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Molecular recognition of bacterial phosphorelay proteins Kottayil Iype Varughese The transfer of the phosphoryl group from a histidine kinase to a response regulator forms the basis of bacterial signal transduction. The critical question of how a component of a signal transduction system specifically associates with its partner to produce the ideal environment for phosphotransfer is addressed in this review in the light of the structure of the Spo0F–Spo0B complex in Bacillus subtilis. Addresses Division of Cellular Biology, Department of Molecular and Experimental Medicine, MEM-116, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA; e-mail: [email protected] Current Opinion in Microbiology 2002, 5:142–148 1369-5274/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations Ala alanine Asp aspartate Gln glutamine Glu glutamic acid Gly glycine His histidin Ile isoleucine Leu leucine Lys lysine Phe phenylalanine Tyr tyrosine

Introduction Bacteria possess the capability to adapt to fluctuating environments that may unexpectedly become hostile. They do so by monitoring the environmental signals and expressing pathways needed for maximal growth under the new conditions. One of the major regulatory mechanisms for signal recognition and response is the two-component system (Figure 1a) and its more sophisticated variant, the phosphorelay system (Figure 1b). Two-component systems consist of a sensor histidine kinase and a response regulator. Sensor kinases can be broadly divided into two parts: an amino-terminal stimulus detection domain and an autokinase domain that consists of a phosphotransferase subdomain containing an active histidine (His) residue and an ATPbinding subdomain (Figure 1a). The autokinase domains are of similar length and show many conserved amino acid motifs, whereas the signal detection domains show considerable variations in size and amino acid sequence, reflecting the diversity of signals. Most of the response regulators are transcription factors and they consist of two domains. The amino-terminal domain receives the phosphoryl group from the kinase and regulates the DNA-binding activity of the carboxy-terminal effector domain. Phosphorelays are used in bacteria to respond to multiple signal inputs [1]. In addition to the histidine kinase and the

response regulator, a phosphorelay has a secondary messenger and a phosphotransferase and the flow of phosphate is His→aspartate (Asp)→His→Asp. The availability of more components gives more points of regulation. Another advantage of having multiple phosphotransfer steps is increased precision of the response as each step reinforces discrimination. All eukaryotic two-component based systems are phosphorelays [2], presumably reflecting the sophistication of eukaryotic organisms. Following the initial analysis of CheY [3,4], a number of proteins in the two-component and phosphorelay systems have been structurally characterized. They include a single domain response regulator, Spo0F [5], and two intact response regulators, a transcription factor (NarL) [6] and a methyl transferase (CheB) [7]. Additionally, regulatory domains of Spo0A [8], NTRC [9], PhoB [10] and FixJ [11], and effector domains of Spo0A [12], OmpR [13] and PhoB [14] have been individually characterized. Furthermore, structures of phosphorelay phosphotransferases Spo0B [15], YPD1 [16,17] and phosphotransferase domains of histidine kinases, CheA [18], EnvZ [19] and ArcB [20] have been elucidated. The catalytic domain of histidine protein kinase, EnvZ [21], and the carboxy-terminal half of CheA, which consists of three domains, have also been characterized [22]. As part of our ongoing project to study the structure and function of the components of the phosphorelay, we initially analyzed the structures of Spo0F [5,23] and Spo0B [15], and more recently derived the structure of the complex between them [24••]. Additionally, we have completed the structural analysis of the effector domain of the transcription factor in complex with the target DNA. This review mainly deals with the interactions between the components of the sporulation phosphorelay, and focuses on the origin of specificity in histidine-kinase–response-regulator recognition that facilitates phosphoryl transfer.

Spo0F structure Spo0F is a single domain protein and is made up of a central β-sheet consisting of five parallel β-strands and five α-helices. Two of the α-helices are located on one side and three on the reverse side of the β-sheet (Figure 2a) [5]. The active site is a small pocket situated at the carboxyterminal end of the β-sheet, and comprises three aspartates, Asp10, Asp11 and Asp54, and the threonine and lysine residues Thr82 and Lys104, respectively. These five residues are crucial for phosphotransfer and are termed catalytic residues. The site of phosphorylation, Asp54, is located at the bottom of the pocket, and is accessible to solvent. The active site Asps are flanked by five loops that connect β-strands to the α-helices. Although the primary amino acid sequences of Spo0F differ significantly from other regulatory domains, it has

Molecular recognition of bacterial phosphorelay proteins Varughese

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Figure 1 Histidine kinase (a)

Signal detection

P

P

H

D

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Histidine kinases (b)

Signal detection

P

P

P

P

H

D

H

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DNA binding Spo0A Current Opinion in Microbiology

Domain organization of two-component/phosphorelay signal transduction systems. (a) In a typical two-component system, signal recognition by the sensor kinase induces the transfer of the ϒ-phosphate of ATP to a histidine residue of the phosphotransferase domain. The phosphoryl group is donated to an aspartate of a

response regulator by the histidine kinase. (b) In the sporulation phosphorelay system of Bacillus, the histidine kinases pass the phosphoryl group to an intermediate response regulator, Spo0F. Subsequently, the phosphotransferase, Spo0B, transfers it to the transcription factor, Spo0A.

the same overall structure as other regulatory domains, such as CheY [3], Spo0A [8], NTRC [9], NarL [6], PhoB [10] and FixJ [11]. The active site geometry of these domains is remarkably similar.

significant change is reorientation of a catalytic Thr in the β4–α4 loop (residue 84 in Spo0A and 82 in FixJ) towards the phosphoryl group to form a hydrogen bond with it.

Spo0B phosphotransferase The crystal structures of the phosphorylated amino-terminal domains of Spo0A [25] and FixJ [11] indicate that the active site undergoes changes upon phosphorylation. The most

Spo0B transfers the phosphoryl moiety from Spo0F to Spo0A and the reaction is reversible. Although Spo0B lacks a signal input domain and ATP-binding subdomain

Figure 2

(a)

(b)

α4

D11 α3

T82 D54 K104 D10

α2

H30

α5 α1

N C C

N Current Opinion in Microbiology

Structure of Spo0F and Spo0B. (a) A ribbon representation of the structure of Spo0F. The central β-sheet consists of five parallel β-strands that are shown in yellow. There are five α-helices: α1 to α5. The five catalytic residues Asp10 (D10), Asp1 (D11), Asp54 (D54), Thr82 (T82) and Lys104 (K104) are shown as ball–stick models.

The regions interacting with Spo0B are shown in green. (b) A ribbon representation of the Spo0B dimer. One protomer is shown in green and the other in blue. His30 (H30), the site of phosphorylation, is shown in red. The portions of the molecule that interact with Spo0F are shown in orange.

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Figure 3

(a)

Spo0F

Spo0F

Asp54 His30

Asp54 His30

His30 Asp54

His30 Asp54

Spo0F

Contrasting views of the Spo0B–Spo0F complex. (a) A stereo view down the axis of the four-helix bundle. The residues His30 of Spo0B and Asp54 of Spo0F are in close proximity for phosphoryl transfer (reproduced, with permission, from [24••]). (b) A view perpendicular to the four-helix bundle.

Spo0F

(b)

H3O

D54

Spo0F

Spo0F

Spo0B four-helix bundle

characteristic of histidine kinases, it has a number of structural and functional similarities with histidine kinases. It accepts the phosphoryl group on a histidine and exists as a dimer. The crystal structure [15] reveals the mode of dimerization (Figure 2b). The monomer is made up of two domains, an amino-terminal α-helical hairpin and a carboxy-terminal domain with an α/β fold. The protein dimerizes by the association of the helical hairpin domains from two protomers to form a four-helix bundle. The site of phosphorylation is His30, and its side chain protrudes from the four-helix bundle toward the solvent. There are two active sites per dimer. NMR studies of this domain in the Escherichia coli histidine kinase EnvZ [19] reveals a structure very similar to the four-helix bundle of Spo0B, formed by dimerization. The Spo0B four-helix bundle superimposes onto the EnvZ four-helix bundle and the histidine, the site of phosphorylation, projects from the bundle in an identical manner.

Similarities of the interacting domains Considerable diversity exists in the signal detection domain of the sensor kinases and the effector domains of

the transcription factors through which the responses are mediated. The phosphotransferase domain of the kinase and the regulatory domain of the response regulator are, on the other hand, remarkably similar. Bacteria expanded the two-component system to perform various specialized tasks by diversifying the signal input and output domains while preserving the salient features of the central components that interact to transmit the signal. The secondary messenger of the sporulation phosphorelay, Spo0F, has virtually the same structure as the regulatory domain of Spo0A or any other response regulator. The majority of histidine kinases in bacteria have the general structure of EnvZ. Therefore, a Spo0B-like four-helix bundle could provide a model for the interaction surface for response regulators.

Spo0F–Spo0B complex The crystal structure of the complex [24••] between Spo0F and Spo0B provides insight into the mode of phosphotransfer and the origin of specificity. The Spo0B molecule has two binding surfaces and a Spo0F molecule binds to each of these sites (Figure 3). The association of the two molecules

Molecular recognition of bacterial phosphorelay proteins Varughese

brings the active His of Spo0B and the active Asp of Spo0A in close proximity and creates the correct geometry for phosphoryl transfer. Figure 4 shows a model of the transition state intermediate obtained by inserting a phosphoryl group between the Asp and the His. The model shows that the Asp and the His are ideally oriented for phosphotransfer. The interactions of Mg2+ and Lys104 neutralize the negative charge of the phosphoryl group and the active site shields the phosphoryl group from external water molecules to prevent hydrolysis. In summary, the model reveals that the association of Spo0F with Spo0B creates an environment for phosphotransfer by ensuring the ideal orientation for the aspartate and histidine residues, stabilizing the transition state intermediate, and preventing hydrolysis by sealing the active site from external water molecules.

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Figure 4

α1 of Spo0B

H30

A83 K104

K56 PO3

T82

Mg2+

The interaction surface of Spo0F is made up of helix α1 and five β–α loops (Figures 2a and 3) that surround the active site. Out of the 22 residues of Spo0F that have significant interactions with Spo0B, 15 of them contact the four-helix bundle. Tzeng and Hoch [26] have carried out alanine-scanning mutagenesis of the surface residues of Spo0F and have documented the in vivo and in vitro consequences of mutation. Proline (Pro), glycine (Gly) and alanine (Ala) residues are not testable by this method. As expected, there is excellent correlation between residues involved in interactions with Spo0B and residues that gave rise to altered phenotypes. Additionally, it has been observed that the regions of Spo0F that interact the phosphotransferase undergo faster internal fluctuations [27].

D11 D54

A model for the transition state intermediate, created by placing a phosphoryl group between the active His30 and Asp54. The phosphorus atom forms partial covalent bonds with O δ of Asp and Nεof His and is in a penta-coordinated state. The negative charges on the phosphoryl oxygens are compensated through interactions with Mg2+ and Lys104 (reproduced, with permission, from [24••]).

Spo0B transfers a phosphoryl group between Spo0F and Spo0A and the interaction surfaces of the two are

Figure 5 The interaction surface of Spo0F. The five residues that form the hydrophobic patch are shown in dark blue. The invariant catalytic residue 104 is also labeled. Four additional residues interacting with the four-helix bundle that are conserved within the OmpR family are colored cyan. Five residues that interact with the four-helix bundle are highly variable within the OmpR family and they are colored green. α1

K56

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Table 1 Conservation and variation of interacting residues of the response regulator.

Spo Family Spo0F Spo0A NarL Family DegU YhcZ YvqC YvfU YocG Ydfl YxjL YflK ComA OmpR Family YcbL YxdJ YtsA YvcP YbdJ YvqA YkoG YrkP YclJ YvrH YycF ResD PhoP YccH A Family YufM YdbG CitT YcbB B Family LytT YesN Che Family CheY CheB Ynel

Residue number in Spo0F 21 56 83 84

12

14

15

18

85

87

104

105

106

Q N

G E

I L

L L

E E

K I

A A

H H H Q Q H Q Q H

L V M M M V L I A

F V V L L V V V V

G G G A A G G G G

R F A S S L Y S T

N S V E E Y Q R N

D S D D D E E D N E E E E Y

S D S R M N K D S A P R S V

I I L I I L I I V I I I I L


M L E L L L V L M M I L L L


H N D G K K L E M R F M Y A

D D D D

M M R A

V V V V

L V I I

E E

L I

A I

A S A

F F F

M M M

107 108

Y F

G G

L E

K K

P P

F F

D D

I M

I S S T T T T T G

H Y F F F Y F F Y

D S I A A N D K E

E Q D P P D E S E

K K K K K K K K K

E D T D D D D D T

M T S G S T T M E

D E K E P S L S S

A P A I S S P A K

M N Q N M M M M M M M M M E

A A S A A A A A A A A A A G

K R R R R R R R K R K K K H

D S D T N D D S D S D G D G

D E P E M D I T E D E E E Y

K K K K K K K K K K K K K K

P P P P P P P P P P P P P P

F F F F F F F F F F F F F Y

S S H H D L E S S N S S S Y

M Y F L P P I E P P T P P A

R D R Q

Y Y Y L

A A A Q

A A A V

S K T E

L K T K

K K K K

P P P P

F F V I

E K T V

F L A L

E G

Y G

D K

A G

Y F

D G

Y F

K K

P P

F C

D S

E L

M M K

D D E

T T E

A A S

M M Q

G R T

Q Q K

K K K

P P P

F F S

Q E L

A E V


These are residues 16 and 19 in YccH. The fifteen residues of Spo0F that interact with the four-helix bundle of Spo0B are listed along with equivalent residues in all the response regulators in B. subtilis. The six residues that are globally conserved are shown in bold. The four residues that are conserved within the OmpR family are shown inside boxes. The other five residues exhibit much higher variability within the OmpR family. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; Y, tyrosine.

well-conserved [24••]. A comparison of the interacting residues of Spo0F with the sequences of Spo0F orthologs from Bacillus anthracis and Bacillus halodurans show that they are 95% identical to each other, whereas the remainder of the residues are only 50% identical to each other, showing that the interaction interface is preserved during evolution [28••]. Spo0B has a rigid structure with a suitable architecture for interaction with Spo0F. Phosphorylation may cause the rearrangement of certain side chains, but is unlikely to have any significant effect on its overall conformation.

Phosphorylation of Spo0F, on the other hand, will cause the rearrangement of the active site, in a similar fashion to that observed in Spo0A–P and FixJ–P, to optimize interactions with the phosphoryl group. Active sites of Spo0A and FixJ take up a more ‘closed’ conformation to resist the hydrolysis of the phosphoryl group. The conformation of Spo0F in the complex is in a more ‘open’ form suitable for stabilizing the transition state and the release of the phosphoryl group. These three snapshots are indicative of the conformational pathway of response regulators during phosphorylation and dephosphorylation.

Molecular recognition of bacterial phosphorelay proteins Varughese

How is discrimination possible in the midst of similarity? B. subtilis cells possess 34 two-component pairs, each dedicated to unique signals and unique responses [29,30]. Some species have even more than a hundred pairs. Hence, it is intriguing how a particular kinase specifically recognizes its partner and activates it to produce the correct response when there are such a large number of pairs. Specificity can be generated through variations in shape and sequence in the response regulator, making the concomitant changes in the phosphotransferase domain to maintain complementarity. The receiver domains of the response regulators are structurally very similar and have identical active sites lined by invariant catalytic residues. The four-helix bundle of Spo0B that mediates most of the interactions with Spo0F should be a prototype for the phosphotransferase domains of the majority of histidine kinases. Hence, sequence variations must play a crucial role in discrimination. On the basis of these sequences, the two-component pairs have been classified into various families. There are two major and several minor families of two-component systems within each bacterium [29,30]. Sequence similarity within a family is high, yet these highly similar systems must process different signals and interact only with their partner to produce the correct response. The following question arises: how does a protein transfer its phosphoryl group only to its unique partner when there is such close sequence similarity?

Core of the binding surface The interaction of Spo0F with the four-helix bundle of Spo0B provides insight into the mechanism of recognition and association. The interaction surface of Spo0F is constituted by the helix α1 and the five β–α loops on the top of the molecule. Five hydrophobic residues, Leu15, Ile18, Pro105, Phe106 and Ile108, form a hydrophobic patch on the interaction surface of Spo0F (Figure 5 and Table 1). This patch interacts with the helix α1 of Spo0B, which contains the site of phosphorylation. With the exclusion of the NarL family, these residues are 88–100% conserved. This hydrophobic patch appears to form the core of the interacting surface and it contacts a hydrophobic region of the four-helix bundle. Out of these five residues, four were tested by alanine-scanning mutagenesis. All of them produced strong deficient phenotypes, and the mutations had serious effects on the kinetics of phosphotransfer [26]. The catalytic residue, Lys104, which interacts with α1 of Spo0B, is invariant. These six residues appear to play a key role in the binding of the two proteins; however, they are unlikely to contribute to specificity in any significant manner. The other interacting residues surrounding these residues however, are not globally conserved. Hence, specificity probably arises from these variable residues.

Peripheral residues and discrimination Out of the six families in B. subtilis, the OmpR family is the largest, consisting of fourteen members. In this family, four

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interacting residues, Gln12, Lys56, Ala83 and Tyr84 are conserved, in addition to the six globally conserved residues mentioned above. These four residues play a role in discrimination of the OmpR family from other families, but they cannot dictate specificity within the family. Of the residues that interact with the four-helix bundle, five residues show high variability within the OmpR family. They are Gly14, Glu21, Gly85, Leu87 and Asp107 (Figure 5 and Table 1) and these residues are located at the periphery of the surface that contact the four-helix bundle. These residues must give rise to discrimination within the family by making the interactions unfavorable for wrong pairing by the lack of complementarity in charge, hydrophobicity and shape.

Conclusions In the interacting interface of the response regulator with the phosphotransferase, conserved and variable residues are arranged in a specific pattern to facilitate binding and specificity. A conserved hydrophobic patch and the invariant Lys104 appear to form the core of the binding interface and they contact the upper portion of the four-helix bundle. Four residues that conserved within the OmpR family contact the four-helix bundle below the surface where the core residues contact. Five residues that are highly variable within the family are distributed along the outer edges of surface interacting with the helix bundle. Therefore, it appears that the binding is initiated through a core and discrimination is accomplished through the variable residues located at the periphery of the interaction surface.

Acknowledgement The original work described in this review was supported by the grant GM54246 from National Institutes of General Medical Sciences, National Institutes of Health, United States Public Health Service.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

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2.

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3.

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4.

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5.

Madhusudan, Zapf J, Whiteley JM, Hoch JA, Xuong NH, Varughese KI: Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis. Structure 1996, 4:679-690.

6.

Baikalov I, Schroder I, Kaczor-Grzeskowiak M, Grzeskowiak K, Gunsalus RP, Dickerson RE: Structure of the Escherichia coli response regulator NarL. Biochemistry 1996, 35:11053-11061.

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Djordjevic S, Goudreau PN, Xu Q, Stock AM, West AH: Structural basis for methylesterase CheB regulation by a phosphorylationactivated domain. Proc Natl Acad Sci USA 1998, 95:1381-1386.

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8.

Lewis RJ, Muchová K, Brannigan JA, Barák I, Leonard G, Wilkinson AJ: Domain swapping in the sporulation response regulator Spo0A. J Mol Biol 2000, 297:757-770.

9.

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22. Bilwes AM, Alex LA, Crane BR, Simon MI: Structure of CheA, a signal-transducing histidine kinase. Cell 1999, 96:131-141. 23. Madhusudan, Zapf J, Hoch JA, Whiteley JM, Xuong NH, Varughese KI: A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis. Biochemistry 1997, 36:12739-12745. 24. Zapf J, Sen U, Madhusudan, Hoch JA, Varughese KI: A transient •• interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction. Structure 2000, 8:851-862. This is a paper on the structure of the complex Spo0F with Spo0B and it describes the details of interactions between the two molecules. Additionally, the paper describes a model for the transition-state intermediate that shows that the association of the two molecules produces an ideal environment for phosphoryl transfer.

14. Okamura H, Hanaoka S, Nagadoi A, Makino K, Nishimura Y: Structural comparison of the PhoB and OmpR DNAbinding/transactivation domains and the arrangement of PhoB molecules on the phosphate box. J Mol Biol 2000, 295:1225-1236.

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16. Xu Q, West AH: Conservation of structure and function among histidine-containing phosphotransfer (HPt) domains as revelated by the crystal structure of YPD1. J Mol Biol 1999, 292:1039-1050.

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28. Hoch JA, Varughese KI: Keeping signals straight in phosphorelay •• signal transduction. J Bacteriol 2001, 183:4941-4949. This review discusses how specificity in phosphotransfer evolved as the bacterium multiplied the two-component pairs to carry out various specialized tasks.

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18. Zhou H, Dahlquist FW: Phosphotransfer site of the chemotaxis-specific protein kinase CheA as revealed by NMR. Biochemistry 1997, 36:699-710. 19. Tomomori C, Tanaka T, Dutta R, Park H, Saha SK, Zhu Y, Ishima R, Liu D, Tong KI, Kurokawa H et al.: Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat Struct Biol 1999, 6:729-734.

Feher VA, Cavanagh J: Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F. Nature 1999, 400:289-293.

29. Fabret C, Feher VA, Hoch JA: Two-component signal transduction in Bacillus subtilis: how one organism sees its world. J Bacteriol 1999, 181:1975-1983. 30. Mizuno T: Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res 1997, 4:161-168.