Flagellar motor-switch binding face of CheY and the biochemical basis of suppression by CheY mutants that compensate for motor-switch defects in Escherichia coli

CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.     

Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.     

Collagen-induced arthritis in nonhuman primates: multiple epitopes of type II collagen can induce autoimmune-mediated arthritis in outbred cynomolgus monkeys

A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY approximately P) and the switch of the flagellar motor, located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY approximately P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM. CheY approximately P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY approximately P. It also competed with full-length FliM on CheY approximately P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding.     

Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis

CheY is a signal transduction protein of the bacterial chemotaxis system that acts as a molecular switch to alter the swimming behavior of the bacterium. When CheY becomes phosphorylated at Asp57, CheY-Pi interacts with flagellar motor proteins, including FliM, to increase the likelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dephosphorylated (inactive) state has been intensively investigated. The short lifetime ( approximately 20 s) of the aspartyl phosphate has precluded the complete structural determination of CheY-Pi. We have synthesized an analogue of CheY-Pi by alkylating an aspartate-to-cysteine mutant at position 57 of CheY to add a phosphonomethyl group at Cys57. This analogue, phosphono-CheY, is stable for months. Phosphono-CheY binds to two of the targets of CheY-Pi, FliM and CheZ, in a manner similar to that of CheY-Pi and much better than either unphosphorylated CheY or the unmodified form of D57C CheY. Phosphono-CheY also binds Mg(II) with a dissociation constant of approximately 6 mM at neutral pH and moderate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-Pi.     

Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.     

Isolation and in vitro characterization of CheZ suppressors for the Escherichia coli chemotactic response regulator mutant CheYN23D

The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein. We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sann, M.G., Swanson, R.V., Bourret, R.B., and Simon, M.I. (1995) Mol. Microbiol. 15, 1069-79). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.     

Electrostatic interactions between rotor and stator in the bacterial flagellar motor

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.     

Fumarate modulates bacterial flagellar rotation by lowering the free energy difference between the clockwise and counterclockwise states of the motor

Switching flagellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to flagella of cytoplasm-free, CheY-containing bacterial envelopes the ability to switch directions and to increase the probability of reversal in intact cells. Neither the target of fumarate action nor the mechanism of function is known. To distinguish between the two potential targets of fumarate, the response regulator CheY and the flagellar switch-motor complex, we compared flagellar rotation between isogenic strains that lacked CheY and had either low or high levels of fumarate. The difference in the fumarate levels was due to a deletion of the genes encoding the enzymes that synthesize and metabolize fumarate; succinate dehydrogenase and fumarase, respectively. The strains were in a gutted background (i.e. a background deleted for the cytoplasmic chemotaxis proteins and some of the receptors), and switching was achieved by carrying out the measurements at 2.5 degreesC, where it has been demonstrated that gutted cells switch spontaneously. The flagellar rotation of the strain with the highest level of fumarate was the most clockwise-biased and had the highest reversal frequency, indicating that fumarate is effective even in the absence of CheY. Fumarate reduced the free energy difference of the counterclockwise-to-clockwise transition and had no appreciable effect on the activation energy of this transition. Similar observations were made at room temperature, provided that intracellular CheY was present. In a wild-type background, both mutants made rings on semi-solid agar typical of normal chemotaxis. Taken together, the results suggest that the target of fumarate is the switch-motor complex, that fumarate acts by increasing the probability of the clockwise state, and that a fumarate level as low as that found in succinate dehydrogenase mutants is sufficient for normal chemotaxis.     

Kinetic characterization of phosphotransfer between CheA and CheY in the bacterial chemotaxis signal transduction pathway

Phosphorylation of the CheY protein is a crucial step in the chemotaxis signal transduction pathway of Escherichia coli. CheY becomes phosphorylated by acquiring a phosphoryl group from CheA, an autophosphorylating protein kinase. In this study, we utilized a rapid-quench instrument to investigate the kinetics of phosphotransfer in single-turnover experiments. Our results are consistent with a three-step mechanism for the CheA-to-CheY phosphotransfer reaction: (i) reversible binding of CheY to P-CheA; (ii) rapid, reversible phosphotransfer to CheY; (iii) reversible dissociation of the resulting CheA x CheY-P complex. Investigation of the effect of CheY concentration on the observed rate of phosphotransfer demonstrated saturation kinetics; the extrapolated limiting rate constant for phosphotransfer was 650 +/- 200 s(-1), while the Km value indicated from this work was 6.5 +/- 2 microM. We demonstrated that the CheA-CheY phosphotransfer reaction was reversible by observing partial transfer of [32P]phosphate from CheY-P to CheA and by observing the effect of high concentrations of unphosphorylated CheA on the equilibrium: P-CheA + CheY <--> CheA + CheY-P. We found that the rate of phosphotransfer from P-CheA to CheY can be inhibited by unphosphorylated CheA as well as by a fragment of CheA (CheA124-257) that contains the CheY binding site; these results suggest that the unphosphorylated form of CheA can effectively compete with P-CheA for available CheY (Kd approximately 1.5 +/- 0.6 microM for the CheY x CheA124-257 complex and for the CheY x CheA complex).     

Management of parvovirus infection in pregnancy and outcomes of hydrops: a survey of members of the Society of Perinatal Obstetricians

Escherichia coli strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.     

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.     

Phosphotransfer between CheA, CheY1, and CheY2 in the chemotaxis signal transduction chain of Rhizobium meliloti

The soil bacterium Rhizobium meliloti responds to chemotactic stimuli by modulating the rotary speed of its flagella. Unlike in Escherichia coli, the signal transduction chain of R. meliloti contains two different response regulators, CheY1 and CheY2, but no CheZ phosphatase. Phosphorylation of CheY1 and CheY2 by the central ATP-dependent autokinase, CheA, is the crucial step in signal transduction. In vivo, phospho-CheY2 (CheY2-P) is the chief regulator of flagellar rotation, its action being modulated by CheY1 [Sourjik, V., and Schmitt, R. (1996) Mol. Microbiol. 22, 427-436]. In this study, we have investigated these phosphotransfer reactions in vitro using the radiolabeled recombinant proteins, CheA (labeled via [gamma-32P]ATP), CheY1, and CheY2 (labeled via acetyl [32P]phosphate). Our results are consistent with the following four-step phosphotransfer: (i) ATP-dependent autophosphorylation of CheA (with a limiting rate constant of 0.008 s-1 at saturating ATP concentrations); (ii) rapid phospho transfer from phospho-CheA to CheY1 and CheY2; (iii) autodephosphorylation of CheY1-P and CheY2-P with half-lives of 12 +/- 1 s and 10.5 +/- 1 s, respectively; and (iv) reversible phosphotransfer from CheY2-P to CheA. In the three-component mixture, CheA/CheY1/CheY2, we observed rapid phosphotransfer from CheY2-P via CheA to CheY1. Thus, CheY1 assumes the role of a "phosphatase" of CheY2-P by acting as a sink for phosphate, whenever unphosphorylated CheA is present. The intracellular concentrations of CheA/CheY1/CheY2 determined immunochemically were 1.5 microM:20 microM:20 microM, a range that was adopted for in vitro assays. The results reflect a unique control by CheY1 of the active, phosphorylated state of the main response regulator, CheY2-P. This mechanism appears to be a new twist to signal transduction among members of the alpha-subgroup of proteobacteria.     

Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.     

Response regulator output in bacterial chemotaxis

Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response-regulator protein P-CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P-CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P-CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P-CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient approximately 2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P-CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free-swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P-CheY. Thus the level of intracellular P-CheY can be estimated from behavior determinations: approximately 30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild-type cells.     

Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli

Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins, defined by the photoreceptor cell-specific protein, recoverin. Recoverin, which regulates the desensitization of photo-excited rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch. Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding. In this work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin delta. I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner. Both myristoylated and unmyristoylated proteins bind three calcium ions. However, the unmyristoylated form exhibits a higher affinity for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium. These data support the model for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor, A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J. B. (1993) Science 259, 829-832). Using point mutations, I have investigated the relative importance of each of the three functional EF hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin. Calcium and membrane binding properties of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein to membranes.     

Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p

Previously, we have shown that the yeast gamma-tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.     

A new class of Caulobacter crescentus flagellar genes

Eight Caulobacter crescentus flagellar genes, flmA, flmB, flmC, flmD, flmE, flmF, flmG, and flmH, have been cloned and characterized. These eight genes are clustered in pairs (flmAB, flmCD, flmEF, and flmGH) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the flm genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the flmAB, flmEF, and flmGH operons was shown to occur primarily in predivisional cells. In contrast, the flmCD operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the flm genes. Furthermore, the flm genes did not affect the expression of class II or class III flagellar genes. However, mutations in the flm genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the flm operons belong to a new class of flagellar genes.     

Functional analysis of the placenta-specific enhancer of the human glycoprotein hormone alpha subunit gene. Emergence of a new element

Placental expression of the alpha subunit gene of the human glycoprotein hormones requires a multicomponent enhancer composed of tandem cAMP-response elements and an adjacent upstream regulatory element. Based on recent studies indicating that the upstream regulatory element includes binding sites for more than one protein, we investigated how functional activity correlated with these binding sites. Through extensive replacement mutagenesis of the native promoter regulatory region, we provide the first functional map of the upstream regulatory element. Within this region, we find that distinct proteins interact with three overlapping binding sites. While each site is functionally significant, no single site is essential or displays clear dominance. This is surprising since one of the sites binds a placenta-specific protein that heretofore has been regarded as essential for activity of the human alpha subunit placenta-specific enhancer. Consequently, our refined functional map of the upstream regulatory element reveals a complex combinatorial code that directs expression of the human alpha subunit gene to placenta.