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A common dimerization interface in bacterial response regulators KdpE and TorR

¹ Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, USA
² Center for Advanced Biotechnology and Medicine and ³ Howard Hughes Medical Institute, Piscataway, New Jersey 08854, USA
⁴ Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA

Reprint requests to: Ann M. Stock, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA; e-mail: stock{at}cabm.rutgers.edu; fax: (732) 235-5289.

(RECEIVED July 23, 2005; FINAL REVISION September 1, 2005; ACCEPTED September 1, 2005)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Bacterial response regulators are key regulatory proteins that function as the final elements of so-called two-component signaling systems. The activities of response regulators in vivo are modulated by phosphorylation that results from interactions between the response regulator and its cognate histidine protein kinase. The level of response regulator phosphorylation, which is regulated by intra-or extracellular signals sensed by the histidine protein kinase, ultimately determines the output response that is initiated or carried out by the response regulator. We have recently hypothesized that in the OmpR/PhoB subfamily of response regulator transcription factors, this activation involves a common mechanism of dimerization using a set of highly conserved residues in the 4–5–5 face. Here we report the X-ray crystal structures of the regulatory domains of response regulators TorR (1.8 Å), Ca²⁺-bound KdpE (2.0 Å), and Mg²⁺/BeF₃^–-bound KdpE (2.2 Å), both members of the OmpR/ PhoB subfamily from Escherichia coli. Both regulatory domains form symmetric dimers in the asymmetric unit that involve the 4–5–5 face. As observed previously in other OmpR/PhoB response regulators, the dimer interfaces are mediated by highly conserved residues within this subfamily. These results provide further evidence that most all response regulators of the OmpR/ PhoB subfamily share a common mechanism of activation by dimerization.

Keywords: phosphorylation; transcription regulation; response regulator; TMAO respiratory system; Kdp K⁺ transport system

Abbreviations: TCS(s), two-component system(s) • RR(s), response regulator(s) • KdpE_N, KdpE regulatory domain • TorR_N, TorR regulatory domain • NCS, noncrystallographic symmetry • RMSD, root mean square deviation • TMAO, trimethylamine N-oxide • SeMet, seleno-methionine • ME, -mercaptoethanol • PDB, Protein Data Bank

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051722805.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Two-component systems (TCSs) that function as phosphotransfer pathways are among the most commonly used signaling mechanisms in bacteria. These TCSs allow bacteria to respond to and survive changes in their environments by regulating a variety of cellular activities/events such as chemotaxis, quorum sensing, and stress responses (Lukat and Stock 1993; Kleerebezem et al. 1997; Raivio and Silhavy 1997). A TCS in its simplest form consists of a histidine protein kinase and a response regulator (RR) protein, but systems can be further expanded to involve multiple phosphorelay reactions. RRs, which contain a conserved regulatory domain capable of accepting a phosphoryl group at an active site aspartate, serve as phosphorylation-dependent molecular "switches" and are generally found at the ends of these signaling pathways. The lifetime of phosphorylated RRs ranges from seconds to hours, a function of the autophosphatase activity of RRs and the inherent instability of the acyl phosphate modification. Beryllofluoride (BeF₃^–), a noncovalent complex that mimics the phosphoryl group (Chabre 1990; Yan et al. 1999; Cho et al. 2001), has been used extensively to lock RRs in their active states for structural and biochemical analyses.

Multiple-domain RRs can be classified by their effector domain architecture with the major group consisting of transcription factors (for reviews, see Robinson et al. 2000 and Stock et al. 2000). The largest subclass of RR transcription factors is the OmpR/PhoB subfamily, which, in Escherichia coli, accounts for almost half of the RRs in the genome (Mizuno 1997). The most extensively characterized RRs of this subfamily are known to bind in tandem to DNA direct repeats (Harlocker et al. 1995; Simon et al. 1995; Blanco et al. 2002). Structural studies of two full-length members of this subfamily have indicated that the recognition helix of the effector domain is exposed to solvent in the inactive state and is not sterically hindered by the regulatory domain (Buckler et al. 2002; Robinson et al. 2003). We recently reported the crystal structure of the BeF₃^–-activated regulatory domain of E. coli ArcA (ArcA_N–BeF₃^–), the first structure of an active regulatory domain from the OmpR/PhoB subfamily. Additionally, the structure of the BeF₃^–-activated regulatory domain of E. coli PhoB (PDB ID 1ZES [PDB] ) has recently been solved (Bachhawat et al. 2005). Conserved features at the dimer interfaces of these RRs led us to postulate a common mechanism of activation for members of the OmpR/PhoB subfamily involving symmetric dimerization of the regulatory domains via their 4–5–5 faces (Bachhawat et al. 2005; Toro-Roman et al. 2005).

Two other members of the OmpR/PhoB subfamily, KdpE (Polarek et al. 1992; Walderhaug et al. 1992) and TorR (Simon et al. 1994), are the RRs of the E. coli TCSs KdpD–KdpE and TorS–TorR, respectively. The KdpD–KdpE TCS is involved in osmoregulation and K⁺ sensing. Under K⁺-limiting conditions of growth, KdpD and KdpE induce expression of the kdpFABC operon, which encodes the K⁺-translocating Kdp-ATPase involved in high affinity K⁺ uptake (Laimins et al. 1978; Rhoads et al. 1978). The TorS–TorR TCS is responsible for the tight regulation of the torCAD operon, which encodes the tri-methylamine N-oxide (TMAO) reductase respiratory system in response to anaerobic conditions and the presence of TMAO (Jourlin et al. 1996). Sensing of TMAO by the unorthodox histidine kinase TorS regulates a four-step phosphorelay that results in phosphorylation of TorR and transcription of the torCAD operon (Jourlin et al. 1997). Induction of this operon allows E. coli to utilize TMAO as an additional electron acceptor during anaerobic respiration. In addition to regulating the torCAD operon, the TorS–TorR TCS has been shown to activate alkaline-stress defenses and repress acid-stress mechanisms in order to cope with alkalinization of the medium resulting from the reduction of TMAO to trimethylamine (Bordi et al. 2003). In this article we report the X-ray crystal structures of the regulatory domains of KdpE bound to Ca²⁺ (KdpE_N-Ca²⁺) and Mg²⁺/BeF₃^– (KdpE_N–BeF₃^–), and of TorR (TorR_N). Both KdpE_N and TorR_N form twofold rotationally symmetric dimers by juxtaposing their 4–5–5 faces. These dimerization interfaces have been analyzed and compared to those of two other OmpR/PhoB subfamily members for which structural information is available in the literature, E. coli ArcA_N–BeF₃^– (Toro-Roman et al. 2005) and Streptococcus pneumoniae MicA_N (Bent et al. 2004).

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Overall structures of KdpE_N and TorR_N
The structures of KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– were solved to a resolution of 2.0 Å and 2.2 Å, respectively, by molecular replacement methods, starting from a model of ArcA_N–BeF₃^– (see Materials and Methods). Initially we tried to activate KdpE_N in solution using BeF₃^–, but the protein precipitated at concentrations higher than 1 mg/mL, making this an obstacle for successful crystallization. Consequently, unactivated KdpE_N was used in crystallization trials. Crystals were obtained in the presence of Ca²⁺ and were subsequently used for solving the structure. Analysis of the structure revealed Ca²⁺ coordinated at the active site and the dimer conformation that we recently showed to be linked to activation in the OmpR/PhoB subfamily of RRs (Bachhawat et al. 2005; Toro-Roman et al. 2005). Indeed, trapping of OmpR/ PhoB subfamily members in this dimer conformation in the absence of an activating agent during crystallization was reported for ArcA_N and MicA_N (Bent et al. 2004; Toro-Roman et al. 2005). Knowing that the crystals contained KdpE_N dimers in an active conformation, we soaked them in solutions containing BeF₃^– with the Ca²⁺ exchanged for Mg²⁺ in order to get a complete picture of the active dimer. As expected, the crystals tolerated the addition of activating agents, and the structure was solved by molecular replacement using an alanine model of KdpE_N–Ca²⁺. The KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– structures contain one dimer per asymmetric unit, corresponding to a solvent content of ~50%. The two structures superimpose with an RMSD value of 0.46 Å for all C atoms in the dimer (see Materials and Methods).

The structure of TorR_N was solved at a resolution of 1.8 Å by calculating experimental phases from a single-wavelength anomalous diffraction experiment, using seleno-methionine (SeMet)-derivatized protein (see Materials and Methods). Four TorR_N molecules were found in the asymmetric unit, corresponding to two dimers, and a solvent content of ~29%. One TorR_N dimer corresponds to protomers with chain IDs A and B (dimer A–B), while the second dimer corresponds to protomers C and D (dimer C–D). Just as with KdpE_N, the TorR_N dimers are found in active conformations in the crystal structure. The two dimers in the asymmetric unit have an aligned RMSD value of 0.86 Å for all C atoms. TorR_N was activated using BeF₃^– and Mg²⁺ prior to crystallization, but after solving the structure and analyzing the active sites, we noticed that no BeF₃^– and no divalent metal ion were present. We suspect that either the crystallization conditions or crystal packing might have displaced BeF₃^– from the active site.

Upon examination of the crystal packing it is apparent that most of the loops in TorR_N, in addition to other secondary structural elements, are involved in lattice contacts. Specifically, contacts to other molecules are made by the 1–1 loop and the 3–3 loop of protomer A; the 1–1 loop, the 3–3 loop, and the 5–5 loop of protomer B; the 1–1 loop, the 3–3 loop, and the 5–5 loop of protomer C; and the 1–1 loop and the 3–3 loop of protomer D. These contacts result in small alterations in the positions of active site residues with the exception of Asp10, which is significantly reoriented with its side chain pointing away from the active site, incompatible with coordination to the divalent cation. Given the fact that a number of important residues within some of these loops form an integral part of the pocket where phosphorylation of the conserved aspartate occurs and that the BeF₃^– interaction is noncovalent, it is plausible that a combination of all these perturbations results in a TorR_N protein that is unable to bind BeF₃^– and Mg²⁺ at the active site. We believe, nonetheless, that the overall backbone and dimer conformation is reflective of the active state of the TorR regulatory domain.

KdpE_N and TorR_N have the traditional ()₅ fold of RR regulatory domains that consists of a central five-stranded parallel -sheet surrounded by five amphipathic helices (Volz 1993) and both form symmetric dimers mediated by the 4–5–5 faces of their regulatory domains (Fig. 1). Each of the structures has an unusual feature that results from crystal contacts with symmetry-related molecules. In KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– the active site of one protomer (chain ID A) is distorted due to the insertion of the Glu83 side chain of a symmetry-related molecule into the active site. Two conformations are observed for this Glu83: an extended conformation that reaches into the active site and another that turns away from the active site. The extended conformation of Glu83 appears to have a higher occupancy in the crystal. In the extended conformation, a carboxylate oxygen of Glu83 is positioned ~4.8 Å from the active site divalent cation, precluding the presence of a water molecule to serve as a ligand for the metal ion. In the alternate conformation, the side chain of Glu83 turns away from the active site, allowing a water molecule to coordinate to the divalent metal ion. Although weak density is observed for such a solvent molecule, it has not been included in the KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– models of protomer A, as it is incompatible with the extended conformation of Glu83. In the case of TorR_N each of the two dimers (A–B and C–D) contains a protomer (B and C) in which Thr80 and Tyr99 are found in conformations associated with an inactive state, even though the dimer is believed to mimic the active conformation (Fig. 1D). These two highly conserved residues (Thr/Ser and Tyr/Phe) comprise a molecular switch that modulates between inward and outward conformations primarily in response to phosphorylation or other activating influences. This crystal-induced TorR_N feature will be discussed in detail in the following section.

View larger version (56K): [in this window] [in a new window]   Figure 1. (A,B) Ribbon diagrams of the regulatory domains of KdpEN–BeF3– (gold) and TorRN (protomers A and B, teal). The two proteins form symmetric dimers mediated by the 4–5–5 faces. In KdpEN–BeF3– the side chains of Asp53, Ser79, and Tyr98 (gray and red), and BeF3– (magenta and salmon) are shown as stick models, and the Mg2+ ion (orange) is shown in sphere representation. BeF3– is noncovalently bound to the site of phosphorylation, Asp52, and serves as one of the ligands for the catalytic Mg2+. Ser79 and Tyr98 are conserved residues involved in the "switch" mechanism of activation associated with phosphorylation of the conserved Asp52. The equivalent residues Asp53, Thr80, and Tyr99 are shown for TorRN. (C) Alignment of KdpEN–Ca2+ (protomer B, green) vs. KdpEN–BeF3– (protomer B, gold) showing the conserved residues involved in propagation of the activation signal from the active site aspartate to the 4–5–5 face. The side chains of Asp52, Ser79, and Tyr98 (oxygens in red), and BeF3– (magenta and salmon) are shown in stick representation, with the Mg2+ (orange) and Ca2+ (green) ions shown as spheres. The 4–4 loops are further stabilized into a fixed conformation by interacting with Tyr98. Minimal differences are seen between the two structures. (D) Alignment of the four protomers found in the asymmetric unit of the TorRN crystals. Side chains of Asp53, Thr80, and Tyr99 (oxygens in red) are shown as sticks. Two dimers are formed between protomers A–B and C–D. Protomers A and D (teal) have the switch residues Thr80 and Tyr99 in an inward active conformation, while in protomers B and C (brown) they adopt an outward conformation associated with the inactive state. The conformation of the 4–4 loops in protomers B and C differs from that of protomers A and D because side chains of residues in these loops are used for crystal contacts.

View larger version (61K): [in this window] [in a new window]   Figure 2. Stereo views of the active sites of KdpEN–Ca2+ (A), KdpEN–BeF3– (B), and TorRN (C). Carbon, oxygen, and nitrogen atoms from the protein chains are shown in gray, red, and blue, respectively. The Ca2+ ion, Mg2+ ion, and water molecules are shown as green, orange, and red spheres, respectively. (A) In the active site of KdpEN–Ca2+, the Ca2+ ion shows a sevenfold coordination to the side-chain oxygens of Glu8, Asp9, and Asp52; the main-chain oxygen of Gly54; and three water molecules. Lys101, which would form an ion pair with a phosphate oxygen in a phosphorylated RR, is found in a conformation pointing toward the active site and interacting with Glu8 through a water molecule bridge. (B) The active site of KdpEN–BeF3– exhibits the same pattern of coordination observed in other activated structures of RR regulatory domains. The Mg2+ ion is octahedrally coordinated to the side-chain oxygens of Asp9 and Asp52, the main-chain oxygen of Gly54, and two water molecules. The presence of BeF3–, which is noncovalently bound to the phosphorylatable Asp52, induces changes in the 4–4 loop, locking it into a particular conformation by interacting with the main-chain nitrogens of Gly54 and Ala80, and the side-chain oxygen and N atom of Ser79 and Lys101, respectively. (C) The active site of TorRN, which has the phosphorylatable Asp53 side chain rotated ~90° in relation to Asp52 of KdpEN, exhibits the fewest number of interactions between conserved residues. The four protomers in the asymmetric unit present slightly different interactions between Asp53 and its neighboring residues (discussed in the main text). A common feature of all four protomers is that the side chain of the conserved acidic residue Asp10 points away from the active site. The active site of protomer B is shown, in which Asp53 directly interacts with a side-chain oxygen of Glu9, the N atom of Lys102, the main-chain nitrogen of Asn55, and a water molecule.

TorR_N has a somewhat different environment at the active site, since there is no BeF₃^– or metal present (Fig. 2C). The site of phosphorylation, Asp53, is found in a fixed conformation forming a hydrogen bond to the conserved active site residue Glu9, while other interactions involving Asp53 vary depending on the protomer in the asymmetric unit. Asp53 forms a hydrogen bond with the main-chain nitrogen of Asn55 in protomers A, B, and C. Salt bridges occur between Asp53 and Lys102 in protomers B and D, and another one between Glu9 and Lys102 of protomer A. A water molecule is found at hydrogen bonding distance of one of the side-chain oxygens of Asp53 in protomers A, B, and D. The other conserved acidic residue at the active site, Asp10, is found in a conformation that points away from the active site. Asp10 is involved in the coordination of the metal cation at the active site. In protomer A Asp10 forms a hydrogen bond with the main-chain carbonyl oxygen of Ala33 (2–2 loop), while in protomer B Asp10 forms a hydrogen bond with the main-chain nitrogen of Pro2 from a neighboring molecule (the initiator methionine had been post-translationally removed). In protomers C and D, Asp10 does not make any contacts and is simply pointing away to the solvent. A similar orientation for this residue was seen in the crystal structure of Spo0F (PDB ID 1NAT [PDB] ; Madhusudan et al. 1997). In the structure of Spo0F, the corresponding Asp11 residue points away from the active site and into the solvent without making any crystal contacts. Binding of a metal cation promotes movement of the Asp11 side chain of Spo0F into the active site to form the cation cavity. The absence of the metal cation could be a reason the side chain of Asp10 is oriented away from the active site in TorR_N.

In the previous section it was mentioned that protomers B and C of the TorR_N A–B and C–D dimers contain the two switch residues, Thr80 and Tyr99, in conformations representative of the inactive state, while in protomers A and D they exist in an active conformation. Examination of the environment around these residues, mainly the 4–4 loop, provides some clues as to why these residues in one of the protomers of each dimer are found in an inactive conformation. The 4–4 loop, which is mainly composed of residues 81–85, is clearly being pulled away from its expected active conformation in order to participate in contacts with symmetry-related molecules in the lattice. These interactions move the loop to a conformation away from the active site (Fig. 1D). The displacement is especially critical for the C atom of Thr80, which moves ~2.0 Å away from the active site and toward Tyr99. This appears to promote a change in the conformation of Thr80 to an outward position, with its side chain oriented away from the active site. Consequently, Tyr99 must adopt an outward conformation to avoid steric collision with the outward conformation of Thr80. It is likely that in the absence of crystal contacts, the 4–4 loop and the switch residues, Thr80 and Tyr99, would be found in an active conformation as observed in the other two protomers in the asymmetric unit.

Common dimer interface
Both KdpE_N and TorR_N form symmetric dimers that are mediated by their 4–5–5 faces. In both cases the dimers are found in the asymmetric units of the crystals. The average total surface area buried at the dimer interface for KdpE_N and TorR_N is 1800 Ų (900 Ų per monomer) and 2000 Ų (1000 Ų), respectively. As in the recently reported structure of ArcA_N–BeF₃^–, the interfaces of KdpE_N–BeF₃^– and TorR_N are mediated by a hydrophobic patch that packs the 4 helix of one monomer against the 5 helix of the other. A network of salt bridges and other electrostatic interactions confer additional stability to the interface. The majority of these interactions involve residues that are highly conserved in the OmpR/PhoB subfamily of RRs. Since KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– have identical interfaces, we will use KdpE_N to refer to both structures within this section.

In KdpE_N the core of the hydrophobic patch is formed by Ile88 (4), Leu91 (4), Ala110 (5), and Val114 (5), with additional contributions from the aliphatic portions of the side chains Glu84 (4), Lys87 (4), Glu107 (5), and Arg111 (5) (Fig. 3A). Val114 is not part of the group of highly conserved residues, and, as a result, extends the hydrophobic patch further down the helices when compared to ArcA_N–BeF₃^–, which has an asparagine at that site. The conserved intermolecular electrostatic interactions are formed between Asp97 (5) and Arg111 (5), Asp96 (4–5 loop) and Arg118 (5), and Asp92 (4) and Arg113 (5) (Fig. 3B). Arg111 is further stabilized by an additional intramolecular salt bridge to Glu107 (5). In addition, the guanidinium group of Arg117 (5) forms two intermolecular hydrogen bonds to the main-chain carbonyl oxygens of Ala95 (4–5 loop) and Leu91 (4). This interaction of Arg117 is equivalent to the one achieved by the side-chain nitrogen of Asn116 (5) in ArcA_N–BeF₃^– with the exception that Asn116 is found one helical turn above Arg117 of KdpE_N, compensating for its shorter side chain. In both cases the interaction results in additional stabilization of the 4–5 loop.

View larger version (65K): [in this window] [in a new window]   Figure 3. Conserved intermolecular interactions of KdpEN–BeF3– (A,B) and TorRN (C,D) at the dimer interface. KdpEN–BeF3– protomers are distinguished by the colors gold and green, and TorRN, by the colors teal and brown. Hydrophobic and electrostatic interactions are represented by sphere and stick models, respectively. (A,C) In both KdpEN–BeF3– and TorRN, the 4 and 5 helices are packed together through a conserved hydrophobic patch formed in KdpEN–BeF3– by Ile88 (4), Leu91 (4), Ala110 (5), and Val114 (5). Analogous conserved interactions are seen in TorRN except for an additional nonconserved hydrophobic residue in KdpEN–BeF3– (Val114) that further extends the hydrophobic patch down the path of the helices. (B,D) The interface of these dimers is further stabilized by a network of inter- and intramolecular salt bridges formed in KdpEN–BeF3– by Glu107 (5) and Arg111 (5), Asp97 (5) and Arg111 (5), Asp96 (4–5 loop) and Arg118 (5), Asp92 (4) and Arg113 (5), and Ala95 (4–5 loop)/Leu91 (4) and Arg117 (5). In TorRN the interactions are formed between Glu108 (5) and Arg88 (4), Glu108 (5) and Arg112 (5), Asp98 (5) and Arg112 (5), Asp97 (4–5 loop) and Arg119 (5), and Ala96 (4–5 loop)/Leu92 (4) and Asn115 (5). On the right side of the TorRN interface the Glu108–Arg88 interaction is bridged by the side-chain oxygen of Tyr99, which is found in an outward conformation in protomers B and C (see Fig. 1D). The side chains of Glu93 and Lys114 are involved in crystal contacts, and thus do not form the analogous salt bridge seen in KdpEN–BeF3–.

Another common feature of the dimer interfaces, including those of ArcA_N and MicA_N, is that the 5 arginine residue involved in the lower salt bridge to the 4–5 loop aspartate is stabilized in its position by a small, highly electrostatic pocket (Fig. 4). This pocket is formed by main-chain atoms of Ala72, Val73, Val75 (all part of the 3–4 loop), and Gly94 (4–5 loop), and side chain atoms of Arg68 (3) and Asp96 (4–5 loop), using the sequence numbering of KdpE (Fig. 4A). The long, positively-charged side chain of Arg68 is also highly conserved as either arginine or lysine. Superimposing all four dimers together shows an identical environment for the main-chain and side-chain atoms involved in electrostatic interactions at this site (Fig. 4B).

View larger version (65K): [in this window] [in a new window]   Figure 4. Stereo views of the environment surrounding the conserved arginine involved in the lower intermolecular salt bridge. Protein atoms are shown in stick representation with oxygen and nitrogen atoms in red and in blue, respectively. (A) A region of KdpEN–BeF3– (gold) showing the Arg118 of one protomer (superimposed with white spheres) positioned within an electrostatic pocket formed by the second protomer of the dimer. The pocket is mainly created by electrostatic interactions between atoms of residues Arg68, Ala72, Val73, Val75, Gly94, and Asp96. (B) Structural alignment of dimers of KdpEN–BeF3– (gold), TorRN (A–B dimer, teal), E. coli ArcAN–BeF3– (PDB ID 1XHF [PDB] , gray) and S. pneumoniae MicAN (PDB ID 1NXW [PDB] , salmon). The tertiary structure of the pocket is highly conserved, with identical positioning of the atoms involved in electrostatic interactions. The position of the side chain of the arginine from the adjacent protomer is also conserved.

View larger version (47K): [in this window] [in a new window]   Figure 5. Alignment of single protomers showing the conservation of residues that mediate the 4–5–5 dimer interface in active RRs of the OmpR/PhoB subfamily. The 4–5–5 faces of KdpEN–BeF3– (protomer A, gold), TorRN (protomer B, teal), E. coli ArcAN–BeF3– (protomer A, PDB ID 1XHF [PDB] , gray), and S. pneumoniae MicAN (PDB ID 1NXW [PDB] , salmon) are depicted as C traces. Sequence numbering is for KdpE. (A) Conservation of hydrophobic residues (spheres) that make up the hydrophobic patch. Dotted spheres represent hydrophobic residues that extend the conserved hydrophobic patch along the two helices but are not fully conserved in E. coli OmpR/PhoB subfamily RRs, among which nine of 15 have a hydrophobic residue at this position. (B) Conservation of residues involved in formation of electrostatic interactions (sticks). All residue side chains superimpose well with the exception of those that are situated at the periphery of the proteins and are more susceptible to solvation or formation of crystal contacts. In TorRN and MicAN, the equivalent residues of the KdpEN Asp92–Arg113 salt bridge are used for crystal contacts.

In summary, we have determined the structures of the activated regulatory domains of KdpE and TorR, both members of the highly populated OmpR/PhoB subfamily of RRs. These two structures provide further evidence for a common mechanism of activation and regulation by dimerization for members of this subfamily. Variations on the basic scheme can provide complexity to specific systems. Greater complexity can presumably be achieved by introduction of additional nonconserved intermolecular interactions during oligomerization in either inactive or active states, variation in the number and sequences of binding sites that regulate an operon, the presence of additional binding sites for heterologous transcription factors, and variation in the length of the linkers that tether the regulatory and DNA-binding domains of the RRs.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Expression and purification
A DNA fragment encoding KdpE_N, residues 1–121 with an A121Q substitution at the C-terminal residue, was PCR amplified from pEF33, which contains full-length E. coli kdpE, and subcloned into the T7 vector pJES307 (Tabor and Richardson 1985) at the NdeI and BamHI polylinker sites to create pEF27. Plasmid pEF27 was transformed into E. coli BL21(DE3) (Novagen). Cells were grown at 37°C in Luria-Bertani medium with the addition of ampicillin to a final concentration of 100 µg/mL. KdpE_N was overexpressed by induction with 0.5 mM isopropyl--D-thiogalactopyrano-side followed by incubation for 3 h. All subsequent steps were carried out at 4°C. Cells were harvested by centrifugation, washed with 50 mM Tris-Cl, 300 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA (pH 8.0), and lysed by sonication in the same buffer. The lysate was clarified by ultracentrifugation for 60 min at 80,000g. The soluble portion of the lysate was fractionated by addition of a saturated solution of (NH₄)₂SO₄ to 40% saturation. The pellet was resuspended and dialyzed overnight in 25 mM Tris-Cl (pH 8.5) (buffer A). The protein sample was loaded onto two tandem 5-mL HiTrap Q Sepharose columns (GE Health Care) and eluted with a 150-mL gradient of 0.10–0.50 M NaCl in buffer A. Fractions containing KdpE_N were pooled and the HiTrap Q chromatographic step was repeated a second time as described. After we pooled the fractions containing KdpE_N, the sample was precipitated by addition of a saturated solution of (NH₄)₂SO₄ to 40% saturation, resuspended in 25 mM Tris-Cl and 100 mM NaCl (pH 7.5) (buffer B), and subjected to gel filtration chromatography on a Superdex75 26/60 gel filtration column (GE Health Care) equilibrated in buffer B.

A DNA fragment encoding TorR_N, residues 1–122 with a L122Q substitution at the C-terminal residue, was PCR-amplified from pEF32, which contains full-length E. coli torR, and subcloned into pJES307 at the NdeI and Bam-HI polylinker sites to create pEF26. Plasmid pEF26 was transformed into the methionine auxotroph strain B834(DE3)-pLysS (Novagen) for production of SeMet-substituted TorR_N. The cell growth procedure was modified from Hendrickson et al. (1990) and described elsewhere (Robinson et al. 2002) with the cell culture being incubated overnight at 25°C after induction with 0.5 mM isopropyl--D-thiogalactopyranoside. Sonication and ultracentrifugation of SeMet TorR_N was performed as described above for KdpE_N with inclusion of 10 mM -mercaptoethanol (ME) in all solutions. The lysate was fractionated by addition of a saturated solution of (NH₄)₂SO₄ to 25% saturation. The pellet was resuspended and dialyzed overnight in 25 mM Tris-Cl and 10 mM ME (pH 8.0) (buffer C). The protein sample was loaded onto two tandem 5-mL HiTrap Q columns and eluted with a 400-mL gradient of 0–2.0 M NaCl in buffer C. Fractions containing SeMet TorR_N were pooled, precipitated with a saturated solution of (NH₄)₂SO₄ to 40% saturation, resuspended in 25 mM Tris-Cl, 100 mM NaCl, and 10 mM ME (pH 8.0) (buffer D), and subjected to gel filtration on a Superdex75 26/60 column equilibrated in buffer D.

Crystallization and data collection
Purified KdpE_N and SeMet TorR_N were dialyzed overnight into 50 mM Tris-Cl (pH 8.4), with the addition of 10 mM ME to SeMet TorR_N. Both protein samples were concentrated at 4°C with Biomax-10 concentrators to 25–35 mg/mL as determined by absorbance at 280 nm. All crystals were grown at 20°C using the hanging drop vapor diffusion method. Orthorhombic KdpE_N crystals containing one dimer in the asymmetric unit grew in 10% polyethylene glycol 8000, 0.1 M 2-morpholino-ethanesulfonic acid (pH 6.0), and 0.1 M calcium acetate. Cryo-protection was achieved by sequential transfer of crystals into six solutions containing reservoir solution in addition to ethylene glycol in increments of 5% to a final concentration of 30% ethylene glycol. Crystals were frozen in a 100 K nitrogen stream. KdpE_N was found to precipitate when BeF₃^– was added to the solution at protein concentrations higher than 1 mg/mL. To obtain the BeF₃^–-bound data set, apo-KdpE_N crystals were transferred into a fresh reservoir solution containing 5.3 mM BeCl₂ and 33 mM NaF, in which the 0.1 M calcium acetate had been replaced with 0.1 M MgCl₂, and were incubated for 3 h. Monoclinic SeMet TorR_N crystals containing two dimers per asymmetric unit grew in 26% polyethylene glycol 2000 monomethyl ether, 0.4 M (NH₄)₂SO₄, 0.1 M sodium acetate trihydrate (pH 6.0), and 1%–3% glycerol. Crystals were cryo-protected by sequential transfer into four solutions containing reservoir solution in addition to glycerol in increments of 5% to a final concentration of 20% glycerol. Crystals were frozen in a 100 K nitrogen stream. Although SeMet TorR_N crystals were grown in the presence of BeF₃^–, there was no indication of incorporation into the active site (see Results and Discussion).

KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– native data sets were each collected from a single crystal grown over a period of 2–3 d. A second KdpE_N–Ca²⁺ data set was collected from the same crystal with the use of a beam attenuator to properly measure overloaded low-resolution reflections obtained in the first data set. A SeMet TorR multiwavelength anomalous diffraction data set was collected from a single crystal grown in ~1 wk. Data sets were collected at beamline X4A at the National Synchrotron Light Source at Brookhaven National Laboratory. All data were processed and scaled with DENZO and SCALEPACK (Otwinowski and Minor 1997). Details and statistics for each data set are listed in Table 1.

The structure of TorR_N was solved by single-wavelength anomalous diffraction phasing using CNS with 11 out of 16 possible selenium sites found corresponding to four molecules in the asymmetric unit. The reflections were phased to a resolution of 1.8 Å using CNS, and an initial interpretable map was obtained through density modification by phase extension with solvent flattening, histogram matching, and twofold NCS averaging (two dimers as NCS units) as implemented in DM. A single protomer was manually traced using XtalView and subsequently was used to place the remaining three protomers in the asymmetric unit using Phaser. Model building and refinement were completed as in KdpE_N-Ca²⁺. Analysis of the structure revealed that the initiator methionine had been post-translationally removed, as the N terminus of the second amino acid in the sequence, Pro2, was found too close to and interacting with other symmetry-related molecules. The final model of TorR_N includes residues 2–122, 2–121, 2–121, and 2–121 of chains A, B, C, and D, respectively, with a final R_cryst of 0.192 and R_free of 0.244.

Data quality and structure stereochemistry for the three structures were validated using the programs SFCHECK (Vaguine et al. 1999) and PROCHECK (Laskowski et al. 1993), respectively. All structures show no residues in disallowed regions of the Ramachandran plot. Details and statistics for each data set are listed in Table 1. All figures were created using the molecular visualization program PyMOL (http://pymol.sourceforge.net/).

Structural alignments and calculation of RMSD values
Structural alignments shown in Figures 1(C and D), 4 (B), and 5 (A and B) were produced using PyMOL’s built-in fitting algorithm. Reported RMSD values for alignment of C atoms between KdpE_N–Ca²⁺ and KdpE_N–BeF₃^– dimers and between TorR_N dimers in the asymmetric unit were calculated using the program LSQKAB (Kabsch 1976) from the CCP4 package.

Protein Data Bank data deposition
Atomic coordinates and structure factors have been deposited in the Protein Data Bank (Berman et al. 2000) with PDB ID codes 1ZH2, 1ZH4, and 1ZGZ for the KdpE_N–Ca²⁺, KdpE_N–BeF₃^–, and TorR_N structures, respectively.

	Acknowledgments

 
We thank E. Fox for technical assistance with construction of expression vectors and the staff at beamline X4A at the National Synchrotron Light Source at Brookhaven National Laboratory for technical assistance. This work was supported by grant R37GM047958 from the NIH. A.T.R. was supported by NIH grants T32GM08319 and 5F31GM070142 (NRSA-MARC). A.M.S. is an investigator of the Howard Hughes Medical Institute.

	References

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