"Frozen" dynamic dimer model for transmembrane signaling in bacterial chemotaxis receptors.

The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor.     

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.     

Engineered single‐ and multi‐cell chemotaxis pathways in E. coli

We have engineered the chemotaxis system of Escherichia coli to respond to molecules that are not attractants for wild‐type cells. The system depends on an artificially introduced enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor, thereby enabling the cells to respond to the new attractant. Two systems were designed, and both showed robust chemotactic responses in semisolid and liquid media. The first incorporates an asparaginase enzyme and the native E. coli aspartate receptor to produce a response to asparagine; the second uses penicillin acylase and an engineered chemoreceptor for phenylacetic acid to produce a response to phenylacetyl glycine. In addition, by taking advantage of a ‘hitchhiker’ effect in which cells producing the ligand can induce chemotaxis of neighboring cells lacking enzymatic activity, we were able to design a more complex system that functions as a simple microbial consortium. The result effectively introduces a logical ‘AND’ into the system so that the population only swims towards the combined gradients of two attractants.     

Signal transduction in neutrophil chemotaxis

This review discusses current knowledge on signal transduction pathways controlling chemotaxis of neutrophils and similar cells. Most neutrophil chemoattractants bind to seven-transmembrane-helix receptors. These receptors activate trimeric G proteins of the Gi class in neutrophils to initiate chemotaxis. Phospholipases Cbeta, phosphoinositide 3-kinase gamma, and PH domain-containing proteins play various roles in signaling further downstream. The actin cytoskeleton is crucial for cell motility, and is controlled by Rho family GTP-binding proteins. PIP 5-kinase, LIM kinase, myosin light chain kinase and phosphatase, or WASP-like proteins may be important links between Rho GTPases and actin during chemotaxis. Newly emerging ideas on the regulation of the "compass" of chemotaxing cells, which may involve Cdc42 and certain PH domain-containing proteins, are also presented.     

Stochastic control of spontaneous signal generation for gradient sensing in chemotaxis

Chemotaxis is characterized by spontaneous cellular behavior. This spontaneity results, in part, from the stochasticity of intracellular reactions. Spontaneous and random migration of chemotactic cells is regulated by spontaneously generated signals, namely transient local increases in the level of phosphoinositol-3,4,5-triphosphate (PIP3 pulses). In this study, we attempted to elucidate the mechanisms that generate these PIP3 pulses and how the pulses contribute to gradient sensing during chemotaxis. To this end, we constructed a simple biophysical model of intracellular signal transduction consisting of an inositol phospholipid signaling pathway and small GTPases. Our theoretical analysis revealed that an excitable system can emerge from the non-linear dynamics of the model, and that stochastic reactions allow the system to spontaneously become excited, which was corresponded to the PIP3 pulses. Based on these results, we framed a hypothesis of the gradient sensing; a chemical gradient spatially modifies a potential barrier for excitation and then PIP3 pulses are preferentially generated on the side of the cell exposed to the higher chemical concentration. We then validated our hypothesis using stochastic simulations of the signal transduction.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Signal transduction in chemotaxis mediated by the bacterial phosphotransferase system

Gram-negative bacteria are able to respond chemotactically to carbohydrates which are substrates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mechanism of signal transduction in PTS-mediated chemotaxis is different from the well-studied mechanism involving methyl-accepting chemotaxis proteins (MCPs). In PTS-mediated chemotaxis, carbohydrate transport is required, and phosphorylation seems to be involved in both excitation and adaptation. In this review the roles of the components of the PTS in chemotactic signal transduction are discussed. © 1993 Wiley-Liss, Inc.     

The physics of flagellar motion of E. coli during chemotaxis

Flagellar motion has been an active area of study right from the discovery of bacterial chemotaxis in 1882. During chemotaxis, E. coli moves with the help of helical flagella in an aquatic environment. Helical flagella are rotated in clockwise or counterclockwise direction using reversible flagellar motors situated at the base of each flagellum. The swimming of E. coli is characterized by a low Reynolds number that is unique and time reversible. The random motion of E. coli is influenced by the viscosity of the fluid and the Brownian motion of molecules of fluid, chemoattractants, and chemorepellants. This paper reviews the literature about the physics involved in the propulsion mechanism of E. coli. Starting from the resistive-force theory, various theories on flagellar hydrodynamics are critically reviewed. Expressions for drag force, elastic force and velocity of flagellar elements are derived. By taking the elastic nature of flagella into account, linear and nonlinear equations of motions are derived and their solutions are presented.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli

Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.     

心脏中不同β肾上腺素受体亚型的信号体系及其病理生理意义

生理情况下,β肾上腺素受体(βAR)对心肌收缩和舒张活动起至关重要的作用;病理情况下,长期激动βAR可以诱发心肌细胞肥大、凋亡以及细胞坏死等心肌重塑性活动,从而参与了慢性心衰的发病过程。近十年以来,许多资料表明β1和β2肾上腺素受体亚型(β1AR和β2AR)共存于心脏中,且激动不同信号系统。短时间激动β1AR,使Gs蛋白-腺苷酸环化酶-环苷腺酸-蛋白激酶A(Gs-adenyly cyclase-cAMP-PKA)信号体系激活并广布于细胞内,而激动βAR则同时激活G1蛋白而产生空间及功能局限的cAMP信号;长时间激动β1AR和β2AR则对心肌细胞的命运产生不同影响：β1AR诱导细胞肥大和凋亡,β2AR促使细胞存活。β2AR的心肌保护作用是通过激活Gi蛋白-Gβγ-PI3K-Akt途径介导。但出乎意料,β1AR的心肌肥厚和凋亡效应并不依赖于经典的cAMP/PKA信号途径,而是激活钙,钙调素依赖性蛋白激酶Ⅱ(caMK Ⅱ)途径。用心肌特异性表达βAR亚型的转基因小鼠进行实验,进一步证实不同βAR亚型在调节心肌重塑和功能方面作用各异。βAR亚型作用不同的新观点不仅为β阻滞剂治疗慢性心衰提供了分子和细胞机制的依据,而且提出了选择性β1AR阻滞和β2AR激动联合治疗慢性心衰的新的治疗思路。     

Co-regulation of acetylation and phosphorylation of CheY, a response regulator in chemotaxis of Escherichia coli

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.     

Effects of the T→R transition on the electrostatic properties of E. coli aspartate transcarbamylase

Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind. The forces that drive this transition are poorly understood. We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs. The results have been compared with calculations for the unliganded protein. While pK_½ values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK_½ values that fall outside the range 0–17. Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops. The pK_½ values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal. As in the unliganded structure, a set of residues forms a network in which ionizable groups with W_ij values greater than 2 kcal-m^-1 are separated by distances greater than 5 Å. Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure. The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule. Proteins 32:200–210, 1998. © 1998 Wiley-Liss, Inc.     

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

Role of hydroxyl containing residues in the intracellular region of rat bradykinin B(2) receptor in signal transduction, receptor internalization, and resensitization

In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.     

Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm

The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63[emsp4 ]kDa GPI-anchored protein and the subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract–speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.     

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.