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R420 Dispatch

Signal transduction: Response regulators on and off Jeff Stock and Sandra Da Re

High resolution structures of the active phosphorylated phosphodiesterase to regulate levels of cAMP and control forms of two-component response regulators have fruiting body development [8]. recently been reported. The results provide a basis for understanding how metabolic energy is coupled to How do these α/β domains convert metabolic energy into signal transduction in cellular regulatory networks. regulatory outputs? The process starts with the binding of a signal molecule to an allosteric site controling a sensor Address: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA. kinase, which acts in turn to control the of a specific response regulator aspartate residue. In ion- Current Biology 2000, 10:R420–R424 motive ATPases, the phosphotransfer from ATP to aspar- tate seems to be direct, whereas in regulators there is an 0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. intermediate –aspartate phosphorelay that pro- vides additional points for sensory regulation. Whatever About 130 years ago James Clerk Maxwell imagined a the details, the end point is a regulatory domain that has ‘finite being’ that could reverse an otherwise favorable dif- acquired the energy it needs to do its job of bringing some fusive process by willfully controlling the passage of differ- sensible order to the world. The is said to undergo ent molecules between two compartments [1]. This little a that leads to the activation of asso- gatekeeper came to be known as Maxwell’s Demon, ciated effector functions. In other words, phosphorylation undoubtedly because of its devilish designs to violate the is thought to cause the α/β domain to change its structure, Second Law of Thermodynamics. We now know that living and this structural change is transmitted to an associated systems are full of Maxwell’s Demons, working hard to gated channel, or factor, or to cause perform seemingly impossible tasks like moving molecules an appropriate output. act to dephosphory- up electrochemical gradients, or magically transmuting one late the aspartate, so that the response can be turned off. kind of or organism into another. But thermodynamics So we have a typical signal transduction pathway in which says that no matter how clever the Demon, all attempts to an input signal impinges on a kinase activity that uses ATP violate the Law will automatically incur a minimum price to to phosphorylate an aspartate, which acts in turn to effect a be paid in full, immediately and without exception. response (Figure 1). Through the combined activity of the kinase and , ATP is hydrolyzed to ADP and Pi The archetypal Maxwellian Demons of biology are the to provide the energy required to satisfy the Second Law. Na+/K+- and Ca2+-ATPases that produce electrochemical gradients across cell membranes [2]. These pumps Over the past several years there has been considerable are composed of integral membrane that serve as effort to fill in the molecular details of response regulator gates, and associated ATPase domains that function as function. Over 400 different paralogues of the response gate keepers. Ion fluxes are coupled to ATP hydrolysis regulator superfamily have been sequenced [9], and X-ray with intermediate phosphorylation of a specific aspartate crystal structures of several have been solved in their inac- side chain. The ATPase domains appear to have an α/β tive unphosphorylated states [10–16]. The domain has a fold that is similar to another family of protein domains highly conserved structure consisting of a five-stranded that are also phosphorylated at aspartate residues, the so- parallel β sheet surrounded by five α helices (Figure 2). called response regulators that control the output activi- The phospho-accepting aspartate, Asp57, is located in the ties of signal transduction networks in microorganisms central β3/α3 loop. There are several highly conserved and [3]. Recent modeling studies also indicate sim- residues surrounding this aspartate, including Asp12 and ilarities in side-chain chemistry around the phospho- Asp13 in the β1/α1 loop, Thr87 in the β4/α4 loop, and accepting aspartates [4]. Response regulator domains Lys109 in the β5/α5 loop. It is clear that many of these usually function to control gene expression through asso- play important roles in catalyzing aspartate phosphoryla- ciated transcription factors [5]. But there are numerous tion and dephosphorylation. For instance, the aspartates exceptions. A response regulator called CheY binds to coordinate the binding of divalent metals required for the motor proteins to generate chemotaxis responses that bias phosphotransfer chemistry [17]. the diffusion-like random walk of motile [6]; a response regulator in functions to control the activ- Having established the structures of inactive response ity of a MAP kinase pathway that regulates growth in regulators, the next logical step was to solve the structures response to osmotic stress [7]; and a response regulator in of the phosphorylated forms to determine the mechanism a slime mold controls the activity of an associated cAMP of activation. At first this seemed an impossible task, as Dispatch R421

Figure 1 Figure 2

Asp

ATP Pi OFF

H O ADP Asp~P 2

ON

Response Structure of a typical response regulator domain. The domain is a α β β Current Biology doubly wound / protein with a central five-stranded parallel sheet surrounded by five α helices. Conserved residues are indicated. The structure is the unphosphorylated form of CheY [17]. The generally accepted view of response regulator function. Sensor kinases act to control the flow of phosphoryl groups from ATP to a specific aspartate in a response regulator protein. Aspartate phosphorylation leads to an activating conformational change that Although the phosphorylated response regulator struc- converts the regulatory domain from an inactive OFF to an active ON tures seem to support the threonine flip–tyrosine tuck conformational state. In the phosphorylated ON conformation the hypothesis, there are several caveats. First, it is not clear domain interacts with a target macromolecule to produce a response. why phosphorylation should cause the critical threonine A sensor phosphatase functions to dephosphorylate the response movements that are seen in phosphorylated Spo0A and regulator to turn it off. FixJN. Second, similar threonine and tyrosine movements have been seen in some crystal forms of unphosphorylated phospho-aspartates are inherently unstable, and the histi- CheY [23,24]. Third, a CheYT87A mutant can still be acti- dine–aspartate phosphorelay involves additional proteins. vated by phosphorylation [25]. Fourth, the structure of Mutagenesis studies of the chemotaxis response regulator, phosphorylated FixJN is significantly different from that CheY, provided indirect evidence that the α4/β5/α5 of unphosphorylated response regulators, whereas the surface was crucial for activation [18], but the X-ray crystal phosphorylated Spo0A structure seems to be relatively structure of a constitutively active mutant, CheYD13K [19], unperturbed [20,21]. Fifth, why were these activating con- did not reveal any significant conformational changes that formational changes not detected in the structure of the could be associated with activation. constitutively active response regulator, CheYD13K [19]? And finally, as outlined below, the whole concept of a Since these results were obtained there have been several phosphorylation-induced conformational change that significant breakthroughs in this field, and within the past turns a response regulator ON is basically incorrect, few months X-ray crystal structures of two phosphorylated because it violates the Second Law of Thermodynamics. response regulator domains have been solved, Spo0A and FixJ [20,21]. Although phosphorylation has little effect on To understand the flaw in the logic of response-regulator the structure of Spo0A, the structure of FixJ is dramatically conformational switching, one must think in terms of altered. Nevertheless, in both phosphorylated proteins the energy rather than structure (Figure 3). In response regu- hydroxyl side chain of the conserved threonine in loop 4 lators, ATP is used to phosphorylate the carboxyl of an moves away from its position pointing toward the critical aspartate side chain. The acylphosphate linkage that is α4/β5/α5 surface to within hydrogen-bonding distance of formed is a high-energy bond, just like the phospho- one of the phosphoryl oxygens, and the hole created by anhydride linkage in ATP; in fact the aspartyl-phosphate this motion is filled by a conserved aromatic residue modification is the highest-energy phosphorylation that moving from an exposed position to a buried position on can exist in a protein [3]. So where is the energy coming the α4/β5/α5 surface. From studies of the effects of CheY from to throw the putative phospho-activated switch? It mutagenesis it had been proposed that the inward move- cannot come from the subsequent hydrolysis of aspartyl- ment of the corresponding residue in CheY, Tyr106, freed phosphate. The Second Law does not decree that the cost up space for binding to a target macromolecule [22]. of any sensible increase in order today is acceptable so R422 Current Biology Vol 10 No 11

Figure 3 a surface of the response-regulator domain with a surface of the associated effector domain that needs to bind a target macromolecule to produce a response [10,13]. The interac- ATP RR tion between the regulatory and effector domains is weak, Asp~P and depends on the domains being tethered together. There is no evidence for one domain binding to the other in the absence of a polypeptide linkage. Phosphorylation G RR relieves this weak inhibitory contact by inducing a much Asp-P stronger competing interaction. In the case of FixJ, a homodimer forms between the β4/α5 surfaces of two sym- ADP metrically opposed phosphorylated FixJN domains. The intensity of the dimer interaction is evidenced by a second- + P i order dependence that is observed in the phosphorylation reaction [29]. The association is so tight that the dimer can Current Biology be purified without any evidence for dissociation, and the Hypothetical free energy (G) profile for elements of an intracellular phospho-aspartyl group is stable for weeks thereafter; this response regulator signaling system. ATP has a high negative free is what allowed its crystallization [21]. energy of hydrolysis, as does the phospho-aspartyl group in a typical phosphorylated response regulator domain. The high negative free Now consider what these results imply about the energet- energy of the phosphorylated response regulator (RR-Asp~P) is used to drive a large activating conformational transition (RR-Asp-P) that ics (Figure 3). The cooperativity observed in the FixJN occurs when it interacts with its regulatory targets (colored circle). phosphorylation reaction, as well as the strength of the dimer interaction, indicate that dimer formation pulls the phosphorylation reaction. It is the formation of the dimer long as it is paid for tomorrow. If you want to throw a that is the switching event, and it is at this step that the switch you have to pay as you throw it, not later when the high energy of the phospho-aspartate bond is used to gen- phosphatase happens to kick in to complete the ATPase erate a conformational transition. The large negative free reaction. Which leads to the question as to why these energy of dimerization must derive from a decrease in the demonic domains use a aspartyl-phosphate rather than a negative free energy of hydrolysis of the phospho-aspartate phospho-serine or phospho-threonine. Clearly, phosphoryl group so that phosphorylation and dimerization are tightly transfer from ATP to a serine or threonine residue gener- coupled. It would not be surprising if, in the presence of ates plenty of energy to cause substantial conformational high concentrations of Pi, the large negative free energy changes in protein structure and activity [26]. that drives the conformational change associated with phospho-FixJ dimerization could be used to actually gener- The high energy of the phospho-aspartate in response reg- ate a phospho-aspartate. Just such a back reaction is, in ulators has been verified by direct measurements of its fact, a well established feature of ion-motive ATPases [2]. reversibility both in vitro and in vivo [5]. Thus, one can argue on thermodynamic grounds that, although response- The X-ray crystal structure of the phosphorylated FixJN regulator phosphorylation may appear to cause some struc- dimer tends to corroborate these suppositions [21]. The tural perturbations in the isolated phosphoproteins, these threonine flip and the tyrosine tuck are a small part of an conformational changes are unlikely to be directly relevant entire restructuring that radiates from the dimer interface to activation. This explains the general lack of any confor- and includes substantial backbone motions in loops 1,3, β α β mational changes seen in structures of phosphorylated or and 4 as well as in 4, 4, and 5. The FixJN dimer inter- mutationally activated response regulators compared to face is very unusual. In a recent study, among over 50 dif- the corresponding unphosphorylated proteins. ferent protein–protein interactions that were analyzed [30], none had so small a change in solvent-accessible surface 2 The large effect of phosphorylation on FixJN is the area as the 880 Å that is seen in the FixJN dimer [21]. exception that proves the rule. The unphosphorylated There is nothing in the phosphorylated dimer structure FixJ protein is a monomer composed of two distinct that would account for its incredible stability, except to modules, an amino-terminal response regulator domain, assume that dimerization generates major stabilizing con- FixJN, linked to a carboxy-terminal , formational changes within each monomer that are FixJC [27]. In its unphosphorylated state, FixJN inhibits reflected in the numerous changes seen in the phospho- FixJC [28]. Thus, a fragment of the fixJ gene encoding FixJ dimer. Dimerization is, after all, the transition that only FixJC is able to activate transcription. constitutes FixJ activation insofar as it both relieves the inhibitory effect of the receiver domain on FixJC and This type of inhibitory effect is a common feature of enhances DNA binding to multiple binding sites in target response regulators that derives simply from interaction of promoter sequences. Dispatch R423

Figure 4 proteins act to carry their high-energy aspartyl-phosphate group to a target macromolecule, and it is only then that the Asp magical event of activation occurs whereby the high-energy acylphosphate bond is used to generate a structural ATP Pi rearrangement that leads to a response (Figure 4).

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23. Volz K, Matsumura P: Crystal structure of Escherichia coli CheY refined at 1.7-A resolution. J Biol Chem 1991, 266:15511-15519. 24. Kato M, Shimizu T, Mizuno T, Hakoshima T: Structure of the histidine- containing phosphotransfer (HPt) domain of the anaerobic sensor If you found this dispatch interesting, you might also want protein ArcB complexed with the chemotaxis response regulator to read the December 1999 issue of CheY. Acta Crystallogr D Biol Crystallogr 1999, 55:1257-1263. 25. Applebury JL, Bourret RB: Proposed signal transduction role for conserved CheY residue Thr87, a member of the response Current Opinion in regulator active-site quintet. J Bacteriol 1998, 180:3563-3569. 26. Johnson LN, O’Reilly M: Control by phosphorylation. Curr Opin Structural Biology Struct Biol 1996, 6:762-769. 27. Kahn D, Ditta G: Modular structure of FixJ: homology of the transcriptional activator domain with the -35 binding domain of which included the following reviews, edited sigma factors. Mol Microbiol 1991, 5:987-997. by Emil F Pai, on Catalysis and regulation: 28. Da Re S, Bertagnoli S, Fourment J, Reyrat JM, Kahn D: Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory Structure and electron transfer mechanism of pyruvate: domain. Nucleic Acids Res 1994, 22:1555-15561. ferredoxin oxidoreductase 29. Da Re S, Schumacher J, Rousseau P, Fourment J, Ebel C, Kahn D: Marie-Helene Charon, Anne Volbeda, Eric Chabriere, Phosphorylation-induced dimerization of the FixJ receiver Laetitia Pieulle and Juan Carlos Fontecilla-Camps domain. Mol Microbiol 1999, 34:504-511. 30. Conte LL, Chothia C, Janin J: The atomic structure of protein-protein Structure and mechanism of iron-only hydrogenases recognition sites. J Mol Biol 1999, 285:2177-2198. John W Peters Catechol oxidase – structure and activity Christoph Eicken, Bernt Krebs and James C Sacchettini Novel ways to prevent proteolysis – prophytepsin and proplasmepsin II Nina Khazanovich Bernstein and Michael NG James Diversity in protein recognition by PTB domains Julie D Forman-Kay and Tony Pawson The structure, organization, activation and plasticity of the erythropoietin receptor Ian A Wilson and Linda K Jolliffe

the same issue also included the following reviews, edited by Edward Baker and Guy Dodson, on Proteins:

Lectins M Vijayan and Nagasuma Chandra β Propellers: structural rigidity and functional diversity Vilmos Fülöp and David T Jones Structural and mechanistic studies on 2-oxoglutarate- dependent oxygenases and related Christopher J Schofield and Zhihong Zhang α/β Hydrolase fold enzymes: the family keeps growing Marco Nardini and Bauke W Dijkstra Cyclin-dependent kinases: inhibition and substrate recognition Jane A Endicott, Martin EM Noble and Julie A Tucker MHC superfamily structure and the immune system Katsumi Maenaka and E Yvonne Jones Signal sequence recognition and protein targeting Robert M Stroud and Peter Walter

The full text of Current Opinion in Structural Biology is in the BioMedNet library at http://BioMedNet.com/cbiology/jstb