sensing domains of receptors from these two organisms to be vastly different. Homology modeling of the sensing domain of the B. subtilis asparagine receptor McpB revealed two tandem PAS domains. McpB mutants having alanine substitutions in key arginine and tyrosine residues of the upper PAS domain but not in any residues of the lower PAS domain exhibited a chemotactic defect in both swarm plates and capillary assays. Thus, binding does not appear to occur across any dimeric surface but within a monomer. A modified capillary assay designed to determine the concentration of attractant where chemotaxis is most sensitive showed that when Arg-111, Tyr-121, or Tyr-133 is mutated to an alanine, much more asparagine is required to obtain an active chemoreceptor. Isothermal titration calorimetry experiments on the purified sensing domain showed a KD to asparagine of 14 μm, with the three mutations leading to less efficient binding. Taken together, these results reveal not only a novel chemoreceptor sensing domain architecture but also, possibly, a different mechanism for chemoreceptor activation. 相似文献
17.
F Kohlhuber N C Rogers D Watling J Feng D Guschin J Briscoe B A Witthuhn S V Kotenko S Pestka G R Stark J N Ihle I M Kerr 《Molecular and cellular biology》1997,17(2):695-706
Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains. 相似文献
18.
Takayama Y Nakasako M Okajima K Iwata A Kashojiya S Matsui Y Tokutomi S 《Biochemistry》2011,50(7):1174-1183
Phototropin, a blue-light receptor protein of plants, triggers phototropic responses, chloroplast relocation, and opening of stomata to maximize the efficiency of photosynthesis. Phototropin is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) that absorb blue light and a serine/theroine kinase domain responsible for light-dependent autophosphorylation leading to cellular signaling cascades. Although the light-activated LOV2 domain is primarily responsible for subsequent activation of the kinase domain, it is unclear how conformational changes in the former transmit to the latter. To understand this molecular mechanism in Arabidopsis phototropin 2, we performed small-angle X-ray scattering analysis on a fragment composed of the LOV2 and kinase domains, which contained an Asp720Asn mutation that led to an absence of ATP binding activity. The scattering data were collected up to a resolution of 25 ?. The apparent molecular weight of the fragment estimated from scattering intensities demonstrated that the fragment existed in a monomeric form in solution. The fragment exhibited photoreversible changes in the scattering profiles, and the radii of gyration under dark and blue-light irradiation conditions were 32.4 and 34.8 ?, respectively. In the dark, the molecular shape restored from the scattering profile appeared as an elongated shape of 110 ? in length and 45 ? in width. The homology modeled LOV2 and kinase domains could be fitted to the molecular shape and appeared to make slight contact. However, under blue-light irradiation, a more extended molecular shape was observed. The changes in the molecular shape and radius of gyration were interpreted as a light-dependent positional shift of the LOV2 domain of approximately 13 ? from the kinase domain. Because the region connecting the LOV2 and kinase domains was categorized as a naturally unfolded polypeptide, we propose that the light-activated LOV2 domain triggers conformational changes in the linker region to separate the LOV2 and kinase domains. 相似文献
19.
G Magistrelli R Bosotti B Valsasina C Visco R Perego S Toma O Acuto A Isacchi 《European journal of biochemistry》1999,266(3):1166-1173
The protein tyrosine kinase ZAP-70, which mediates T-cell antigen receptor (TCR) signalling, contains three distinct functional modules, two tandemly arranged SH2 domains, a kinase domain and a linker region (interdomain B) that connects them. ZAP-70 enzymatic activation is strictly dependent on the binding, via its SH2 domains, to the triggered TCR and on tyrosine phosphorylation. Here we utilized recombinant ZAP-70 and carried out a mutational analysis to understand the structural requirements for its activation. We show that deletion of both SH2 domains corresponding to the first 254 residues moderately increases ZAP-70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP-70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320-619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP-70 to the TCR promotes, through conformational changes, its extensive phosphorylation on tyrosine. However, Tyr292, 315 and 319 do not affect ZAP-70 enzymatic activity and may influence ZAP-70 signalling only indirectly by mediating its association with intracellular transducers. 相似文献
20.
Fedoroff OY Townson SA Golovanov AP Baron M Avis JM 《The Journal of biological chemistry》2004,279(33):34991-35000
WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms. 相似文献