Structure/function studies of S100A8/A9

The functional importance of members of the S100 Ca²⁺-binding protein family is recently emerging. A variety of activities, several of which are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10^-13–10^-11 M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.     

Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion

Motile cells can use and switch between different modes of migration. Here, we use traction force microscopy and fluorescent labeling of actin and myosin to quantify and correlate traction force patterns and cytoskeletal distributions in Dictyostelium discoideum cells that move and switch between keratocyte‐like fan‐shaped, oscillatory, and amoeboid modes. We find that the wave dynamics of the cytoskeletal components critically determine the traction force pattern, cell morphology, and migration mode. Furthermore, we find that fan‐shaped cells can exhibit two different propulsion mechanisms, each with a distinct traction force pattern. Finally, the traction force patterns can be recapitulated using a computational model, which uses the experimentally determined spatiotemporal distributions of actin and myosin forces and a viscous cytoskeletal network. Our results suggest that cell motion can be generated by friction between the flow of this network and the substrate.     

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.     

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.     

An allosteric model for transmembrane signaling in bacterial chemotaxis

Bacteria are able to sense chemical gradients over a wide range of concentrations. However, calculations based on the known number of receptors do not predict such a range unless receptors interact with one another in a cooperative manner. A number of recent experiments support the notion that this remarkable sensitivity in chemotaxis is mediated by localized interactions or crosstalk between neighboring receptors. A number of simple, elegant models have proposed mechanisms for signal integration within receptor clusters. What is a lacking is a model, based on known molecular mechanisms and our accumulated knowledge of chemotaxis, that integrates data from multiple, heterogeneous sources. To address this question, we propose an allosteric mechanism for transmembrane signaling in bacterial chemotaxis based on the "trimer of dimers" model, where three receptor dimers form a stable complex with CheW and CheA. The mechanism is used to integrate a diverse set of experimental data in a consistent framework. The main predictions are: (1) trimers of receptor dimers form the building blocks for the signaling complexes; (2) receptor methylation increases the stability of the active state and retards the inhibition arising from ligand-bound receptors within the signaling complex; (3) trimer of dimer receptor complexes aggregate into clusters through their mutual interactions with CheA and CheW; (4) cooperativity arises from neighboring interaction within these clusters; and (5) cluster size is determined by the concentration of receptors, CheA, and CheW. The model is able to explain a number of seemingly contradictory experiments in a consistent manner and, in the process, explain how bacteria are able to sense chemical gradients over a wide range of concentrations by demonstrating how signals are integrated within the signaling complex.     

Necessary conditions for a minimal model of receptor to show adaptive response over a wide range of levels of stimulus

Sensory systems respond to temporal changes in the stimulus and adapt to the new level when it persists, this pattern of response being maintained in a wide range of levels of stimulus. Here we use a simple model of adaptation developed by Segel et al. (J. Theor. Biol. 120 (1986) 151-179) and extended by Hauri and Ross (Biophys. J. 68 (1995) 708-722) to study the conditions in which it shows wide range of response. The model consists of a receptor that switches between a variable number of states, either by mass action law or by covalent modification. Using a global optimization procedure, we have optimized the adaptive response of the alternatives of the model with different number of states. We find that it is impossible to obtain a wide range of response if the receptor switches between states following mass-action laws, irrespective of the number of states. Instead, a wide range (of five orders of magnitude of ligand concentration) can be obtained if the receptor switches between several states by irreversible covalent modification, in agreement with previous models. Therefore, in this model, expenditure of energy to maintain a large number of covalent modification cycles operating outside equilibrium is necessary to achieve a wide range of response. The optimal values of the parameters present similar patterns to those reported for specific receptors, but there is no quantitative agreement. For instance, ligand affinity varies several orders of magnitude between the different states of the receptor, what is unlikely to be fulfilled by real systems. To see if the minimal model can show adaptive response and range with quantitatively plausible parameter values a sub-optimal receptor was studied, finding that adaptive response of high intensity can still be obtained in at least three orders of magnitude.     

Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.     

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.     

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells. (Mol Cell Biochem 271: 29–41, 2005)