运动细胞自适应性过程中双相性现象的分子机制及动态数值模拟

盘基网柄菌细胞(Dictyostelium)和白细胞(leukocyte)等真核运动细胞受到外界信号刺激时,在最初的1～2 min内,胞内信号转导的首要成员PI(3,4,5)P3的浓度随时间变化呈现"双相性"(biphasic adaptation),即先后出现一大一小两个峰值,然后平息。为解释这一现象,特别是第二个峰值产生的原因,根据已有实验资料,分析了有关分子机制,建立了相应的数学模型。其中,PI(3,4,5)P3及其激活酶和抑制酶的浓度变化由一组耦合的非稳态反应-扩散方程描述,外界刺激及效应因子(如Rac和Scar/WAVE)的相互激励包含在源项中,并由蒙特-卡诺(Monte-Carlo)法处理,数值模拟结果与已有实验一致。研究发现,质膜上处于激活态的效应因子Scar/WAVE是影响PI(3,4,5)P3第二个峰值的关键,起正反馈作用。在受到胞外信号刺激后的前期,Scar/WAVE的激活态浓度受到小G蛋白Rac活性的抑制,后期反过来受到PI(3,4,5)P3的抑制,从而始终处于较低水平,这使得第二个峰值较小;当Scar/WAVE的总浓度低于0.005μmol/L后,PI(3,4,5)P3不会出现第二个峰值。由于Scar/WAVE是肌动蛋白结合蛋白,可以推测:许多经肌动蛋白合成抑制剂处理过的盘基网柄菌细胞在实验中仍然出现"双相性",应与此时的细胞骨架活性未被完全抑制有关。     

Druggability Simulations and X-Ray Crystallography Reveal a Ligand-Binding Site in the GluA3 AMPA Receptor N-Terminal Domain

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Correlation of Local Changes in the Temperature-Dependent Conformational Flexibility of Thioredoxins with Their Thermostability

For the development of a method capable of predicting single point mutations substantially affecting protein thermostability, we studied the effect of the E85R and R82E mutations on the thermostability of thioredoxins from Escherichia coli (Trx) andBacillus acidocaldarius (BacTrx), respectively. The basic method of investigation was the molecular dynamics simulation of 3D protein models in an explicit solvent at different temperatures (300 and 373 K). Some thermolabile regions in Trx, BacTrx, and their mutants were revealed by analyzing the temperature effect on the molecular dynamics of the protein molecule. The effect of single point mutations on the temperature changes of the protein conformation flexibility in several thermolabile regions was found. The results of the simulations are in accord with experimental data indicating that the mutation E85R increases Trx thermostability, whereas the mutation R82E decreases BacTrx thermostability. The thermostability of these proteins was revealed to depend on ionic interactions between the thermolabile regions. The single point mutations change the parameters of these interactions and make them more favorable in the E85R-Trx mutant and less favorable in the R82E-BacTrx mutant.     

Molecular Dynamics Study of the Structure,Flexibility and Dynamics of Thermostable L1 Lipase at High Temperatures

Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn²⁺-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn²⁺-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control

The PhoQ/PhoP two-component signal transduction system is conserved in various Gram-negative bacteria and is often involved in the expression of virulence in pathogens. The small inner membrane protein SafA activates PhoQ in Escherichia coli independently from other known signals that control PhoQ activity. We have previously shown that SafA directly interacts with the sensor domain of the periplasmic region of PhoQ (PhoQ-SD) for activation, and that a D179R mutation in PhoQ-SD attenuates PhoQ activation by SafA. In this study, structural comparison of wild-type PhoQ-SD and D179R revealed a difference in the cavity (SD (sensory domain) pocket) found in the central core of this domain. This was the only structural difference between the two proteins. Site-directed mutagenesis of the residues surrounding the SD pocket has supported the SD pocket as a site involved in PhoQ activity. Furthermore, the SD pocket has also been shown to be involved in SafA-mediated PhoQ control.     

Conformational Plasticity of the Immunoglobulin Fc Domain in Solution

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赤霉素信号转导的分子生物学研究进展

近年来在拟南芥、水稻等模式植物中对赤霉素信号转导途径进行了广泛的研究,主要是通过对赤霉素相关突变体的生理及分子生物学研究,鉴定出一些介入赤霉素信号转导途径的重要基因,并根据相应蛋白的特征结构域,推导了它们的功能．利用双突变体分析相关基因,提出了赤霉素信号转导途径的基本机理,但植物激素的网状交互调控机制仍需进一步研究．     

Molecular modelling of the mass density of single proteins

Using molecular dynamics (MD) simulations, the density of single proteins and its temperature dependence was modelled starting from the experimentally determined protein structure and a generic, transferable force field, without the need of prior parameterization. Although all proteins consist of the same 20 amino acids, their density in aqueous solution varies up to 10% and the thermal expansion coefficient up to twofold. To model the protein density, systematic MD simulations were carried out for 10 proteins with a broad range of densities (1.32–1.43?g/cm³) and molecular weights (7–97?kDa). The simulated densities deviated by less than 1.4% from their experimental values that were available for four proteins. Further analyses of protein density showed that it can be essentially described as a consequence of amino acid composition. For five proteins, the density was simulated at different temperatures. The simulated thermal expansion coefficients ranged between 4.3 and 7.1?×?10^?4?K^?1 and were similar to the experimentally determined values of ribonuclease-A and lysozyme (deviations of 2.4 and 14.6%, respectively). Further analyses indicated that the thermal expansion coefficient is linked to the temperature dependence of atomic fluctuations: proteins with a high thermal expansion coefficient show a low increase in flexibility at increasing temperature. A low increase in atomic fluctuations with temperature has been previously described as a possible mechanism of thermostability. Thus, a high thermal expansion coefficient might contribute to protein thermostability.     

A Refined Open State of the Glycine Receptor Obtained via Molecular Dynamics Simulations

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Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr⁹²⁵ facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr⁹²⁵ phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr⁸⁶¹, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser⁹¹⁰ by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.     

Molecular Dynamics Simulations of the Bacterial UraA H+-Uracil Symporter in Lipid Bilayers Reveal a Closed State and a Selective Interaction with Cardiolipin

The Escherichia coli UraA H⁺-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations. Simulations were performed in the absence of uracil, and resulted in a closed state of the transporter, due to relative movement of the gate and core domains. Anionic lipids, including POPG and especially CL, were found to associate with UraA, involving interactions between specific basic residues in loop regions and phosphate oxygens of the CL head group. In particular, three CL binding sites were identified on UraA: two in the inner leaflet and a single site in the outer leaflet. Mutation of basic residues in the binding sites resulted in the loss of CL binding in the simulations. CL may play a role as a “proton trap” that channels protons to and from this transporter within CL-enriched areas of the inner bacterial membrane.     

The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.     

Mutational Analysis of the Histidine-containing Phosphotransfer (HPt) Signaling Domain of the ArcB Sensor in Escherichia coli

The Escherichia coli ArcB sensor is involved in anaerobic phosphotransfer signal transduction. ArcB is a hybrid sensor that contains three types of phosphotransfer signaling domains in its primary amino acid sequence, namely, transmitter (or His-kinase), receiver, and histidine-containing phosphotransfer (HPt) domains. However, examination of the function of the newly-discovered HPt domain (named ArcB^c) is still at a very early stage. To gain a general insight into the structure and function of the widespread HPt domains, on the basis of its three-dimensional crystal structure, in this study we constructed a certain set of mutants each having a single amino acid substitution in the HPt domain of ArcB. These ArcB^c mutants were characterized and evaluated, based on the in vivo ability to signal the OmpR receiver via trans-phosphorylation.     

Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance‐dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation‐induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation‐dependent and five stimulation‐independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation‐induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.     

Molecular dynamics simulations of trehalose as a 'dynamic reducer' for solvent water molecules in the hydration shell

Systematic computational work for a series of 13 disaccharides was performed to provide an atomic-level insight of unique biochemical role of the alpha,alpha-(1-->1)-linked glucopyranoside dimer over the other glycosidically linked sugars. Superior osmotic and cryoprotective abilities of trehalose were explained on the basis of conformational and hydration characteristics of the trehalose molecule. Analyses of the hydration number and radial distribution function of solvent water molecules showed that there was very little hydration adjacent to the glycosidic oxygen of trehalose and that the dynamic conformation of trehalose was less flexible than any of the other sugars due to this anisotropic hydration. The remarkable conformational rigidity that allowed trehalose to act as a sugar template was required for stable interactions with hydrogen-bonded water molecules. Trehalose made an average of 2.8 long-lived hydrogen bonds per each MD step, which was much larger than the average of 2.1 for the other sugars. The stable hydrogen-bond network is derived from the formation of long-lived water bridges at the expense of decreasing the dynamics of the water molecules. Evidence for this dynamic reduction of water by trehalose was also established based on each of the lowest translational diffusion coefficients and the lowest intermolecular coulombic energy of the water molecules around trehalose. Overall results indicate that trehalose functions as a 'dynamic reducer' for solvent water molecules based on its anisotropic hydration and conformational rigidity, suggesting that macroscopic solvent properties could be modulated by changes in the type of glycosidic linkages in sugar molecules.     

Characterization and Molecular Genetic Complementation of Mutants Affecting Dimorphism in the FungusUstilago maydis

Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1⁻background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Conformational Flexibility of the Peptide Hormone Ghrelin in Solution and Lipid Membrane Bound: A Molecular Dynamics Study

Abstract

Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short α-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.     

类受体激酶FER调节植物与病原菌相互作用的分子机制

植物细胞依赖细胞质膜上的受体感知并传递环境信号,而受体通过与配体特异结合启动一系列下游信号转导途径,维持植物正常的生命活动及其对外界环境变化的适应。类受体激酶是其中一类重要受体,通常由胞外结合结构域、跨膜结构域和胞内激酶结构域3部分组成,是植物适应外界环境变化的重要调节枢纽。FER属于CrRLK1L类受体蛋白激酶家族,...     

Molecular cloning and tissue-specific expression of the mouse homologue of the rat brain 14-3-3 θ protein: Characterization of its cellular and developmental pattern of expression in the male germ line

The highly conserved 14-3-3 family of proteins, originally reported as brain-specific and then found in various somatic cells and oocytes, interacts with several important signal transduction kinases so that actually the 14-3-3 proteins are considered as modulators of multiple signal transduction pathways. Here we show that a 14-3-3 protein is also expressed in the male germ cells, thus extending the protein cellular distribution to a cell line never reported to express 14-3-3 proteins. Screening of a mouse spermatogenic cells λgt11 cDNA library with affinity-purified polyclonal antibodies to the tyrosine kinase SP42 allowed the isolation of several positive clones. Sequencing of a positive cDNA clone revealed a 735-nucleotide open reading frame encoding a protein of 245 amino acids (27,778 Da). The predicted protein was found to be identical to the most recently discovered 14-3-3 isoform, the θ subtype from a rat brain. Here we demonstrate that 14-3-3 θ mRNA is highly expressed in testis and brain only. Western immunoblot analyses confirm the Northern blot data. Developmental Northern and Western blot analyses are consistent with an expression and translation of the 14-3-3 θ gene throughout spermatogenesis. However, analysis of RNA from purified populations of spermatogenic cells at different developmental stages and immunohistochemistry on adult testis sections reveal that within the testis the 14-3-3 θ gene products are most abundant in meiotic prophase spermatocytes, and, above all, in differentiating spermatids. Both testicular and epididymal spermatozoa are negative. The present study is the first report on the presence and molecular characterization of the 14-3-3 θ gene product in the male germ line. Our observations suggest that this specific member of the 14-3-3 protein family could play distinct modulatory roles in the complex development of the mammalian male germ cell lineage. Mol. Reprod. Dev. 47:370–379, 1997. © 1997 Wiley-Liss, Inc.