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.     

Differences in signalling by directly and indirectly binding ligands in bacterial chemotaxis

In chemotaxis of Escherichia coli and other bacteria, extracellular stimuli are perceived by transmembrane receptors that bind their ligands either directly, or indirectly through periplasmic‐binding proteins (BPs). As BPs are also involved in ligand uptake, they provide a link between chemotaxis and nutrient utilization by cells. However, signalling by indirectly binding ligands remains much less understood than signalling by directly binding ligands. Here, we compared intracellular responses mediated by both types of ligands and developed a new mathematical model for signalling by indirectly binding ligands. We show that indirect binding allows cells to better control sensitivity to specific ligands in response to their nutrient environment and to coordinate chemotaxis with ligand transport, but at the cost of the dynamic range being much narrower than for directly binding ligands. We further demonstrate that signal integration by the chemosensory complexes does not depend on the type of ligand. Overall, our data suggest that the distinction between signalling by directly and indirectly binding ligands is more physiologically important than the traditional distinction between high‐ and low‐abundance receptors.     

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.     

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.     

Spatial organization in bacterial chemotaxis

Spatial organization of signalling is not an exclusive property of eukaryotic cells. Despite the fact that bacterial signalling pathways are generally simpler than those in eukaryotes, there are several well‐documented examples of higher‐order intracellular signalling structures in bacteria. One of the most prominent and best‐characterized structures is formed by proteins that control bacterial chemotaxis. Signals in chemotaxis are processed by ordered arrays, or clusters, of receptors and associated proteins, which amplify and integrate chemotactic stimuli in a highly cooperative manner. Receptor clusters further serve to scaffold protein interactions, enhancing the efficiency and specificity of the pathway reactions and preventing the formation of signalling gradients through the cell body. Moreover, clustering can also ensure spatial separation of multiple chemotaxis systems in one bacterium. Assembly of receptor clusters appears to be a stochastic process, but bacteria evolved mechanisms to ensure optimal cluster distribution along the cell body for partitioning to daughter cells at division.     

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.     

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis‐interactions as well as intercellular trans‐interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand‐induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid–protein interactions important for the spatial organization of signalling at the membrane.     

A spatially extended stochastic model of the bacterial chemotaxis signalling pathway

We have combined two distinct but related stochastic approaches to model the Escherichia coli chemotaxis pathway. Reactions involving cytosolic components of the pathway were assumed to obey the laws of conventional stochastic chemical kinetics, while the clustered membrane receptors were represented in two-dimensional arrays similar to the Ising model. Receptors were assumed to flip between an active and an inactive state with probabilities dependent upon three energy inputs: ligand binding, methylation level due to adaptation, and the activity of neighbouring receptors. Examination of models with different lattice size and geometry showed that the sensitivity to stimuli increases with lattice size and the nearest-neighbour coupling strength up to a critical point, but this amplification was also accompanied by a proportional increase in steady-state noise. Multiple methylation of receptors resulted in diminished signal-to-noise ratio, but showed improved stability to variation in the coupling strength and increased gain. Under the best conditions the simulated output of a coupled lattice of receptors closely matched the time-course and amplitude found experimentally in living bacteria. The model also has some of the properties of a cellular automaton and shows an unexpected emergence of spatial patterns of methylation within the receptor lattice.     

Antigen‐responsive regulation of Cell motility and migration via the signalobodies based on c‐Fms and c‐Mpl

Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single‐chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen‐dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro‐B Ba/F3 cells expressing either a scFv‐Fms or scFv‐Mpl signalobody, and compared their migratory characteristics. We employed a lipid–polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen‐concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv‐Fms underwent directional migration toward a positive antigen‐concentration gradient. Taken together, we successfully demonstrated antigen‐responsive regulation of cell motility and migration via the signalobodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:411–417, 2014     

A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting

Remodeling of the actin cytoskeleton is controlled by signaling pathways that include the Wiskott-Aldrich syndrome protein (WASP). WASP is regulated by autoinhibition, and the intramolecular contacts that inactivate the protein can be relieved through binding to the Rho-family GTPase Cdc42. Here, we show that the allosteric regulation of WASP can be quantitatively described by a two-state equilibrium between an active, largely unfolded conformation that is able to stimulate the Arp2/3 complex, and an inactive, folded conformation. The model is parameterized by the stability of WASP against unfolding and by the Cdc42 affinities of WASP constructs that mimic the unfolded and folded conformations. The model is consistent with NMR spectra of GTPase-bound WASP, and accurately predicts changes of amide hydrogen exchange behavior and Cdc42 affinity as a function of WASP stability. The results provide a thermodynamic rationale for the GTPase-independent recruitment of WASP and other autoinhibited effectors to their sites of activity. They also explain how basal activity is suppressed and confirm that WASP needs to integrate multiple cooperative inputs for maximal activation. Our analysis suggests that, in general, simple modulation of a two-state equilibrium may determine several regulatory functions, allowing the generation of complex signaling behavior in vivo.     

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm.     

Lattice-Boltzmann model for bacterial chemotaxis

We present a new numerical approach for modeling bacterial chemotaxis and the fate and transport of a chemoattractant in bulk liquids. This Lattice-Boltzmann method represents the microorganisms and the chemoattractant by quasi-particles that move, collide, and react with each other on a two-dimensional numerical lattice. We use the model to simulate traveling bands of bacteria along self-generated gradients in substrate concentration in bulk liquids. Particularly, we simulate Pseudomonas putida that respond chemotactically to naphthalene dissolved in water. We find that only a fraction of a bacterial slug injected into a domain containing the chemoattractant at constant concentration forms a traveling band as the slug length exceeds a critical value. An expanding bacterial ring forms as one injects a droplet of bacteria into a two-dimensional domain.     

A theoretical model for the association of amphiphilic transmembrane peptides in lipid bilayers

A theoretical model is proposed for the association of trans-bilayer peptides in lipid bilayers. The model is based on a lattice model for the pure lipid bilayer, which accounts accurately for the most important conformational states of the lipids and their mutual interactions and statistics. Within the lattice formulation the bilayer is formed by two independent monolayers, each represented by a triangular lattice, on which sites the lipid chains are arrayed. The peptides are represented by regular objects, with no internal flexibility, and with a projected area on the bilayer plane corresponding to a hexagon with seven lattice sites. In addition, it is assumed that each peptide surface at the interface with the lipid chains is partially hydrophilic, and therefore interacts with the surrounding lipid matrix via selective anisotropic forces. The peptides would therefore assemble in order to shield their hydrophilic residues from the hydrophobic surroundings. The model describes the self-association of peptides in lipid bilayers via lateral and rotational diffusion, anisotropic lipid-peptide interactions, and peptide-peptide interactions involving the peptide hydrophilic regions. The intent of this model study is to analyse the conditions under which the association of trans-bilayer and partially hydrophilic peptides (or their dispersion in the lipid matrix) is lipid-mediated, and to what extent it is induced by direct interactions between the hydrophilic regions of the peptides. The model properties are calculated by a Monte Carlo computer simulation technique within the canonical ensemble. The results from the model study indicate that direct interactions between the hydrophilic regions of the peptides are necessary to induce peptide association in the lipid bilayer in the fluid phase. Furthermore, peptides within each aggregate are oriented in such a way as to shield their hydrophilic regions from the hydrophobic environment. The average number of peptides present in the aggregates formed depends on the degree of mismatch between the peptide hydrophobic length and the lipid bilayer hydrophobic thickness: The lower the degree of mismatch is the higher this number is. Received: 30 December 1996 / Accepted: 9 May 1997     

An algebra of dimerization and its implications for G-protein coupled receptor signaling

Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Formyl peptide chemotaxis receptors on the rat neutrophil: experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[3H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analysis of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P less than .010). In addition, Hill coefficients of 0.80 +/- 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[3H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[3H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone. In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[3H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[3H]Phe receptor/f Met-Leu-[3H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.     

Local control models of cardiac excitation-contraction coupling. A possible role for allosteric interactions between ryanodine receptors

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation-contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation-contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.