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Structure/function relationships in OmpR and other winged-helix transcription factors Linda J Kenney Response regulators are the output component of two-component regulatory systems, the predominant form of signal transduction systems utilized by prokaryotes. The majority of response regulators function as transcription factors, yet detailed descriptions of their mechanisms of DNA binding and its consequences are lacking. Versatility in the modes of DNA binding is evident with winged helix-turn-helix proteins, raising doubts that mechanisms of DNA binding will be generalizable among members of the family. The current focus of some of the research efforts aimed at understanding activation and DNA binding by response regulators is highlighted in this review.

serine or threonine, a lysine and a tyrosine. Phosphorylation of the aspartate enables the threonine hydroxyl to hydrogen-bond to the phosphoryl group, creating a space that lets the tyrosine side chain swing from the outward hydrophilic environment to the inward hydrophobic environment [3,9–11]. The major differences in response regulator function arise from the subtle ways in which dimerization, autoinhibition by the receiver domain and activation by phosphorylation affect the output function. One question that remains is: how is activation of the receiver domain by phosphorylation transmitted to the carboxyl terminus?

Addresses Department of Molecular Microbiology and Immunology, Oregon Health & Science University, 3181 South West Sam Jackson Park Road, Portland, Oregon 97201, USA; e-mail: [email protected]

The OmpR subfamily

Current Opinion in Microbiology 2002, 5:135–141 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations α CTD carboxy-terminal domain of RpoA IRF-1 interferon regulatory factor 1 RFX1 regulatory factor X1 wHTH winged helix-turn-helix

Introduction Two-component regulatory systems represent the major paradigm for signal transduction in prokaryotes. In lower eukaryotes, two-component phosphorelay systems feed into mitogen-activated protein (MAP) kinase pathways to alter gene expression. The simplest systems utilize a sensor kinase and a response regulator. The sensor is often a membrane protein that senses a change in environmental conditions and is autophosphorylated by ATP on a histidine residue. The phosphoryl group is transferred onto an aspartate of the response regulator, which activates the regulator and alters its output, most often resulting in a change in gene expression (see [1,2] for recent reviews). In this review, the current status of the knowledge of activation by phosphorylation and of mechanisms of DNA binding by response regulators is discussed. In doing so, I highlight areas in which I believe we lack critical information, and emphasize questions that remain unanswered.

Response regulator activation The structures of several single-domain response regulators and amino-terminal phosphorylation or receiver domains have been solved [3–8]. They reveal a doubly wound five-stranded α/β fold; the five-stranded parallel β-pleated sheet is surrounded by five α helices. Highly conserved active site residues include the phosphorylated aspartate, a

OmpR is an extensively studied member of a subfamily of response regulators, such as PhoB, VirG, ResD and CpxR, that has fourteen homologues in Escherichia coli alone [12]. A detailed description of DNA binding has not been determined for any member of the OmpR subfamily of transcription factors. However, two co-crystal structures of response regulators bound to their targets in other subfamilies have been determined and will contribute substantially to our rudimentary knowledge of DNA–response-regulator interactions [13••,14••]. The EnvZ/OmpR system regulates the porin genes ompF and ompC in response to changes in osmolarity (Figure 1). Earlier studies localized the OmpR DNA-binding domain to the carboxyl terminus [15]. The isolated carboxyterminal domain binds DNA with significantly lower affinity than the full-length protein. In contrast to response regulators Spo0A and PhoB, the carboxy-terminal domain of OmpR alone cannot activate transcription [16–19]. It is worth noting that OmpR requires α CTD (the carboxyterminal domain of the alpha subunit [RpoA] of RNA polymerase), whereas the others do not [20,21].

Structure of OmpRc The carboxy-terminal domains of the OmpR subfamily contain an amino-terminal four-stranded β sheet, a central three-helical bundle and a carboxy-terminal β hairpin [22,23,24••]. Residues that form the hydrophobic core are the most highly conserved throughout the subfamily (Figure 2). The binding motif is a winged helix-turn-helix (wHTH). The turn of the HTH in OmpR is unusually long — 10 amino acids compared to the more usual 3–4 in other HTH proteins (Figure 3). The turn has been referred to as the α loop — an unfortunate designation, as it has not yet been demonstrated to interact directly with the RNA polymerase α subunit. The recognition helix is also long and both ends are implicated in contacting DNA, as mutations at either end

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Figure 1

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Osmoregulation of ompF and ompC by OmpR. OmpR binds in a hierarchical fashion to three sites between –100 and –40 upstream from the transcriptional start site of ompF and ompC [39,41,60] and an upstream site between –380 and –350 at ompF [61]. At low osmolarity, OmpR–P is bound at F4, F1, F2, F3 and C1, and OmpF is expressed. At high osmolarity, OmpR–OmpR interactions stimulate formation of a loop that represses ompF. OmpR–P binds to C2 and C3, leading to activation of ompC. The different shapes of OmpR at low and high osmolarity signify a change in OmpR conformation. Reproduced with permission from [34•].

ompC

affect porin phenotypes [25–27]. However, it was not possible to model the helix with both ends in contact with DNA [23]. Proposed amino-acid–base contacts are S200 and V203 [28,29]. V203M is the original OmpR2 mutant (OmpR472, F+ C–) [30,31], deficient in high affinity binding to the C1 site but not to F1 [32]. Substitution of a single base in C1 with that of F1 (Figure 4) enables V203M to bind with high affinity, making V203 a good candidate to contact DNA, or be very close to a contact site [32].

Rather, phosphorylation drives a conformational change in OmpR that promotes interaction between OmpR molecules at adjacent sites and also with RNA polymerase, leading to the activation or repression of transcription. In contrast, in the two-domain response regulator NarL (in the FixJ subfamily), its recognition helix is blocked by the amino terminus and phosphorylation results in a conformational change that exposes the recognition helix and relieves this autoinhibition [13••,37].

A proposal that the recognition helix makes different amino-acid–base contacts at ompF and ompC sites suggests that the turn alters its conformation to accommodate two binding modes [32]. High temperature factors for residues in the turn imply that it may be conformationally flexible [23]. Limited proteolysis studies identified this region as being surface-exposed; its exposure was unaffected by protein phosphorylation [33]. Our view is that steps subsequent to phosphorylation are important for signaling and the ompF-bound form of OmpR is not equivalent to the ompC-bound form [34•]. Phosphorylation alters the binding affinity of OmpR, but its affinity at ompF and ompC is too similar to allow differential expression [35], as required by the affinity model of porin regulation [36].

Binding specificity The structure of the DNA-binding domain of PhoB has been determined by NMR and is similar to that of OmpR [24••]. The major differences lie in the turn of the HTH, which, in PhoB, is three amino acids shorter, with one consequence that the PhoB recognition helix is longer than that of OmpR (Figure 2) [24••]. It was proposed that the conformation of the PhoB turn is different from that of OmpR. This is significant, as substitutions W184R, G185R, V190M and D192G, which alter the PhoB interaction with σ70 (the sigma subunit [RpoD] of RNA polymerase), map to this region [18]. The turn structure is relatively poorly defined, indicating that it is flexible [24••]. DNA causes the largest chemical shift changes in the PhoB recognition

Figure 2 Sequence alignment and secondary structure assignments of the DNA-binding and transactivation domains of PhoB and OmpR. The amino acids that form the hydrophobic cores of PhoB and OmpR are highlighted in yellow. Around the turn between helix 2 and helix 3, the residues that may affect the interaction with RNA polymerase are indicated by red filled circles. The figure is based on a figure from [24••], with the authors’ permission.

Structure/function relationships in OmpR and other winged-helix transcription factors Kenney

Figure 3

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The secondary structure of OmpRc. The wings of the wHTH motif are loops that extend out from the recognition helix in the threedimensional structure of OmpRc, labeled W1 and W2. The helix-turnhelix is α2–α–loop–α3. The OmpR DNA-binding domain of E. coli from [23] (PDB ID# 1OPC) was modeled using the SwissPdb Viewer software package. The graphical structure was then rendered using POV-Ray 3.1g.r2 software on a Macintosh G4 computer.

helix, suggesting that the amino-terminal residues of the PhoB recognition helix R193 and T194 are responsible for specific DNA recognition [24••]. Specificity in hepatocyte nuclear factors (HNF-3/forkhead, HFH) is determined by a 20-amino-acid segment adjacent to the recognition helix [38]. This region may also be important for OmpR and PhoB specificity, as many of the amino acids in their recognition helices are identical or conservative replacements, even though OmpR and PhoB clearly recognize different DNA sequences. Another difference between OmpR and PhoB is in the loop between the recognition helix and the carboxy-terminal β hairpin. OmpR is a monomer in solution, though it presumably binds to DNA as a dimer [39–41]. DNA binding may drive dimerization. NarLc is also a monomer, but binds DNA as an antiparallel dimer in a tail-to-tail fashion, burying 800Å 2 of surface area [13••]. This type of mechanism has also been proposed for UhpA [42]. In contrast, the transcription regulatory factor X1 (RFX1) binds co-operatively to DNA. In the co-crystal structure, however, two copies of the protein make a symmetric complex in which the two monomers have no intermolecular interactions, and co-operativity appears to be mediated by protein-induced DNA deformation [43••].

Mechanism of transcriptional activation Response regulators from the same subfamily can interact with different subunits of RNA polymerase. Truncations in the α CTD inhibit expression from an OmpR-dependent promoter. In contrast, PhoB stimulates transcription in the absence of the α CTD [20]. Mutations in rpoD (and

F1 F2 F3 F4 C1 C2 C3

∗ ACTTTTGGTTACATATTT TCTTTTTGAAACCAAATC TATCTTTGTAGCACTTTC TTACATTGCAACATTTAC ACATTTTGAAACATCTAT GATAAATGAAACATCTTA AGTTTTAGTATCATATTC

CTGTCATA(A/T)A(T/A)CTGTCA(C/T) Current Opinion in Microbiology

An alignment of OmpR-binding sites from the ompF and ompC regulatory regions. The sites are approximately 18 base pairs, composed of two half-sites, and an OmpR molecule binds to each half-site. The sites are poorly conserved but share some features, most notably the GXXXC that is present in all of the binding sites. The two sets of AC base pairs (underlined) were found to be the most critical for OmpR binding in an in vivo analysis of binding-site mutants [62]. The asterisk (*) indicates the position where mutation of the C1 base (A) to the F1 base (T) enabled OmpRV203M to bind to C1 with high affinity [32]. In contrast, the promoter regions of the pho genes share a common regulatory element, a pho box of approximately 18 nucleotides. PhoB binds to a TGTCA sequence and each pho box contains two binding sites. The binding sites are located 10 nucleotides upstream from the –10 region [63].

not in rpoA) are specifically defective in the expression of the pho genes, suggesting that PhoB interacts with the σ subunit [44,45]. Mutations in rpoA that decreased activity of ompF–lacZ or ompC–lacZ fusions, altered ompF expression, but had only minor effects on ompC [46]. Perhaps RNA polymerase interactions with OmpR are distinct at ompF and ompC. OmpR activation mutants repressed ompF (therefore, the mutants could bind to DNA), but were unable to activate ompC [29]. These mutants, G191S and E193K, map to the turn of the HTH, hence the designation ‘α loop’. The underlying assumption is that the binding interactions at ompF and ompC are equivalent events. Recent biochemical characterization of OmpR mutants suggests that this is not so [32,34•]. These ‘positive control’ mutants may, in fact, be binding mutants at ompC. In the family member ToxR, an α loop substitution, W68L, prevented binding to the toxT promoter [47]. Additional support for the turn being involved in DNA binding and not the RNA polymerase interaction site comes from a co-crystal structure of the related interferon regulatory factor 1 (IRF-1) and DNA. IRF-1 interacts with DNA via three large loops between β2–α2, α2–α3 (the turn) and β3–β4 [48]. Activation mutants that affect the interaction of PhoB with σ70 are also located in the turn, the location that in OmpR may be required for interaction with α. Although the OmpR mutants may not be true activation mutants, there is evidence that PhoB substitutions in the turn are altered in their interaction with RNA polymerase. Addition of RNA polymerase to wild-type PhoB extended the DNase I

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Figure 5 (a) 225

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Model of the positions of the two OmpRc monomers on linear B-form DNA in an arrangement consistent with DNA affinity cleavage data reported in [40] using o-phenanthroline-copper. Side view (a) and top view (b). The top strands are light gray and the bottom strands are dark gray. Reproduced, with permission, from [40].

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footprint. However, with the PhoB activation mutants, it did not [18]. Other potential candidates for OmpR interaction with α are P179 and S181, between α-helices 1 and 2 [25,28,49,50]. In this region of PhoB, changes in chemical shifts were also observed upon addition of DNA [24••].

The amino-terminal β-sheet The effector domains of OmpR subfamily members differ significantly from other wHTH proteins by the presence of an amino-terminal four-stranded antiparallel β-sheet. The β sheet packs against α helices 1 and 3 (the recognition helix) and contributes six amino acids to the hydrophobic core [22,23]. No function has been ascribed to this region of OmpR yet, and knowledge of interdomain orientations is hampered by the lack of full-length structures. A recently determined structure of DrrD (in the OmpR subfamily from Thermatoga) indicates only a low level of direct interdomain interaction [51] (see Update).

The role of the wings It was originally assumed that wHTH proteins shared a common DNA-binding mode. However, wHTH proteins are remarkable in the versatility that their wings exhibit with respect to DNA contacts. In the first structures determined, wHTH proteins co-crystallized with DNA contained wings that make DNA backbone contacts, with the recognition helix presented to the major groove [52]. In contrast, RFX1 makes most of its major groove DNA contacts using wing 1 [43••]. The recognition helix overlies the minor groove and makes one DNA contact. In IRF-1, the recognition helix tracks the major groove tangentially, as opposed to the more common perpendicular orientation.

Contacts to bases within the major groove occur at a GAA core sequence within the binding element [48]. This GAA motif is present in three out of seven OmpR binding sites from the porin regulatory regions (Figure 4). The variations in the mode of DNA binding by wHTH proteins raise the question of how OmpR binds to DNA. Is it via recognition helix contacts in the major groove, and what is the orientation of OmpR at its binding site? Site-directed DNA cleavage at the OmpR binding site F1 with OmpR mutants S163C and V225C led to the proposal that OmpR binds asymmetrically as a tandemly arranged dimer with the recognition helix of each monomer in the major groove (Figure 5) [40]. However, S163C has reduced affinity for ompF DNA ([40]; K Mattison, LJ Kenney, unpublished data). The model also made the then reasonable assumption that the recognition helix would make major-groove contacts. In light of the range of DNA binding and recognition observed with wHTH proteins, a re-examination of the OmpR orientation at its binding site(s) should prove worthwhile.

The linker Receiver and effector domains are connected by a variablelength linker that is disordered in reported structures [37,53,54]. The OmpR linker is sensitive to proteolysis, which is altered by phosphorylation and DNA binding, indicating a role for the linker in communication between domains [33,55]. Alterations in the linkers of the two-domain response regulators NarL and CheB affect the output domains ([56]; M Jarvis, RP Gunsalus, personal communication). The OmpR linker (15 amino

Structure/function relationships in OmpR and other winged-helix transcription factors Kenney

acids long) is longer than the PhoB linker (nine amino acids long). Chimeras containing the receiver domain of PhoB and the carboxy terminus of OmpR bind to F1 DNA when they contain the OmpR linker, but not with the PhoB linker (DM Walthers, V Tran, LJ Kenney, unpublished data). Linker substitutions in OmpR containing only nine amino acids are unable to activate ompF or ompC expression, highlighting the flexibility required in this region for OmpR function (K Mattison, R Oropeza, LJ Kenney, unpublished data).

New roles for response regulators: priming proteins, overlapping sites and co-regulators? New roles for response regulators are becoming apparent. In E. coli, the curli operon contains the csgDEFG genes, which are responsible for the transcription, transport and assembly of curli fibers (aggregative fimbriae or adhesins), the products of the csgBA genes. A recent study reported that both OmpR and CpxR bind to the same regulatory region of csgD [57]. In Salmonella typhimurium, OmpR and SsrB bind to srfH, a type-III-secreted effector (X Feng, R Oropeza, LJ Kenney, unpublished data). At the toxT promoter in Vibrio cholerae, the OmpR-like regulator TcpP binds downstream of the ToxR activator. TcpP appears to be the direct activator, whereas ToxR provides a supportive role [47]. In Bacillus subtilis, the response regulator DegU (in the FixJ subfamily) is responsible for expression of the transcription factor ComK. The DegU-binding site overlaps the ComK-binding site and DegU binding stimulates ComK binding. It was proposed that DegU acts as a priming protein to prime the autoregulatory expression of comK [58].

Conclusions The identification of the phosphorelay and its components and their interactions increased our understanding of the complexity of the two-component system [59]. The combinations of response regulators and their dual functions at promoters increases this complexity further. New structures of response regulators bound to DNA will shed light on the mechanisms of transcriptional regulation in this important group of DNA-binding proteins. Perhaps signal transduction in prokaryotes will prove to have as complex multiple interactions as those existing in eukaryotes.

Update The crystal structure of the transcription factor DrrD has been determined to 1.5 Å resolution [51,54]. This report provides the first full-length structural information for the OmpR subfamily of response regulators. A small interdomain interface (245 Å2) occurs between α5 of the amino terminus and the antiparallel sheet of the carboxyl terminus (Figure 3). The orientation of the amino-terminal α4 helix is novel, implicating conformational mobility in a key segment in phosphorylation-induced activation. This orientation is not present in the PhoBN structure [64]. A comparison of key residues that contribute to this altered orientation among the family members suggests that this unusual orientation is unlikely to be prevalent in OmpR/PhoB family members. It is worth noting that DrrB,

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DrrC and DrrD have short linkers (five amino acids), whereas DrrA, the homologue most like OmpR in terms of its linker length (12 amino acids), was difficult to crystallize. The difficulty in crystallizing OmpR subfamily members is in keeping with a general view that they show considerable interdomain flexibility [33] and are likely to display limited interdomain interfaces [54].

Acknowledgements LJK is grateful to the following for helpful discussions: Ann Maris (UCLA), Vic DiRita (University of Michigan) and KI Varughese (Scripps Research Institute). LJK thanks Jack H Kaplan for providing a high hurdle while striving for clarity, and Barry Wanner (Purdue University) and laboratory members X Feng, K Mattison, R Oropeza and DM Walthers for their comments on the manuscript. Ted Michelini (OHSU) created Figure 3. Supported by National Institutes of Health GM58746 and National Science Foundation MCB9904658 to LJK.

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

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Sola M, Gomis-Ruth FX, Serrano L, Gonzalez A, Coll M: Three-dimensional crystal structure of the transcription factor PhoB receiver domain. J Mol Biol 1999, 285:675-687.

7.

Stock AM, Mottonen JM, Stock JB, Schutt CE: Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature 1989, 337:745-749.

8.

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10. Lewis RJ, Brannigan JA, Muchova K, Barak I, Wilkinson AJ: Phosphorylated aspartate in the structure of a response regulator protein. J Mol Biol 1999, 294:9-15. 11. Zhu X, Amsler CD, Volz K, Matsumura P: Tyrosine 106 of CheY plays an important role in chemotaxis signal transduction in Escherichia coli. J Bacteriol 1996, 178:4208-4215. 12. Mizuno T: Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res 1997, 4:161-168. 13. Maris AE, Sawaya MR, Grzeskowiak M, Jarvis MR, Kopka ML, •• Gunsalus RP, Dickerson RE: Domain rearrangement facilitates DNA recognition in the NarL response regulator/DNA complex. VI Conference on Bacterial Locomotion and Signal Transduction: 2001 Jan 14-19; Cuernavaca, Mexico. This report represents one of only two co-crystal structures of response regulators bound to their target DNA. It indicates that NarLc binds as a dimer in a tail-to-tail fashion. The DNA has a 42° bend induced by DNA backbone contacts. V189, in the recognition helix, causes large distortions of a GC base pair in the major groove. This work is described in [51].

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14. Zhao H, Hoch JA, Varughese KI: Crystal structure of the •• Spo0A:DNA complex. VI Conference on Bacterial Locomotion and Signal Transduction: 2001; Cuernavaca, Mexico. The X-ray structure of Spo0Ac bound to DNA is only the second example of the structure of a response regulator bound to DNA. The fold of Spo0Ac consists of six alpha helices and is not homologous to other responseregulator–DNA-binding domains. Two Spo0A molecules bind in a head-to-tail manner and most interactions with the DNA are via the recognition helix. This work is also described in [51]. 15. Kato M, Aiba H, Tate S, Nishimura Y, Mizuno T: Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in the activation of the osmoregulatory genes of Escherichia coli. FEBS Lett 1989, 249:168-172. 16. Grimsley JK, Tjalkens RB, Strauch MA, Bird TH, Spiegelman GB, Hostomsky Z, Whiteley JM, Hoch JA: Subunit composition and domain structure of the Spo0A sporulation transcription factor of Bacillus subtilis. J Biol Chem 1994, 269:16977-16982. 17.

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28. Kato N, Tsuzuki M, Aiba H, Mizuno T: Gene activation by the Escherichia coli positive regulator OmpR: a mutational study of the DNA-binding domain of OmpR. Mol Gen Genet 1995, 248:399-406. 29. Russo FD, Slauch JM, Silhavy TJ: Mutations that affect separate functions of OmpR the phosphorylated regulator of porin transcription in Escherichia coli. J Mol Biol 1993, 231:261-273. 30. Nara F, Matsuyama S, Mizuno T, Mizushima S: Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli. Mol Gen Genet 1986, 202:194-199. 31. Slauch J, Silhavy TJ: Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12. J Mol Biol 1989, 210:291-292.

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