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The NMR Solution Structure of BeF3−-Activated Spo0F Reveals the Conformational Switch in a Phosphorelay System
doi:10.1016/S0022-2836(03)00733-2
J. Mol. Biol. (2003) 331, 245–254
The NMR Solution Structure of BeF2 3 -Activated Spo0F Reveals the Conformational Switch in a Phosphorelay System Alexandra K. Gardino1, Brian F. Volkman2, Ho S. Cho3 Seok-Yong Lee3, David E. Wemmer3 and Dorothee Kern1* 1
Department of Biochemistry Brandeis University, Waltham MA 02453, USA 2
Department of Biochemistry Medical College of Wisconsin Milwaukee, WI 53226, USA 3 Physical Biosciences Division Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, CA 94720 USA
Two-component systems, which are comprised of a single histidineaspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidineaspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF2 3 -activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF2 3 activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators. q 2003 Elsevier Ltd. All rights reserved
*Corresponding author
Keywords: response regulator; two-component systems; phosphorelay; Spo0F; NMR spectroscopy
Introduction Protein phosphorylation is one of the most common mechanisms of signal transduction. In bacteria, signaling is dominated by the phosphorylation-mediated two-component paradigm in which a histidine kinase autophosphorylates a histidine residue in response to an external Present address: H. S. Cho, Xencor, Monrovia, CA 91016, USA. Abbreviations used: NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy. E-mail address of the corresponding author: [email protected]
stimulus and subsequently transfers the phosphate moiety in a magnesium-dependent manner to an aspartate residue in the receiver domain of the second component, the response regulator. Phosphorylation activates the response regulator, which is then capable of eliciting a cellular response, usually as regulation of gene expression, as many response regulators are transcription factors. Phosphatases as well as the intrinsic phosphatase activity of the response regulators themselves control the duration of the signaling event, and the lifetime of this chemical signal correlates strongly with the biological role that the response regulator plays in the cell. More than one two-component signaling module can be connected in tandem to create a complex signal transduction pathway called a
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved
246 phosphorelay.1 This extended two-component signaling pathway was first identified in the sporulation of Bacillus subtilis and has also been found in eukaryote.2,3 The first components of this His-Asp-His-Asp phosphotransfer sequence are the sensor histidine kinases, KinA and KinB, which both autophosphorylate in response to an environmental stimulus. This phosphate moiety is then transferred to the first of two response regulators in the pathway, Spo0F (Scheme 1). Spo0B, the second histidine kinase and phosphotransferase, receives the phosphate group from phosphorylated Spo0F and transfers it to the final response regulator in the pathway Spo0A, which transcriptionally represses the AbrB gene, allowing for the expression of various genes that become activated at the onset of stationary phase.1 Two phosphatases, RapA and RapB, can catalyze the dephosphorylation of Spo0F, and Spo0E has been shown to dephosphorylate Spo0A should the event of sporulation become inappropriate or superfluous. Thus, the phosphorelay employs several points of regulation before eliciting a final cellular response, which is desirable for a pathway that controls the energetically costly process of sporulation. The focus of this study is to characterize the structural basis of activation in the phosphorelay response regulator, Spo0F. While a number of receiver domain structures from response regulators have been solved in their inactive states, a detailed structural comparison with regard to their activated states has proven to be difficult due to the short lifetime of the phosphorylated state. Recently, this obstacle has been overcome by the use of beryllofluoride as a phosphate analogue.4 Comparison of inactive –active receiver domain pairs from different two-component
BeF2 3 -Activated Spo0F
systems has revealed that they use a common signaling surface; however, different types of structural rearrangements have been found to occur upon activation.5 – 8 Recent NMR dynamics data measured in the response regulator NtrC in different functional states9 as well as Spo0F in its inactive state10 in combination with biochemical data11,12 suggested a pre-existing equilibrium between the inactive and active states before phosphorylation as a possible general feature of response regulators. To date, structures of receiver domain pairs in their inactive and active states have been solved only for simple His-Asp two-component signaling pathways. In contrast, Spo0F is a receiver domain that functions as a phosphorelay protein, transferring its phosphate group to a second histidine kinase, unlike multidomain response regulators that activate a downstream partner through protein– protein interactions. While receiver domains of response regulators from twocomponent and phosphorelay systems are structurally similar in their inactive, non-phosphorylated state, we address the question of whether the additional interaction in a phosphorelay has associated distinct structural changes. To address this, we have solved the NMR solution structure of the BeF2 3 -activated form of Spo0F, and report here the first structure of an activated response regulator from a phosphorelay system.
Results and Discussion NMR structure determination of activated Spo0F Spo0F is a 124 residue single-domain response
Scheme 1. Sporulation signal transduction pathway in B. subtilis. The His-Asp-His-Asp phosphate transfer is termed a phosphorelay. Sporulation is finally initiated by phosphorylated Spo0A, which represses the AbrB gene. Genes involved in sporulation that are kept silent by the AbrB repressor during vegetative conditions are now upregulated. Arrows represent the phosphate transfer.
247
BeF2 3 -Activated Spo0F
regulator consisting of just a conserved receiver domain. The NMR solution structure of inactive Spo0F solved by Feher et al. revealed the characteristic b5/a5-barrel scaffold architecture found for the receiver domains of all response regulators of known structure to date.10 The active site is composed of residues Asp10 and Asp11 (Mg2þ coordination), Asp54 (the site of phosphorylation), and the invariant residue Lys104. Although the half-life of phosphorylated Spo0F (Spo0F~P) is relatively long (five or more hours in vitro) among the superfamily of response regulators,13,14 detailed structural analysis of Spo0F~P was further complicated by the inability to phosphorylate Spo0F completely. Therefore, we chose an alternative approach to determine the three-dimensional structure of Spo0F on the basis of the finding that beryllofluoride can act as an acyl phosphate analogue by forming a tight complex with the site of phosphorylation (Asp54). Beryllofluoride has been shown to fully activate several response regulators,4 and this chemical modification has aided in solving structures of response regulators in their activated states, including CheY,6 NtrC (D.E.W., unpublished results), DctD,8 and a complex of CheY with its phosphatase CheZ.15 First, the experimental conditions for BeF2 3 activation of Spo0F were screened using 1H15NHSQC spectra in order to evaluate the yield of BeF2 3 labeling. It was found that Spo0F could be converted quantitatively into a stable, active form using 50 mM MgCl2, 6 mM BeCl2, and 60 mM NaF. Standard triple-resonance NMR experiments using uniformly 15N-labeled or 15N/13C-labeled BeF2 3 -Spo0F were performed to fully assign the backbone and 90% of the side-chain resonances. The results were compared to previously published chemical shifts for the inactive form of Spo0F16 (Figure 1). The Lys104-Pro105 peptide bond was found to be in a cis conformation, as verified by the typical 1Ha – 1Ha sequential nuclear Overhauser effect (NOE) in a 13C-edited NOESY.
Distance and torsion angle constraints obtained from NMR data were used for calculating 60 structures using DYANA 1.5 and TALOS.17,18 The 20 lowest-energy conformers with a target function of less than 5 and minimal violations are shown in Figure 2A, and structural statistics are presented ˚ was in Table 1. An r.m.s. deviation value of 0.77 A reached for secondary structure backbone elements ˚ for the as well as an r.m.s. deviation value of 0.83 A backbone excluding the N and C termini (residues 5– 120). Conformational changes in Spo0F upon activation After backbone assignments were completed, chemical shift mapping provided initial identification of regions in Spo0F that were altered upon BeF2 3 activation (Figure 1). In addition to amide chemical shift changes detected in regions previously identified in other response regulators, including helix 3, strand 4, helix 4 and strand 5 (known as the “3445 face”) and expected changes 7 in close proximity to the site of BeF2 3 modification, additional changes were detected in helix 1. The 13 a C chemical shift changes were smaller than 1 ppm for most regions of the protein, indicative of no significant changes in secondary structure upon activation,19 consistent with previous observations for other inactive/active response regulator structures.6 Larger 13Ca chemical shift changes were seen for residues in close proximity to the binding site of beryllofluoride as well as the N-terminal region of helix 4. Significant rearrangements of secondary structure elements were observed in BeF2 3 -activated Spo0F compared to the NMR structure of the inactive form. A superposition of the structures ˚ for parts resulted in an r.m.s. deviation of 1.19 A of the protein involved in secondary structure elements (Figure 2B). While helix 1 is displaced by half a helical turn downwards in BeF2 3 -Spo0F,
Figure 1. Chemical shift mapping to identify conformational changes upon BeF2 3 activation. Backbone amide chemical shift changes between Spo0F and BeF2 3 -Spo0F DvNH were calculated according to the following formula: ððvNinactive 2 vNBeF3 Þ2 þ ðvHNinactive 2 vHNBeF3 Þ2 Þ1=2 : Secondary structure elements are shown symbolically.
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BeF2 3 -Activated Spo0F
Table 1. Structural statistics for BeF2 3 -Spo0F Number of NOEs Number of upper distance constraints Total Intraresidual Short-range Medium-range Long-range Dihedral angle restraintsa Total restraints ˚ 2) DYANA target function (A Maximum violations Upper limits Van der waals Torsion angles (radian) ˚ )b r.m.s.d. to the mean for N, Ca, and C0 (A ˚ )b r.m.s.d. to the mean for heavy atoms (A r.m.s.d. to the mean for N, Ca, C0 secondary ˚ )c structure elements (A Ramachandran analysis (%)d Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions a b c d
4221 1835 602 540 310 383 165 2000 4.20 ^ 0.42 0.44 ^ 0.10 0.24 ^ 0.05 0.09 ^ 0.04 0.83 ^ 0.17 1.48 ^ 0.18 0.77 ^ 0.19 82.4 14 3.2
Calculated using TALOS.18 For 20 conformers excluding residues 1–4 and 121–124. Residue numbers are same as reported in legend of Figure 2. Analysis was done by PROCHECK-NMR.33
towards the active site and to have an extra helical turn at the N terminus, which was confirmed by the presence of typical aNi, iþ3 NOEs in residues 87 –97 in BeF3-activated Spo0F instead of residues 90 –97 found to comprise helix 4 in the inactive form of Spo0F. The increase in 13Ca chemical shifts for residues 87 – 89 further supports the N-terminal extension of helix 4 in activated Spo0F. Comparison of activation mode in Spo0F with other response regulators
Figure 2. The BeF2 3 -activated structure of Spo0F. (A) The bundle of structures represent the 20 lowestenergy conformers calculated from DYANA 1.5, which were superimposed using the backbone atoms of residues in secondary structure elements. Secondary structure, color-coded blue for b strands and red for a helices, are as follows: b1, 5– 9; a1, 15 – 24; b2, 29 – 33; a2, 36 – 45; b3, 50– 53; a3, 62 – 72; b4, 77 –81; a4, 87 – 96; b5, 100– 103; a5, 108– 118. Loops and the N and C termini are colored in gray. (B) Superposition of the inactive conformation of Spo0F in blue (1FSP) with BeF2 3 -activated Spo0F in orange (1PUX). Residues that did not display backbone chemical shift changes between both forms were used for superposition: 5 – 9, 29 –33, 50 – 53, 36 – 45, and 108– 118, RMSD¼ 1.194. (C) Conserved Thr82 and His101 side-chains are shown in the inactive (blue) and active (orange-red) conformations.
helices 3 and 4 show an upward displacement by half a helical turn upon activation. In addition, helix 4 was found to reorient slightly
How do the structural changes upon activation in Spo0F compare to those found in other response regulators? Structural changes induced by activation already identified in the four known inactive/active receiver domain pairs are not identical, but there seems to be a common region including helix 4 and the loops close to the active site that are always affected by activation. However, the character of the conformational change in helix 4 differs from one receiver domain to another, ranging from a relatively small movement up and towards the active site (CheY, Spo0F)6 to small movements away from the active site (FixJ, DctD),5,8 and finally to a large rearrangement in helix 4 away from the active site and a rotation around the helical axis (NtrC).7 Additional changes in secondary structure elements surrounding helix 4 (helix 3 and strands 4 and 5) have been observed for several receiver domains (Figure 3). Two highly conserved residues among the family of response regulators are a hydroxylcontaining residue (Thr82 in Spo0F) and an aromatic residue (His101 in Spo0F)20 whose sidechain positions have been functionally linked to
BeF2 3 -Activated Spo0F
249
Figure 3. Comparison of inactive versus active structures of receiver domains among various response regulators. Inactive forms are colored in blue and active forms are colored in orange for: (A) CheY; (B) NtrC; (C) DctD; and (D) FixJ. Regions of the protein previously determined not to change in structure upon activation were used for superposition as follows: CheY: 8 – 11, 15 – 26, 33 – 34, 39 – 46, and 53– 56, RMSD¼ 0.85; NtrC: 4 – 9, 14 – 53, and 108–121, RMSD¼ 1.74; FixJ: 5 –8, 16 – 47, 49 – 52, 107– 120, RMSD¼0.31; DctD: 6 – 10, 14– 26, 30 – 34, 37 – 42, 51 –55, 63 – 73, RMSD¼0.3. PDB accession codes for all structures shown are as follows: 3CHY (inactive CheY), 1DJM (BeF2 3 -CheY), 1DC7 (inactive NtrC), 1DC8 (P-NtrC), 1DBW (inactive FixJ), 1D5W (P-FixJ), 1QKK (inactive DctD), and 1L5Y (BeF2 3 -DctD).
the signaling state of the protein.21,22 In CheY, reorientation of Thr87 through a direct hydrogen bond to the phosphorylated Asp in the active site was suggested to be the trigger for a reorientation of Tyr106 from a solvent-exposed position to a buried conformation, based on the results of structural studies.6 While reorientation of the hydroxylcontaining residue seems to be a common feature for response regulators, the different rotameric conformations of the conserved aromatic residue do not display a recognizable pattern. In NtrC, different conformations of Tyr101 could not be related directly to different functional states of the protein.9 In the NMR structure of inactive Spo0F, His101 is in a buried conformation, with the imidazole ring within hydrogen bonding distance of the backbone amide group of Thr82.10 A crystal structure of the Tyr13Ser mutant of Spo0F identified His101 in a solvent-exposed conformation; however, the crystals were grown at pH 4.5, which causes protonation of the imidazole ring, removing the ability to act as a hydrogen-bond acceptor.10,23 BeF2 activation of Spo0F produces a partially 3 solvent-exposed His101 and movement of Thr82 towards the active site, increasing the distance between them so they can no longer hydrogen bond (Figure 2C). Perhaps disruption of this
critical hydrogen bond is coupled to the conformational change in the 3445 face of Spo0F. This idea is supported by the observation that the His101Ala mutant of Spo0F is known to hypersporulate in vivo.24 Presumably, this mutation would stabilize the active form of the protein, thus shifting the equilibrium towards the active state even without phosphorylation. Strikingly, structural changes are also seen in helix 1, located on the opposite side of the protein with respect to the common activation surface, the first observation of changes in this region for a response regulator. Although the primary sequence of the N terminus of helix 1 is conserved or highly similar among many response regulators, some residues found in the b1 –a1 loop are not. This loop contains the invariant active-site residues Asp10 and Asp11, involved in Mg2þ binding, which in Spo0F are followed by Tyr13 and Gly14. The two latter residues are conserved in homologous Spo0F genes from Bacillus thuringiensis and Bacillus halodurans but are not conserved in response regulators from two-component systems. Tyr13 is known to provide strong specificity for the phosphatase RapB and upon mutation leads to a phosphatase-resistant form of Spo0F.13 Upon phosphorylation, the two Asp residues are
250
BeF2 3 -Activated Spo0F
observed to turn in towards the active site in Spo0F, thus pulling on the b1– a1 loop. Gly14 provides conformational flexibility in this region allowing for the rearrangement observed in helix 1. The link between activation surface and function in different receiver domains
Figure 4. Molecular recognition in Spo0F. (A) The direct phosphorelay partner, Spo0B, is displayed according to the X-ray co-crystal structure of Spo0F (green) and Spo0B (gray) (1F51);28 the side-chains of the phosphorelay residues Asp54 in Spo0F and His30 in Spo0B are highlighted in purple. The other partners, KinA and RapB, are represented symbolically due to lack of structural information. Residues implicated in protein – protein interactions between Spo0F and its interaction partners colored in red were found via alanine-scanning mutagenesis31: 12, 13, 14, 15, 16, 18, 19, 21, 34, 35, 37, 38, 56, 57, 58, 59, 81, 83, 84, 85, 86, 87, 104, 105, 106, 107, and 108. Superposition of Spo0F from the co-crystal complex (green) and (B) inactive Spo0F (blue) or (C) BeF2 3Spo0F (orange) suggests that Spo0F was crystallized in
While receiver domains use the same chemistry of phosphoryl transfer and have very similar structures, the structural changes caused by phosphorylation, or more generally by activation, are more diverse when compared in detail. These variations are essential to preserve fidelity and provide specificity given the over 30 or more twocomponent signaling pathways in a cell that follow the same basic rules of phosphoryl transfer between a His residue of a histidine kinase and an Asp residue of a response regulator.25 Upon activation of receiver domains, their function is to propagate the signal to a downstream target via protein– protein interactions and in the case of a phosphorelay component such as Spo0F, to transfer the phosphate moiety to another histidine kinase. CheY, NtrC, FixJ, and DctD interact with their downstream partners through parts of the 3445 face. The area of helix 4, strand 5 and helix 5 of activated CheY has been shown to bind the N16-FliM peptide of its downstream target FliM, a component of the flagellar switch used to reverse the direction and phenotype of swimming in bacteria.26 NtrC, a multidomain response regulator, has been shown to undergo a much larger conformational change upon activation (Figure 3B). Helix 4 was found to undergo a rotation about its helical axis as a result of activation, which together with a change in helix tilt causes the exposure of hydrophobic residues thought to contact and activate the downstream transcriptional activation domain.7 The hypothesis of signal transmission through helix 4 in NtrC was further supported by electron spin resonance experiments.27 In contrast to this positive activation in NtrC, a negative activation mechanism is seen in DctD, another response regulator that belongs to the same subfamily of s54-transcriptional activators as NtrC. BeF2 3 activation of DctD results in the disruption of the inactive dimeric interface, involving an extended helix 5, creating an active dimeric interface with contacts in helix 4 instead.8 This rearrangement releases the autoinhibition of the transcriptional activation domain by its own receiver domain. A subtle rearrangement of the b4 –a4 loop, helix 4, and strand 5 in the two-domain response regulator FixJ (Figure 3D) creates a surface for homodimerization between two receiver
its inactive conformation in complex with Spo0B. Regions of superposition for each pair of structures are residues 5 – 9, 29 –33, 36 –45, 50– 53, and 108– 118.
BeF2 3 -Activated Spo0F
domains. This dimerization relieves the repression of the C-terminal DNA-binding domain by its own receiver domain.5 Spo0F performs a function distinct from the response regulators described above, in that it
251
transfers the phosphate moiety to a second histidine kinase, Spo0B. For this function, Spo0F uses a very different part of the protein than CheY, NtrC, DctD, or FixJ. In the co-crystal structure of a Rap phosphatase-resistant mutant of Spo0F (Tyr13Ser)
Figure 5. Correlation between conformational changes upon activation and backbone dynamics. (A) 15N Amide chemical shift perturbation upon activation (in ppm); (B) conformational exchange dynamics in Spo0F previously reported as chemical exchange term Rex calculated from NMR relaxation experiments;30 and (C) structural displacements ˚ upon BeF2 3 activation (in A) shown as a continuous color scale. The correlation supports a pre-existing equilibrium model.
252
and Spo0B, helix 1 and all five b –a loops of Spo0F were found to contact Spo0B (Figure 4A).28 Interestingly, the residues found to make contacts to Spo0B map to regions of Spo0F that exhibit structural displacements when activated with beryllofluoride. The orientation between Spo0F and Spo0B in the co-crystal structure revealed that the side-chain of Asp54 in Spo0F is in close proximity to the phosphate-accepting residue His30 in Spo0B. The crystals were grown in the presence of aluminum fluoride that was not seen in the electron density, and the question remains of whether this complex represents the active or inactive form of Spo0F. A superposition of the NMR structures of inactive and BeF2 3 -Spo0F with the X-ray structure of Spo0F in the complex clearly reveals that Spo0F is in the inactive conformation in the crystal complex (Figure 4B and C). The affinities of nonphosphorylated and phosphorylated Spo0F for Spo0B are not known. However, the fact that the complex crystallized with proteins in their inactive states suggests that they have a substantial affinity before phosphorylation. Similarly, the affinity of active CheY for the N16-FliM peptide increases by only 20-fold upon activation of CheY.29 In the Spo0F/Spo0B co-crystal structure, an extra turn at the top of helix 428 is present that is seen also upon BeF2 3 activation. This region of the protein is known to exhibit conformational exchange in inactive Spo0F, as shown by previous 15N relaxation backbone dynamics studies.30 Crystallization may stabilize the species that contains the extended helical arrangement. Spo0F interacts with the first histidine kinase in the phosphorelay, KinA (or KinB) and can be dephosphorylated by RapB. Residues involved in these protein– protein interactions have been identified by alanine scanning mutagenesis (Figure 4A). Spo0F forms a binding site for KinA through a hydrophobic surface created by the N-terminal interface of helices 1 and 5, residues on the b3 –a3 loop directly after the site of phosphorylation (Asp54), and the b4 – a4 loop.31 Spo0F residues important for RapB specificity were identified in helix 1 and in all b –a loops except for the b2– a2 loop.13 Thus, residues of Spo0F shown to be involved in protein– protein interactions, identified via structural and biochemical data, localize to a common face of Spo0F with helix 1 playing an important role. Pre-existing equilibrium between the inactive and active state, a common feature of response regulators? The regions of Spo0F found to experience a change in chemical environment upon activation correspond to regions in inactive Spo0F shown to exhibit motions on the micro- to millisecond timescale (Figure 5A and B). Model-free analysis of 15N relaxation measurements performed by Feher et al. identified a number of residues with significant Rex contributions to R2, indicative of conformation-
BeF2 3 -Activated Spo0F
al exchange dynamics.30 These residues form a semicontiguous surface implicated in protein – protein interactions31 that correspond to the regions of Spo0F that undergo structural changes upon activation (Figure 5C). An analogous correlation was observed for NtrC.9 Regions in NtrC shown to have Rex contributions in the inactive form also experience chemical shift perturbations upon phosphorylation and structural displacements associated with activation. For NtrC, it was further verified through comparative chemical shift analyses of a series of constitutively active mutants that the conformational exchange process detected by NMR relaxation studies is an interconversion between the inactive and active states. Taken together, published data for NtrC and the structure of BeF2 3 -activated Spo0F presented here support the hypothesis put forward by Feher et al.30 of a pre-existing equilibrium in Spo0F in which the population is shifted towards the active state upon phosphorylation. Direct experimental data for NtrC and Spo0F together with the fact that basal levels of activity have been detected for response regulators prior to activation12 suggests an occasional sampling of the active-state conformation prior to chemical modification and is most likely a general feature of response regulators. This dynamic process may be crucial for activation by phosphorylation, since the active-site Asp residue is quite occluded in the inactive state, and access to the active conformation could improve the efficiency of phosphotransfer from the histidine kinase.
Materials and Methods Spo0F was cloned from genomic DNA into pET21a vectors (NOVAGEN). Spo0F proteins were expressed using Escherichia coli BL21(DE3) cells with pACYC in M9 medium supplemented with thiamine. Cells were harvested and sonicated in 25 mM Tris –HCl (pH 8.0), 10 mM KCl, 10 mM EDTA, 5 mM DTT, 0.5 mM PMSF. Lysate was subjected to centrifugation: 4 ml of 6 M GuHCl was added to the pellet and resuspended gently with a magnetic stirrer bar for two hours. Then 36 ml of 25 mM Tris – HCl (pH 8.0) was added to the resuspended pellet. Aggregates and cell debris were removed by centrifugation. The supernatant was dialyzed against 25 mM Tris (pH 8.0), 1 mM EDTA, 2 mM DTT. The protein was purified further by passage through DEAESepharose and gel-filtration columns (Superdex75, Pharmacia). The purified protein was dialyzed extensively against 5 mM ammonium bicarbonate (2 L chamber, three buffer changes for about 24 hours) and then lyophilized. Lyophilized Spo0F was redissolved in 20 mM Hepes (pH 6.85), 50 mM MgCl2, 6 mM BeCl2, 60 mM NaF. The final protein concentration was 1 mM. Uniformly 15N and 15N/13C-labeled samples were prepared by growth in minimal medium adjunct with [15N]ammonium chloride or [15N]ammonium chloride and [13C]glucose. NMR experiments were run on Bruker DMX500, 600 and 750 spectrometers at the National Magnetic Resonance Facility at Madison. Backbone resonances
253
BeF2 3 -Activated Spo0F
were assigned using 15N-1H heteronuclear single quantum coherence (HSQC), HNCA, and C(CO)NH spectra. Side-chain carbon assignments were determined from C(CO)NH and HCCH-total correlation spectroscopy (TOCSY) spectra. Side-chain proton assignments were determined from 3D 15N-TOCSY-HSQC, HCCH-TOCSY, and 13C-1H CT-HSQC data. Side-chain aromatic assignments were determined from a 13C-1H HSQC and 3D 13 C-edited NOESY spectra. NMR data were visualized using XEASY.32 Distance information was collected from a 3D 15N-edited NOESY, a 3D 13C-edited NOESY, and a 3D 13C-edited NOESY optimized for aromatic residues. Backbone f and c torsion angle constraints were generated from 1H, 13C and 15N chemical shift assignments using the program TALOS.18 Structure calculations were performed with DYANA 1.5, and the final structural ensemble consisted of the 20 lowest-energy conformers from a total of 60 calculated structures.17 PROCHECK analysis of 20 structures revealed that 99.5% of the residues fall within the allowed or generously allowed regions of the Ramachandran plot.33 Figures 2 – 5 were generated using MOLMOL.34
Protein Data Bank accession numbers
5.
6. 7.
8.
9. 10.
Coordinates have been deposited in the Protein Data Bank under accession code 1PUX. 11.
Acknowledgements This work was supported by NIH grants GM62117 to D.K. and GM62163 to D.E.W., and by instrumentation grants by the NSF and the Keck foundation to D.K. NMR studies were carried out at the National Magnetic Resonance Facility at Madison with support from the NIH Biomedical Technology Program (RR02301) and additional equipment funding from the University of Wisconsin, NSF Academic Infrastructure Program (BIR-9214394), NIH Shared Instrumentation Program (RR02781, RR08438), NIH Research Collaborations to Provide 900 MHz NMR Spectroscopy (GM66326), NSF Biological Instrumentation Program (DMB-8415048), and US Department of Agriculture.
12.
13.
14.
15.
16.
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18.
19.
response regulators. Proc. Natl Acad. Sci. USA, 96, 14789 – 14794. Birck, C., Mourey, L., Gouet, P., Fabry, B., Schumacher, J., Rousseau, P. et al. (1999). Conformational changes induced by phosphorylation of the FixJ receiver domain. Struct. Fold. Des. 7, 1505 –1515. Cho, H. S., Lee, S. Y., Yan, D., Pan, X., Parkinson, J. S., Kustu, S. et al. (2000). NMR structure of activated CheY. J. Mol. Biol. 297, 543– 551. Kern, D., Volkman, B. F., Luginbuhl, P., Nohaile, M. J., Kustu, S. & Wemmer, D. E. (1999). Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature, 402, 894– 898. Park, S., Meyer, M., Jones, A. D., Yennawar, H. P., Yennawar, N. H. & Nixon, B. T. (2002). Two-component signaling in the AAA þ ATPase DctD: binding Mg2þ and BeF2 3 selects between alternate dimeric states of the receiver domain. FASEB J. 16, 1964 –1966. Volkman, B. F., Lipson, D., Wemmer, D. E. & Kern, D. (2001). Two-state allosteric behavior in a singledomain signaling protein. Science, 291, 2429– 2433. Feher, V. A., Zapf, J. W., Hoch, J. A., Whiteley, J. M., McIntosh, L. P., Rance, M. et al. (1997). Highresolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: implications for phosphorylation and molecular recognition. Biochemistry, 36, 10015– 10025. Silversmith, R. E. & Bourret, R. B. (1999). Throwing the switch in bacterial chemotaxis. Trends Microbiol. 7, 16 – 22. Barak, R. & Eisenbach, M. (1992). Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor. Biochemistry, 31, 1821–1826. Tzeng, Y. L., Feher, V. A., Cavanagh, J., Perego, M. & Hoch, J. A. (1998). Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB. Biochemistry, 37, 16538 – 16545. Zapf, J. W., Hoch, J. A. & Whiteley, J. M. (1996). A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates. Biochemistry, 35, 2926– 2933. Zhao, R., Collins, E. J., Bourret, R. B. & Silversmith, R. E. (2002). Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ. Nature Struct. Biol. 9, 570– 575. Feher, V. A., Zapf, J. W., Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. & Cavanagh, J. (1995). 1H, 15N, and 13 C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR. Protein Sci. 4, 1801– 1814. Guntert, P., Mumenthaler, C. & Wuthrich, K. (1997). Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283– 298. Cornilescu, G., Delaglio, F. & Bax, A. (1999). Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR, 13, 289– 302. Wishart, D. S. & Sykes, B. D. (1994). The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR, 4, 171– 180.
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20. Volz, K. (1993). Structural conservation in the CheY superfamily. Biochemistry, 32, 11741– 11753. 21. Ganguli, S., Wang, H., Matsumura, P. & Volz, K. (1995). Uncoupled phosphorylation and activation ˚ structure of a in bacterial chemotaxis. The 2.1 A threonine to isoleucine mutant at position 87 of CheY. J. Biol. Chem. 270, 17386 –17393. 22. Zhu, X., Rebello, J., Matsumura, P. & Volz, K. (1997). Crystal structures of CheY mutants Y106W and T87I/Y106W. CheY activation correlates with movement of residue 106. J. Biol. Chem. 272, 5000– 5006. 23. Madhusudan, Zapf, J., Whiteley, J. M., Hoch, J. A., Xuong, N. H. & Varughese, K. I. (1996). Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis. Structure, 4, 679–690. 24. Jiang, M., Tzeng, Y. L., Feher, V. A., Perego, M. & Hoch, J. A. (1999). Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation. Mol. Microbiol. 33, 389– 395. 25. Parkinson, J. S. & Kofoid, E. C. (1992). Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26, 71 –112. 26. Lee, S. Y., Cho, H. S., Pelton, J. G., Yan, D., Henderson, R. K., King, D. S. et al. (2001). Crystal structure of an activated response regulator bound to its target. Nature Struct. Biol. 8, 52 – 56. 27. Hwang, I., Thorgeirsson, T., Lee, J., Kustu, S. & Shin, Y. K. (1999). Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC. Proc. Natl Acad. Sci. USA, 96, 4880– 4885.
28. Zapf, J., Sen, U., Madhusudan, Hoch, J. A. & Varughese, K. I. (2000). A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction. Struct. Fold. Des. 8, 851– 862. 29. Bren, A. & Eisenbach, M. (1998). The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY. J. Mol. Biol. 278, 507– 514. 30. Feher, V. A. & Cavanagh, J. (1999). Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F. Nature, 400, 289– 293. 31. Tzeng, Y. L. & Hoch, J. A. (1997). Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis. J. Mol. Biol. 272, 200–212. 32. Bartels, C., Xia, T.-H., Billeter, M., Guntert, P. & Wuthrich, K. (1995). The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR, 5, 1 – 10. 33. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR, 8, 477–486. 34. Koradi, R., Billeter, M. & Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51 – 55. see also pp. 29 – 32.
Edited by M. F. Summers (Received 14 April 2003; received in revised form 5 June 2003; accepted 5 June 2003)
J. Mol. Biol. (2003) 331, 245–254
The NMR Solution Structure of BeF2 3 -Activated Spo0F Reveals the Conformational Switch in a Phosphorelay System Alexandra K. Gardino1, Brian F. Volkman2, Ho S. Cho3 Seok-Yong Lee3, David E. Wemmer3 and Dorothee Kern1* 1
Department of Biochemistry Brandeis University, Waltham MA 02453, USA 2
Department of Biochemistry Medical College of Wisconsin Milwaukee, WI 53226, USA 3 Physical Biosciences Division Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, CA 94720 USA
Two-component systems, which are comprised of a single histidineaspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidineaspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF2 3 -activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF2 3 activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators. q 2003 Elsevier Ltd. All rights reserved
*Corresponding author
Keywords: response regulator; two-component systems; phosphorelay; Spo0F; NMR spectroscopy
Introduction Protein phosphorylation is one of the most common mechanisms of signal transduction. In bacteria, signaling is dominated by the phosphorylation-mediated two-component paradigm in which a histidine kinase autophosphorylates a histidine residue in response to an external Present address: H. S. Cho, Xencor, Monrovia, CA 91016, USA. Abbreviations used: NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy. E-mail address of the corresponding author: [email protected]
stimulus and subsequently transfers the phosphate moiety in a magnesium-dependent manner to an aspartate residue in the receiver domain of the second component, the response regulator. Phosphorylation activates the response regulator, which is then capable of eliciting a cellular response, usually as regulation of gene expression, as many response regulators are transcription factors. Phosphatases as well as the intrinsic phosphatase activity of the response regulators themselves control the duration of the signaling event, and the lifetime of this chemical signal correlates strongly with the biological role that the response regulator plays in the cell. More than one two-component signaling module can be connected in tandem to create a complex signal transduction pathway called a
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved
246 phosphorelay.1 This extended two-component signaling pathway was first identified in the sporulation of Bacillus subtilis and has also been found in eukaryote.2,3 The first components of this His-Asp-His-Asp phosphotransfer sequence are the sensor histidine kinases, KinA and KinB, which both autophosphorylate in response to an environmental stimulus. This phosphate moiety is then transferred to the first of two response regulators in the pathway, Spo0F (Scheme 1). Spo0B, the second histidine kinase and phosphotransferase, receives the phosphate group from phosphorylated Spo0F and transfers it to the final response regulator in the pathway Spo0A, which transcriptionally represses the AbrB gene, allowing for the expression of various genes that become activated at the onset of stationary phase.1 Two phosphatases, RapA and RapB, can catalyze the dephosphorylation of Spo0F, and Spo0E has been shown to dephosphorylate Spo0A should the event of sporulation become inappropriate or superfluous. Thus, the phosphorelay employs several points of regulation before eliciting a final cellular response, which is desirable for a pathway that controls the energetically costly process of sporulation. The focus of this study is to characterize the structural basis of activation in the phosphorelay response regulator, Spo0F. While a number of receiver domain structures from response regulators have been solved in their inactive states, a detailed structural comparison with regard to their activated states has proven to be difficult due to the short lifetime of the phosphorylated state. Recently, this obstacle has been overcome by the use of beryllofluoride as a phosphate analogue.4 Comparison of inactive –active receiver domain pairs from different two-component
BeF2 3 -Activated Spo0F
systems has revealed that they use a common signaling surface; however, different types of structural rearrangements have been found to occur upon activation.5 – 8 Recent NMR dynamics data measured in the response regulator NtrC in different functional states9 as well as Spo0F in its inactive state10 in combination with biochemical data11,12 suggested a pre-existing equilibrium between the inactive and active states before phosphorylation as a possible general feature of response regulators. To date, structures of receiver domain pairs in their inactive and active states have been solved only for simple His-Asp two-component signaling pathways. In contrast, Spo0F is a receiver domain that functions as a phosphorelay protein, transferring its phosphate group to a second histidine kinase, unlike multidomain response regulators that activate a downstream partner through protein– protein interactions. While receiver domains of response regulators from twocomponent and phosphorelay systems are structurally similar in their inactive, non-phosphorylated state, we address the question of whether the additional interaction in a phosphorelay has associated distinct structural changes. To address this, we have solved the NMR solution structure of the BeF2 3 -activated form of Spo0F, and report here the first structure of an activated response regulator from a phosphorelay system.
Results and Discussion NMR structure determination of activated Spo0F Spo0F is a 124 residue single-domain response
Scheme 1. Sporulation signal transduction pathway in B. subtilis. The His-Asp-His-Asp phosphate transfer is termed a phosphorelay. Sporulation is finally initiated by phosphorylated Spo0A, which represses the AbrB gene. Genes involved in sporulation that are kept silent by the AbrB repressor during vegetative conditions are now upregulated. Arrows represent the phosphate transfer.
247
BeF2 3 -Activated Spo0F
regulator consisting of just a conserved receiver domain. The NMR solution structure of inactive Spo0F solved by Feher et al. revealed the characteristic b5/a5-barrel scaffold architecture found for the receiver domains of all response regulators of known structure to date.10 The active site is composed of residues Asp10 and Asp11 (Mg2þ coordination), Asp54 (the site of phosphorylation), and the invariant residue Lys104. Although the half-life of phosphorylated Spo0F (Spo0F~P) is relatively long (five or more hours in vitro) among the superfamily of response regulators,13,14 detailed structural analysis of Spo0F~P was further complicated by the inability to phosphorylate Spo0F completely. Therefore, we chose an alternative approach to determine the three-dimensional structure of Spo0F on the basis of the finding that beryllofluoride can act as an acyl phosphate analogue by forming a tight complex with the site of phosphorylation (Asp54). Beryllofluoride has been shown to fully activate several response regulators,4 and this chemical modification has aided in solving structures of response regulators in their activated states, including CheY,6 NtrC (D.E.W., unpublished results), DctD,8 and a complex of CheY with its phosphatase CheZ.15 First, the experimental conditions for BeF2 3 activation of Spo0F were screened using 1H15NHSQC spectra in order to evaluate the yield of BeF2 3 labeling. It was found that Spo0F could be converted quantitatively into a stable, active form using 50 mM MgCl2, 6 mM BeCl2, and 60 mM NaF. Standard triple-resonance NMR experiments using uniformly 15N-labeled or 15N/13C-labeled BeF2 3 -Spo0F were performed to fully assign the backbone and 90% of the side-chain resonances. The results were compared to previously published chemical shifts for the inactive form of Spo0F16 (Figure 1). The Lys104-Pro105 peptide bond was found to be in a cis conformation, as verified by the typical 1Ha – 1Ha sequential nuclear Overhauser effect (NOE) in a 13C-edited NOESY.
Distance and torsion angle constraints obtained from NMR data were used for calculating 60 structures using DYANA 1.5 and TALOS.17,18 The 20 lowest-energy conformers with a target function of less than 5 and minimal violations are shown in Figure 2A, and structural statistics are presented ˚ was in Table 1. An r.m.s. deviation value of 0.77 A reached for secondary structure backbone elements ˚ for the as well as an r.m.s. deviation value of 0.83 A backbone excluding the N and C termini (residues 5– 120). Conformational changes in Spo0F upon activation After backbone assignments were completed, chemical shift mapping provided initial identification of regions in Spo0F that were altered upon BeF2 3 activation (Figure 1). In addition to amide chemical shift changes detected in regions previously identified in other response regulators, including helix 3, strand 4, helix 4 and strand 5 (known as the “3445 face”) and expected changes 7 in close proximity to the site of BeF2 3 modification, additional changes were detected in helix 1. The 13 a C chemical shift changes were smaller than 1 ppm for most regions of the protein, indicative of no significant changes in secondary structure upon activation,19 consistent with previous observations for other inactive/active response regulator structures.6 Larger 13Ca chemical shift changes were seen for residues in close proximity to the binding site of beryllofluoride as well as the N-terminal region of helix 4. Significant rearrangements of secondary structure elements were observed in BeF2 3 -activated Spo0F compared to the NMR structure of the inactive form. A superposition of the structures ˚ for parts resulted in an r.m.s. deviation of 1.19 A of the protein involved in secondary structure elements (Figure 2B). While helix 1 is displaced by half a helical turn downwards in BeF2 3 -Spo0F,
Figure 1. Chemical shift mapping to identify conformational changes upon BeF2 3 activation. Backbone amide chemical shift changes between Spo0F and BeF2 3 -Spo0F DvNH were calculated according to the following formula: ððvNinactive 2 vNBeF3 Þ2 þ ðvHNinactive 2 vHNBeF3 Þ2 Þ1=2 : Secondary structure elements are shown symbolically.
248
BeF2 3 -Activated Spo0F
Table 1. Structural statistics for BeF2 3 -Spo0F Number of NOEs Number of upper distance constraints Total Intraresidual Short-range Medium-range Long-range Dihedral angle restraintsa Total restraints ˚ 2) DYANA target function (A Maximum violations Upper limits Van der waals Torsion angles (radian) ˚ )b r.m.s.d. to the mean for N, Ca, and C0 (A ˚ )b r.m.s.d. to the mean for heavy atoms (A r.m.s.d. to the mean for N, Ca, C0 secondary ˚ )c structure elements (A Ramachandran analysis (%)d Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions a b c d
4221 1835 602 540 310 383 165 2000 4.20 ^ 0.42 0.44 ^ 0.10 0.24 ^ 0.05 0.09 ^ 0.04 0.83 ^ 0.17 1.48 ^ 0.18 0.77 ^ 0.19 82.4 14 3.2
Calculated using TALOS.18 For 20 conformers excluding residues 1–4 and 121–124. Residue numbers are same as reported in legend of Figure 2. Analysis was done by PROCHECK-NMR.33
towards the active site and to have an extra helical turn at the N terminus, which was confirmed by the presence of typical aNi, iþ3 NOEs in residues 87 –97 in BeF3-activated Spo0F instead of residues 90 –97 found to comprise helix 4 in the inactive form of Spo0F. The increase in 13Ca chemical shifts for residues 87 – 89 further supports the N-terminal extension of helix 4 in activated Spo0F. Comparison of activation mode in Spo0F with other response regulators
Figure 2. The BeF2 3 -activated structure of Spo0F. (A) The bundle of structures represent the 20 lowestenergy conformers calculated from DYANA 1.5, which were superimposed using the backbone atoms of residues in secondary structure elements. Secondary structure, color-coded blue for b strands and red for a helices, are as follows: b1, 5– 9; a1, 15 – 24; b2, 29 – 33; a2, 36 – 45; b3, 50– 53; a3, 62 – 72; b4, 77 –81; a4, 87 – 96; b5, 100– 103; a5, 108– 118. Loops and the N and C termini are colored in gray. (B) Superposition of the inactive conformation of Spo0F in blue (1FSP) with BeF2 3 -activated Spo0F in orange (1PUX). Residues that did not display backbone chemical shift changes between both forms were used for superposition: 5 – 9, 29 –33, 50 – 53, 36 – 45, and 108– 118, RMSD¼ 1.194. (C) Conserved Thr82 and His101 side-chains are shown in the inactive (blue) and active (orange-red) conformations.
helices 3 and 4 show an upward displacement by half a helical turn upon activation. In addition, helix 4 was found to reorient slightly
How do the structural changes upon activation in Spo0F compare to those found in other response regulators? Structural changes induced by activation already identified in the four known inactive/active receiver domain pairs are not identical, but there seems to be a common region including helix 4 and the loops close to the active site that are always affected by activation. However, the character of the conformational change in helix 4 differs from one receiver domain to another, ranging from a relatively small movement up and towards the active site (CheY, Spo0F)6 to small movements away from the active site (FixJ, DctD),5,8 and finally to a large rearrangement in helix 4 away from the active site and a rotation around the helical axis (NtrC).7 Additional changes in secondary structure elements surrounding helix 4 (helix 3 and strands 4 and 5) have been observed for several receiver domains (Figure 3). Two highly conserved residues among the family of response regulators are a hydroxylcontaining residue (Thr82 in Spo0F) and an aromatic residue (His101 in Spo0F)20 whose sidechain positions have been functionally linked to
BeF2 3 -Activated Spo0F
249
Figure 3. Comparison of inactive versus active structures of receiver domains among various response regulators. Inactive forms are colored in blue and active forms are colored in orange for: (A) CheY; (B) NtrC; (C) DctD; and (D) FixJ. Regions of the protein previously determined not to change in structure upon activation were used for superposition as follows: CheY: 8 – 11, 15 – 26, 33 – 34, 39 – 46, and 53– 56, RMSD¼ 0.85; NtrC: 4 – 9, 14 – 53, and 108–121, RMSD¼ 1.74; FixJ: 5 –8, 16 – 47, 49 – 52, 107– 120, RMSD¼0.31; DctD: 6 – 10, 14– 26, 30 – 34, 37 – 42, 51 –55, 63 – 73, RMSD¼0.3. PDB accession codes for all structures shown are as follows: 3CHY (inactive CheY), 1DJM (BeF2 3 -CheY), 1DC7 (inactive NtrC), 1DC8 (P-NtrC), 1DBW (inactive FixJ), 1D5W (P-FixJ), 1QKK (inactive DctD), and 1L5Y (BeF2 3 -DctD).
the signaling state of the protein.21,22 In CheY, reorientation of Thr87 through a direct hydrogen bond to the phosphorylated Asp in the active site was suggested to be the trigger for a reorientation of Tyr106 from a solvent-exposed position to a buried conformation, based on the results of structural studies.6 While reorientation of the hydroxylcontaining residue seems to be a common feature for response regulators, the different rotameric conformations of the conserved aromatic residue do not display a recognizable pattern. In NtrC, different conformations of Tyr101 could not be related directly to different functional states of the protein.9 In the NMR structure of inactive Spo0F, His101 is in a buried conformation, with the imidazole ring within hydrogen bonding distance of the backbone amide group of Thr82.10 A crystal structure of the Tyr13Ser mutant of Spo0F identified His101 in a solvent-exposed conformation; however, the crystals were grown at pH 4.5, which causes protonation of the imidazole ring, removing the ability to act as a hydrogen-bond acceptor.10,23 BeF2 activation of Spo0F produces a partially 3 solvent-exposed His101 and movement of Thr82 towards the active site, increasing the distance between them so they can no longer hydrogen bond (Figure 2C). Perhaps disruption of this
critical hydrogen bond is coupled to the conformational change in the 3445 face of Spo0F. This idea is supported by the observation that the His101Ala mutant of Spo0F is known to hypersporulate in vivo.24 Presumably, this mutation would stabilize the active form of the protein, thus shifting the equilibrium towards the active state even without phosphorylation. Strikingly, structural changes are also seen in helix 1, located on the opposite side of the protein with respect to the common activation surface, the first observation of changes in this region for a response regulator. Although the primary sequence of the N terminus of helix 1 is conserved or highly similar among many response regulators, some residues found in the b1 –a1 loop are not. This loop contains the invariant active-site residues Asp10 and Asp11, involved in Mg2þ binding, which in Spo0F are followed by Tyr13 and Gly14. The two latter residues are conserved in homologous Spo0F genes from Bacillus thuringiensis and Bacillus halodurans but are not conserved in response regulators from two-component systems. Tyr13 is known to provide strong specificity for the phosphatase RapB and upon mutation leads to a phosphatase-resistant form of Spo0F.13 Upon phosphorylation, the two Asp residues are
250
BeF2 3 -Activated Spo0F
observed to turn in towards the active site in Spo0F, thus pulling on the b1– a1 loop. Gly14 provides conformational flexibility in this region allowing for the rearrangement observed in helix 1. The link between activation surface and function in different receiver domains
Figure 4. Molecular recognition in Spo0F. (A) The direct phosphorelay partner, Spo0B, is displayed according to the X-ray co-crystal structure of Spo0F (green) and Spo0B (gray) (1F51);28 the side-chains of the phosphorelay residues Asp54 in Spo0F and His30 in Spo0B are highlighted in purple. The other partners, KinA and RapB, are represented symbolically due to lack of structural information. Residues implicated in protein – protein interactions between Spo0F and its interaction partners colored in red were found via alanine-scanning mutagenesis31: 12, 13, 14, 15, 16, 18, 19, 21, 34, 35, 37, 38, 56, 57, 58, 59, 81, 83, 84, 85, 86, 87, 104, 105, 106, 107, and 108. Superposition of Spo0F from the co-crystal complex (green) and (B) inactive Spo0F (blue) or (C) BeF2 3Spo0F (orange) suggests that Spo0F was crystallized in
While receiver domains use the same chemistry of phosphoryl transfer and have very similar structures, the structural changes caused by phosphorylation, or more generally by activation, are more diverse when compared in detail. These variations are essential to preserve fidelity and provide specificity given the over 30 or more twocomponent signaling pathways in a cell that follow the same basic rules of phosphoryl transfer between a His residue of a histidine kinase and an Asp residue of a response regulator.25 Upon activation of receiver domains, their function is to propagate the signal to a downstream target via protein– protein interactions and in the case of a phosphorelay component such as Spo0F, to transfer the phosphate moiety to another histidine kinase. CheY, NtrC, FixJ, and DctD interact with their downstream partners through parts of the 3445 face. The area of helix 4, strand 5 and helix 5 of activated CheY has been shown to bind the N16-FliM peptide of its downstream target FliM, a component of the flagellar switch used to reverse the direction and phenotype of swimming in bacteria.26 NtrC, a multidomain response regulator, has been shown to undergo a much larger conformational change upon activation (Figure 3B). Helix 4 was found to undergo a rotation about its helical axis as a result of activation, which together with a change in helix tilt causes the exposure of hydrophobic residues thought to contact and activate the downstream transcriptional activation domain.7 The hypothesis of signal transmission through helix 4 in NtrC was further supported by electron spin resonance experiments.27 In contrast to this positive activation in NtrC, a negative activation mechanism is seen in DctD, another response regulator that belongs to the same subfamily of s54-transcriptional activators as NtrC. BeF2 3 activation of DctD results in the disruption of the inactive dimeric interface, involving an extended helix 5, creating an active dimeric interface with contacts in helix 4 instead.8 This rearrangement releases the autoinhibition of the transcriptional activation domain by its own receiver domain. A subtle rearrangement of the b4 –a4 loop, helix 4, and strand 5 in the two-domain response regulator FixJ (Figure 3D) creates a surface for homodimerization between two receiver
its inactive conformation in complex with Spo0B. Regions of superposition for each pair of structures are residues 5 – 9, 29 –33, 36 –45, 50– 53, and 108– 118.
BeF2 3 -Activated Spo0F
domains. This dimerization relieves the repression of the C-terminal DNA-binding domain by its own receiver domain.5 Spo0F performs a function distinct from the response regulators described above, in that it
251
transfers the phosphate moiety to a second histidine kinase, Spo0B. For this function, Spo0F uses a very different part of the protein than CheY, NtrC, DctD, or FixJ. In the co-crystal structure of a Rap phosphatase-resistant mutant of Spo0F (Tyr13Ser)
Figure 5. Correlation between conformational changes upon activation and backbone dynamics. (A) 15N Amide chemical shift perturbation upon activation (in ppm); (B) conformational exchange dynamics in Spo0F previously reported as chemical exchange term Rex calculated from NMR relaxation experiments;30 and (C) structural displacements ˚ upon BeF2 3 activation (in A) shown as a continuous color scale. The correlation supports a pre-existing equilibrium model.
252
and Spo0B, helix 1 and all five b –a loops of Spo0F were found to contact Spo0B (Figure 4A).28 Interestingly, the residues found to make contacts to Spo0B map to regions of Spo0F that exhibit structural displacements when activated with beryllofluoride. The orientation between Spo0F and Spo0B in the co-crystal structure revealed that the side-chain of Asp54 in Spo0F is in close proximity to the phosphate-accepting residue His30 in Spo0B. The crystals were grown in the presence of aluminum fluoride that was not seen in the electron density, and the question remains of whether this complex represents the active or inactive form of Spo0F. A superposition of the NMR structures of inactive and BeF2 3 -Spo0F with the X-ray structure of Spo0F in the complex clearly reveals that Spo0F is in the inactive conformation in the crystal complex (Figure 4B and C). The affinities of nonphosphorylated and phosphorylated Spo0F for Spo0B are not known. However, the fact that the complex crystallized with proteins in their inactive states suggests that they have a substantial affinity before phosphorylation. Similarly, the affinity of active CheY for the N16-FliM peptide increases by only 20-fold upon activation of CheY.29 In the Spo0F/Spo0B co-crystal structure, an extra turn at the top of helix 428 is present that is seen also upon BeF2 3 activation. This region of the protein is known to exhibit conformational exchange in inactive Spo0F, as shown by previous 15N relaxation backbone dynamics studies.30 Crystallization may stabilize the species that contains the extended helical arrangement. Spo0F interacts with the first histidine kinase in the phosphorelay, KinA (or KinB) and can be dephosphorylated by RapB. Residues involved in these protein– protein interactions have been identified by alanine scanning mutagenesis (Figure 4A). Spo0F forms a binding site for KinA through a hydrophobic surface created by the N-terminal interface of helices 1 and 5, residues on the b3 –a3 loop directly after the site of phosphorylation (Asp54), and the b4 – a4 loop.31 Spo0F residues important for RapB specificity were identified in helix 1 and in all b –a loops except for the b2– a2 loop.13 Thus, residues of Spo0F shown to be involved in protein– protein interactions, identified via structural and biochemical data, localize to a common face of Spo0F with helix 1 playing an important role. Pre-existing equilibrium between the inactive and active state, a common feature of response regulators? The regions of Spo0F found to experience a change in chemical environment upon activation correspond to regions in inactive Spo0F shown to exhibit motions on the micro- to millisecond timescale (Figure 5A and B). Model-free analysis of 15N relaxation measurements performed by Feher et al. identified a number of residues with significant Rex contributions to R2, indicative of conformation-
BeF2 3 -Activated Spo0F
al exchange dynamics.30 These residues form a semicontiguous surface implicated in protein – protein interactions31 that correspond to the regions of Spo0F that undergo structural changes upon activation (Figure 5C). An analogous correlation was observed for NtrC.9 Regions in NtrC shown to have Rex contributions in the inactive form also experience chemical shift perturbations upon phosphorylation and structural displacements associated with activation. For NtrC, it was further verified through comparative chemical shift analyses of a series of constitutively active mutants that the conformational exchange process detected by NMR relaxation studies is an interconversion between the inactive and active states. Taken together, published data for NtrC and the structure of BeF2 3 -activated Spo0F presented here support the hypothesis put forward by Feher et al.30 of a pre-existing equilibrium in Spo0F in which the population is shifted towards the active state upon phosphorylation. Direct experimental data for NtrC and Spo0F together with the fact that basal levels of activity have been detected for response regulators prior to activation12 suggests an occasional sampling of the active-state conformation prior to chemical modification and is most likely a general feature of response regulators. This dynamic process may be crucial for activation by phosphorylation, since the active-site Asp residue is quite occluded in the inactive state, and access to the active conformation could improve the efficiency of phosphotransfer from the histidine kinase.
Materials and Methods Spo0F was cloned from genomic DNA into pET21a vectors (NOVAGEN). Spo0F proteins were expressed using Escherichia coli BL21(DE3) cells with pACYC in M9 medium supplemented with thiamine. Cells were harvested and sonicated in 25 mM Tris –HCl (pH 8.0), 10 mM KCl, 10 mM EDTA, 5 mM DTT, 0.5 mM PMSF. Lysate was subjected to centrifugation: 4 ml of 6 M GuHCl was added to the pellet and resuspended gently with a magnetic stirrer bar for two hours. Then 36 ml of 25 mM Tris – HCl (pH 8.0) was added to the resuspended pellet. Aggregates and cell debris were removed by centrifugation. The supernatant was dialyzed against 25 mM Tris (pH 8.0), 1 mM EDTA, 2 mM DTT. The protein was purified further by passage through DEAESepharose and gel-filtration columns (Superdex75, Pharmacia). The purified protein was dialyzed extensively against 5 mM ammonium bicarbonate (2 L chamber, three buffer changes for about 24 hours) and then lyophilized. Lyophilized Spo0F was redissolved in 20 mM Hepes (pH 6.85), 50 mM MgCl2, 6 mM BeCl2, 60 mM NaF. The final protein concentration was 1 mM. Uniformly 15N and 15N/13C-labeled samples were prepared by growth in minimal medium adjunct with [15N]ammonium chloride or [15N]ammonium chloride and [13C]glucose. NMR experiments were run on Bruker DMX500, 600 and 750 spectrometers at the National Magnetic Resonance Facility at Madison. Backbone resonances
253
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were assigned using 15N-1H heteronuclear single quantum coherence (HSQC), HNCA, and C(CO)NH spectra. Side-chain carbon assignments were determined from C(CO)NH and HCCH-total correlation spectroscopy (TOCSY) spectra. Side-chain proton assignments were determined from 3D 15N-TOCSY-HSQC, HCCH-TOCSY, and 13C-1H CT-HSQC data. Side-chain aromatic assignments were determined from a 13C-1H HSQC and 3D 13 C-edited NOESY spectra. NMR data were visualized using XEASY.32 Distance information was collected from a 3D 15N-edited NOESY, a 3D 13C-edited NOESY, and a 3D 13C-edited NOESY optimized for aromatic residues. Backbone f and c torsion angle constraints were generated from 1H, 13C and 15N chemical shift assignments using the program TALOS.18 Structure calculations were performed with DYANA 1.5, and the final structural ensemble consisted of the 20 lowest-energy conformers from a total of 60 calculated structures.17 PROCHECK analysis of 20 structures revealed that 99.5% of the residues fall within the allowed or generously allowed regions of the Ramachandran plot.33 Figures 2 – 5 were generated using MOLMOL.34
Protein Data Bank accession numbers
5.
6. 7.
8.
9. 10.
Coordinates have been deposited in the Protein Data Bank under accession code 1PUX. 11.
Acknowledgements This work was supported by NIH grants GM62117 to D.K. and GM62163 to D.E.W., and by instrumentation grants by the NSF and the Keck foundation to D.K. NMR studies were carried out at the National Magnetic Resonance Facility at Madison with support from the NIH Biomedical Technology Program (RR02301) and additional equipment funding from the University of Wisconsin, NSF Academic Infrastructure Program (BIR-9214394), NIH Shared Instrumentation Program (RR02781, RR08438), NIH Research Collaborations to Provide 900 MHz NMR Spectroscopy (GM66326), NSF Biological Instrumentation Program (DMB-8415048), and US Department of Agriculture.
12.
13.
14.
15.
16.
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Edited by M. F. Summers (Received 14 April 2003; received in revised form 5 June 2003; accepted 5 June 2003)