1 This manuscript has been accepted for publication in Annual Review of Microbiology (2019, vol. 73).

2

3 STRUCTURAL BASIS OF RESPONSE REGULATOR FUNCTION

4

5 Rong Gao, Sophie Bouillet, Ann M. Stock

6

7 Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular

8 Biology, Rutgers – Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA;

9 email: [email protected], [email protected], [email protected]

10

11 Author ORCID Numbers:

12 Gao: 0000-0002-9245-4952

13 Bouillet: 0000-0002-0113-3169

14 Stock: 0000-0002-0446-8079

15

16 Corresponding Author: Ann Stock

17 CABM

18 679 Hoes Lane West

19 Piscataway, NJ 08854

20 +1 848-445-9812

21

22 Shortened Title: Response Regulator Structure Function

23

1 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 24 Keywords

25 histidine kinase, phosphorylation, response regulator, signal transduction, transcription factor, two-

26 component system

2 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 27 Abstract

28 Response regulators function as the output components of two-component systems, which couple the

29 sensing of environmental stimuli to adaptive responses. Response regulators typically contain

30 conserved receiver (REC) domains that function as phosphorylation-regulated switches to control

31 the activities of effector domains that elicit output responses. This modular design is extremely

32 versatile, enabling different regulatory strategies tuned to the needs of individual signaling systems.

33 This review summarizes functional features that underlie response regulator function. An abundance

34 of atomic resolution structures and complementary biochemical data have defined the mechanisms

35 for response regulator enzymatic activities, revealed trends in regulatory strategies utilized by

36 response regulators of different subfamilies and provided insights into interactions of response

37 regulators with their cognate histidine kinases. Among the hundreds of thousands of response

38 regulators identified, variations abound. This article provides a framework for understanding

39 structural features that enable function of canonical response regulators and a basis for

40 distinguishing non-canonical configurations.

41

3 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 42 1. INTRODUCTION

43 Two-component systems (TCSs) are the predominant multi-step signaling pathways in bacteria. Two

44 conserved proteins, a histidine protein kinase (HK) and response regulator (RR), constitute the core

45 system (Figure 1a). Autophosphorylation of the HK at a conserved His provides a high-energy

46 phosphoryl group that is transferred to a conserved Asp in the RR, resulting in RR activation (99).

47 Stimuli, sensed either directly or indirectly by the HK, regulate opposing

48 autophosphorylation/phosphotransfer and RR phosphatase activities of the HK, thus determining the

49 level of phosphorylation of the RR and the output response. A large variety of input domains in HKs

50 and output domains in RRs allows the coupling of an almost limitless array of chemical or physical

51 stimuli to diverse output responses. The conserved proteins and the systems themselves are

52 extremely versatile. An enormous range of variations in protein activities, domain architectures and

53 system configurations allows adaptation of TCSs to the needs of specific signaling systems.

54 TCSs are found in bacteria, archaea (41) and eukaryotes such as slime molds, yeast, and

55 plants (2). While great diversity exists among all TCSs it should be noted that some specific trends

56 in system configurations are found in different organisms. The Pfam database (35) lists >342,000

57 entries of proteins containing the conserved receiver (REC, named Response_reg in Pfam) domain

58 that characterizes RRs and >650 structures of such proteins in the Protein Data Bank (PDB) (18). In

59 addition to their presence in RRs, REC domains are also found in hybrid HKs that function within

60 phospho-relay systems involving multiple phosphotransfer steps, accounting for ~10% of REC

61 domain-containing proteins. The scope of this review will focus on bacterial RRs that mediate output

62 of TCSs. Among these RRs, variations abound. The plasticity of the REC domain and the versatility

63 of TCS architecture has driven the evolution of RRs that are uniquely adapted and fine-tuned for

64 function in specific pathways. Out of necessity, this review will focus on canonical RRs with full

4 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 65 understanding, but lack of specific acknowledgement of individual cases, that for every feature noted,

66 exceptions exist.

67 This review provides an overview of the structure of RRs and features that provide conserved

68 enzymatic activities in REC domains. Beyond the core domain structures and common enzymatic

69 mechanisms, RRs display great variation in domain arrangements that provide a variety of

70 mechanistically distinct regulatory mechanisms. However, as structures have accumulated, trends in

71 regulatory strategies used by different subfamilies of RRs have begun to emerge and these will be

72 explored in this review.

73

74 2. RESPONSE REGULATOR ARCHITECTURE

75 REC Domains

76 RRs are defined by the presence of a conserved REC domain. The REC domain fold is composed of

77 five α helices surrounding a central five-stranded parallel β-sheet with a 21345 topology (Figure 1b).

78 Sequence identity between REC domains is usually 20-30%, showing great variations (Figure 1c).

79 The central hydrophobic β-strands, β1, β3, β4 and β5, are more conserved than the peripheral

80 helices and loops. The most conserved residues include the site of phosphorylation, an Asp at the C-

81 terminus of β3, and several other residues in the β-α loops, which constitute the active site. The

82 highly conserved phosphorylation site and the variable peripheral sequences allow RRs to couple

83 phosphorylation to diverse effector functions.

84 Effector Domains

85 RRs typically function as the output components of signaling pathways. Regulatory REC domains

86 can be linked either covalently or non-covalently to a great diversity of effectors and thus control

87 numerous diverse responses. Figure 2 summarizes the distribution of the major RR effector domains

5 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 88 identified in the Pfam database. Approximately one fourth of RRs (23%) consist solely of a REC

89 domain. These single-domain RRs (SDRRs) regulate intermolecular effectors such as the chemotaxis

90 system regulator CheY, which binds to the flagellar motor component FliM controlling flagellar

91 rotation. CheY-like proteins found in chemotaxis-like or chemosensory systems mediate responses

92 other than chemotaxis, for example biofilm formation regulated by Cle proteins (77). Other

93 functions for SDRRs include acting as intermediates in phosphorelays (e.g. the general stress

94 response proteins SdrG and MrrA, and the sporulation protein Spo0F) (45, 63), as phosphate sinks to

95 control phosphate flux within phosphorelays (e.g. Rhizobium meliloti CheY1) (97), as allosteric

96 regulators of HKs (e.g. Caulobacter crescentus DivK) (24, 117) or as protease adapters (e.g. C.

97 crescentus CpdR) (56).

98 The largest class of RRs (64.5%) is composed of RRs containing a DNA-binding domain

99 (DBD) with subfamilies defined by different DBD folds. The OmpR subfamily (29% of RRs)

100 contains a winged-helix effector domain (68), the NarL subfamily (19%) a four-helix DNA-binding

101 HTH domain (73), the NtrC subfamily (7%) a Fis-type HTH domain fused to an AAA+ ATPase

102 domain and the LytTR (5.5%) a predominantly β fold (94). The abundance of RRs in this class

103 likely reflects the importance of transcriptional regulation as a response to environmental change. A

104 small class of RRs (1%) harbors an RNA-binding domain belonging to the ANTAR subfamily of

105 anti-termination factors such as AmiR (79).

106 RRs with enzymatic domains account for ~8% of RRs. A major group within this class (3%

107 of RRs) is the regulators of cyclic di-GMP including cyclases (GGDEF) and phosphodiesterases

108 (EAL, HD-GYP) (92). A second major group (2%) is the chemotaxis methylesterase CheB proteins

109 (31). A variety of different enzymatic domains have been identified in other RRs (3%) such as the

110 PP2C phosphatase domain of RsbY and the hybrid kinase domain of FrzE (30, 54). RRs with protein

6 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 111 binding domains account for ~1% of RRs. Examples in this class include CheV, with a CheW-like

112 domain that connects chemoreceptors to the chemotaxis histidine kinase CheA; RssB, which

113 regulates turnover of the stress response sigma factor RpoS; and PhyR, a regulator of the general

114 stress response that contains an extracytoplasmic function (ECF) sigma factor-like domain (1, 11,

115 66). While RRs often contain a simple REC-effector domain architecture they can have complex

116 domain organizations with additional signaling domains including PAS, GAF, HisKa, HATPase, etc.

117 The large variety of RRs identified to date emphasizes the versatility of the REC domain with no

118 apparent limits on the types of effector domains that can be controlled by this phosphorylation-

119 activated switch.

120

121 3. ENZYMATIC ACTIVITIES OF RRs

122 Conserved Phosphorylation Site

123 One of the defining characteristics of the REC domain is its highly conserved phosphorylation site.

124 Due to the lability of the high-energy acyl phosphate, structural characterization of the active site

- 125 often relies on beryllofluoride (BeF3 ) that noncovalently binds to the phosphorylation site Asp and

- 126 serves as a mimic of the phosphoryl group (119). Different BeF3 -bound RR structures reveal a

127 conserved active site with a network of hydrogen bonds (Figure 3a). The carboxylate side chains of

128 the acidic residue duo (DD) at the β1-α1 loop participate in coordinating a Mg2+ required for

129 catalysis. Sidechains of two additional residues, a Thr/Ser (T) at the β4-α4 loop and a Lys (K) at the

130 β5-α5 loop, together with backbone atoms of non-conserved active site residues, coordinate

131 phosphate oxygens in the phosphorylated REC domain.

132 RR phosphorylation level, the ultimate determining factor of TCS output for canonical RRs,

133 is regulated by multiple enzyme activities, including phosphotransfer from HKs, dephosphorylation

7 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 134 by auxiliary phosphatases or bifunctional HKs that also possess RR phosphatase activity,

135 autophosphorylation by small-molecule phosphodonors such as phosphoramidate and acetyl

136 phosphate (AcP) (67, 115), and autodephosphorylation activities. Similar phosphorylation sites with

137 almost identical positioning of active site residues have been observed in numerous RR or RR

138 complex structures (21, 81, 86, 107). A pentavalent phosphorus intermediate is believed to be a

139 common transition state for different activities of the REC domain (Figure 3b). Both

140 phosphorylation and dephosphorylation reactions can proceed through either an associative or a

141 dissociative mechanism depending on how tight or extended the transition state is. A phosphodonor

142 or water molecule needs to be in line with the acyl phosphate bond that is to be formed or broken.

143 The phosphotransferase or phosphatase helps position these molecules to further enhance the

144 reaction rates. In all cases, the majority of residues involved in coordinating the phosphorus

145 intermediate are from the REC domain. Thus, most RRs are catalytically competent of

146 autophosphorylation and autodephosphorylation in the absence of any enzymatic protein partner and

147 in vitro analyses of these reactions often provide insights into RR regulatory mechanisms.

148 Autodephosphorylation and Autophosphorylation

149 Despite the highly conserved active site geometry, RRs show large variations in rates of

150 autophosphorylation and autodephosphorylation (87, 101, 102). For example, autodephosphorylation

151 rate constants of the REC domain range over six orders of magnitude, giving phosphorylation half-

152 lives of seconds to hours and even days (101). Such great diversity is partly attributed to several

153 variable residues surrounding the active site, such as positions D+2 (two residues C-terminal to the

154 conserved D), T+1 and T+2 (one and two residues C-terminal to T) (Figure 3b and 3c). Backbone

155 atoms of D+2 and T+1 directly form hydrogen bonds with the phosphoryl group while the charge,

156 size and hydrophobicity of sidechains at these three positions may affect the energy barrier of the

8 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 157 transition state, facilitate the positioning or block the in-line path of the phosphodonor or attacking

158 water (53, 80, 102, 121). In more than half of RR sequences, a limited number of amino acid

159 combinations are preferred at these positions and the distribution of preferred amino acids correlates

160 with RR effector subfamilies (53, 80). REC enzyme activities are therefore suggested to co-evolve

161 with effector domain regulation and these residues represent functional sites for modulating the

162 stability of RR phosphorylation to match the timescale of individual TCS output responses.

163 Phosphorylation by small molecules, particularly AcP, has long been suggested to be

164 physiologically relevant in some RRs to couple TCS output to global conditions (115). RR

165 phosphorylation, often in the absence of the cognate HK, can be influenced by cellular AcP levels

166 that are sensitive to the metabolic state of cell. Because the phosphorylation rate by AcP is usually

167 much slower than the rate of phosphotransfer by the cognate HK and often offset by the phosphatase

168 activity of bifunctional HKs or other auxiliary phosphatases (43, 57), the contribution of

169 phosphorylation by AcP to TCS output is typically minimal in wild-type cells. However, given the

170 great diversity of TCSs, AcP can play a significant role in some systems, specifically those with a

171 fast RR autophosphorylation rate and/or a slow phosphatase rate (59, 85).

172 HK-mediated Activities

173 RR phosphatase activities mediated by HKs or auxiliary phosphatases are believed to function

174 through positioning a water molecule and stimulating the intrinsic RR autophosphatase activity (17).

175 Structure of CheY3 complexed with the phosphatase CheX (86) reveals an amide side chain

176 inserting into the RR active site and forming a hydrogen bond with the attacking water molecule

177 (Figure 3c). Similar positioning of the amide from a Gln or Asn residue has been observed in

178 different phosphatases with distinct structures, such as CheX (86), CheZ (123) and RapH (81), as

179 well as the phosphatase state of DesK, a bifunctional HK from the second largest HK subfamily,

9 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 180 HisKA_3 (107). The Gln-containing sequence motif, DXXXQ, is located immediately after the

181 phospho-accepting His residue in HKs from the HisKA_3 subfamily. The His residue does not

182 appear to be required for RR dephosphorylation despite its close proximity to the phosphatase-

183 essential Gln (52) . For the largest HK subfamily, HisKA, the His residue may play a role in RR

184 dephosphorylation (62, 125) but the exact phosphatase mechanism is less clear. An EXXN/T motif

185 similar to CheX-like phosphatases has been identified and the conserved Asn/Thr residue is

186 suggested to be the catalytic residue (52). However, among the available structures of RR complexes

187 with HKs of the HisKA subfamily, the side chain of the Asn/Thr residue is not at a similar position

188 as the Asn/Gln in other phosphatases and is distant from the active site Asp. A dual engagement

189 model has been suggested involving both His and Asn/Thr residues positioning the catalytic water

190 molecule (62). Roles of the two residues may differ for individual HisKA proteins depending on

191 structural details and structures unequivocally capturing the phosphatase state are needed to

192 elucidate the mechanism.

193 Loss of RR phosphorylation can result from autodephosphorylation, dephosphorylation by

194 the HK and back-transfer to the cognate HK (34, 98), i.e. the reverse reaction of phosphotransfer.

195 The back-transferred histidyl phosphoryl group can be further transferred to ADP (46, 100) or to

196 other RRs that function as phosphate sinks to modulate phosphorylation levels (3, 98, 103). Back-

197 transfer is suggested to accompany an associative transfer mechanism that has a tight RR

198 phosphorylation transition state and appears to be evolved for bi-directional transfer common in

199 CheA or other HPt-containing phosphorelay proteins (107, 122). Unidirectional phosphotransfer

200 with little back-transfer, observed in several canonical HKs, is linked to a dissociative mechanism

201 and the asymmetry of the Mg2+ position in an extended transition state (107). The dissociative

202 mechanism can be distinguished by a long distance between the conserved Asp and His residues in

10 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 203 an HK-RR complex structure. It remains to be investigated whether there is any domain preference

204 or sequence signature that distinguishes the two mechanisms and determines the relative rates of

205 forward- and back-transfer. Residues affecting the transition state stability, such as non-conserved

206 residues at D+2, T+1 and T+2 positions, also modulate the HK-catalyzed phosphotransfer rate (53,

207 107). Due to multiple phosphorylation and dephosphorylation reactions present simultaneously,

208 quantitation of individual activities is often complicated by interference from other activities.

209 Furthermore, RR activities measured in vitro with truncated cytoplasmic fragments of

210 transmembrane HKs require careful examination and may differ greatly from their full-length

211 counterparts in the cellular environment (43).

212

213 4. REGULATORY MECHANISMS IN RR SUBFAMILIES

214 As a phosphorylation-activated switch between inactive and active conformations, the REC domain

215 mediates effector functions through intramolecular and/or intermolecular interactions. Structures of

216 individual REC and effector domains usually undergo subtle changes upon phosphorylation, but the

217 overall structures may vary greatly because of different domain arrangements. A typical RR

218 regulatory mechanism is exemplified by Staphylococcus aureus VraR (Figure 4a), one of a few RRs

- 219 with full-length protein structures available in both phosphorylated (or BeF3 -bound) and

220 unphosphorylated states (60). The monomeric unphosphorylated VraR adopts a closed conformation

221 with extensive contacts between the REC and DBD effector domains, holding the DNA-recognition

222 helix at a position unfavorable for dimerization on DNA. Phosphorylation results in an extended

223 conformation with a flexible linker between the two domains and an altered REC surface that

224 promotes RR dimerization for DNA binding.

11 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 225 Negative regulation in the inactive state and positive regulation in the active state, as shown

226 for VraR, are two common mechanisms mediated by the REC domain (Figure 4b). For positive

227 regulation, the REC domain facilitates the effector domain function, as shown for many DBD-

228 containing RRs in which dimerization of the phosphorylated REC domains is thought to promote

229 DNA binding and transcription regulation. Effector domains of some RRs, such as NtrC1 (58) and

230 CheB (31), are catalytically competent when alone, but are inhibited by the REC domain in the

231 inactive state. The two mechanisms are not exclusive and many RRs use both. Regulatory details for

232 individual RRs show great variations that may have been evolved to adapt each protein to its unique

233 structure and function. For example, in inactive states, interactions between REC and effector

234 domains can differ dramatically (Figure 4b), even within the same subfamily of RRs (42).

235 Functional sites, such as the DNA-recognition helix for DBDs and the active site for enzymatic

236 effector domains, can be buried within the REC-effector interface or exposed but held at unfavorable

237 positions by tight interactions. Extended conformations with few interdomain contacts have also

238 been observed for a few RRs that are believed to employ a positive regulatory mechanism.

239 Inactive and Active Conformational States

240 The REC interaction surface that mediates effector domain function is usually distant from the

241 phosphorylation site. A dynamic allosteric mechanism allows conformational changes at the

242 phosphorylation site to propagate to the distal interaction surface. The REC domain samples

243 different allosteric conformations and exists in equilibrium between inactive and active

244 conformations, with phosphorylation shifting the equilibrium. A “Y-T coupling” allosteric

245 mechanism was initially described for CheY (25, 124) and several other RRs (14, 47).

246 Rearrangement of the conserved T at the phosphorylation site (Figure 3a) is believed to correlate

247 with the rotameric conformation of a conserved Tyr/Phe (Y) residue in the β5 strand. In the active

12 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 248 state, the aromatic side chain is oriented toward the interior of the REC domain, distinct from the

249 outward position in the inactive state (Figure 5a), resulting in alteration of the α4-β5-α5 face, a

250 surface widely used by many RRs for interdomain interactions. Because of the readily recognizable

251 position of the two residues, they are often used to classify structures as inactive or active, with the

252 caveat that the conformational change involves a broad surface of the REC domain and Y-T

253 coupling is not the only allosteric mechanism (20, 109).

254 X-ray crystallography has been central to understanding RR regulatory mechanisms although

255 conformations trapped in crystals represent only static snapshots of RR conformational dynamics

256 and can be influenced by experimental conditions, crystal lattice contacts and high protein

257 concentrations used in crystallization. A particular interaction interface or

258 dimerization/oligomerization mode observed in crystal structures, such as domain-swapped dimers

259 observed in several RRs (17, 26, 55), may not be physiologically relevant and requires

260 complementary experiments for validation. Nevertheless, an increasing number of RR structure

261 snapshots start to reveal different states of conformational trajectories and trends of prevalent

262 regulatory strategies in RR subfamilies.

263 DBD-containing RRs, the largest class of RRs, have the greatest number of X-ray structures

264 available, making it possible to analyze the allosteric conformational features within individual RR

- 265 subfamilies (Table 1). All structures crystalized with phosphorylated or BeF3 -bound RRs display

266 inward orientations of the side chain at the conserved Y position while conformations of

267 unphosphorylated RRs are diverse. For unphosphorylated RRs in the OmpR and NtrC subfamilies,

268 both outward and inward orientations of Y residues have been observed, with the inward orientation

269 more readily observed for the REC domain alone than for multi-domain proteins (Table 1). This is

270 consistent with NMR studies suggesting that unphosphorylated RRs exist in equilibrium between

13 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 271 inactive and active conformations (110) while interactions with the effector domain shift the

272 equilibrium to the inactive state (27). The inward orientation of Y is predominant for most NarL

273 subfamily members irrespective of phosphorylation status, thus is unlikely indicative of the active

274 state, but rather a result of packing the α4-β5-α5 face with an accessory β6 strand, a structural

275 feature found in many RRs of the NarL subfamily (20, 60, 83, 106). Therefore, the Y-T coupling

276 mechanism is not universal in all RRs. Even for NtrC in which orientation switching has been

277 observed, interconversion of the aromatic side chain has been suggested not to be involved in

278 allosteric regulation because its rate of conversion is faster than the rate of active/inactive state

279 conversion (109). RRs appear to be highly plastic for allosteric regulation with diverse mechanisms

280 matching their sequence and structural features.

281 The Y and T residues are two of many residues that may participate in allosteric regulation.

282 As discussed earlier, the phosphorylation site involves an intricate hydrogen-bond network with

283 several residues from the β1-α1, β3-α3, β4-α4 and β5-α5 loops. It has been recognized that

284 interactions, such as salt bridges and van der Waals contacts, of residues at D+1, T+1, T+2, K and

285 other positions can impact loop conformations and propagate the conformational changes to different

286 areas of the REC domain (5, 14, 20, 44, 70, 106). Conformations with only subsets of active site

287 residues at proper hydrogen-bonding positions have been discovered for many RRs (37, 95, 106) and

288 are often referred to as meta-active states. A single RR can have multiple meta-active states with

289 different combinations of loop conformations, as shown for NtrX (37). Unphosphorylated RRs can

290 exist in equilibrium between the active state and multiple inactive or meta-active conformations and

291 there are multiple pathways for transition to the active state (37, 44, 89). NMR relaxation data also

292 support a model of segmental motions of multiple allosteric residues for activation of CheY, instead

293 of a strict two-state switching model (70).

14 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 294 Just as phosphorylation shifts the equilibrium for effector domain regulation, the reverse

295 scenario, with output domains affecting phosphorylation, has also been observed (8, 37). It has been

296 suggested that adoption of an active conformation precedes phosphorylation (44, 53).

297 Phosphorylation can be accelerated by any effect that increases the population of active

298 conformation, such as relieving inhibition from the output domain by truncation, DNA binding in

299 DBD-containing RRs, dimerization at high protein concentrations and HK-RR interaction (8, 9, 27,

300 28, 37, 66). Surface changes caused by these different interactions can propagate through the

301 connecting βα loops to the phosphorylation site and other regions of the REC domain, increasing the

302 active state population. One extraordinary example is DesR in which the HK-RR interaction can

303 stabilize the active state of DesR and promote dimerization (66). Besides the phosphorylation-

304 activated switch, interaction with the HK also functions as an allosteric switch to increase the

305 population of active conformation for transcription activation. The same principle may be the basis

306 for many phosphorylation-independent regulatory mechanisms.

307 Dimerization Modes and Corresponding Conformational Changes

308 Phosphorylation-promoted conformational changes are routinely identified by comparing X-ray or

- 309 NMR structures obtained for unphosphorylated and BeF3 -bound RRs. As shown in Figure 5a, for

310 the stand-alone RR CheY, significant differences in positions of backbone atoms occur at the β4-α4,

311 β5-α5 loops and part of the α4-β5-α5 surface where CheY binds the FliM effector protein to regulate

312 flagellar rotation. It is unsurprising that structural elements of the REC domain undergoing the

313 largest rearrangements upon phosphorylation often correlate with protein-protein interaction surfaces,

314 particularly, the dimer interface for DBD-containing RRs. Certain dimerization modes are popular

315 for specific RR subfamilies with corresponding conformational changes.

15 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 316 A dimerization mode using the complete α4-β5-α5 surface is predominant in the OmpR

317 subfamily (Figure 5b). Dimer interaction involves salt bridges between pairs of highly conserved

318 charged residues within the α4-β5-α5 region (104, 105). For the DrrB protein shown in Figure 5b,

319 conformational changes span the entire dimer surface. A Tyr residue is prevalent at the Y position

320 (111) and switching of Tyr orientations has been observed. In several RRs (40, 78, 91), the hydroxyl

321 group of Tyr is in close proximity to a polar residue from the DBD (Figure 5b right), suggesting a

322 potential role in effector inhibition. This is unlikely to be a common mechanism given the wide

323 diversity of domain arrangements within the subfamily (8). RRs from the OmpR subfamily often

324 recognize tandem DNA sites and two DBDs bind DNA in a head-to-tail manner (15, 48, 64, 76). The

325 translational symmetry of DBDs coupled with the rotational symmetry of the REC domain predicts a

326 flexible linker or different REC-DBD interfaces for individual RR monomers within the RR-DNA

327 complex. Such asymmetry of REC-DBD interfaces has been observed for KdpE and PmrA (65, 76).

328 However, NMR studies suggest that the REC-DBD interaction observed for PmrA in the crystal

329 structure is transient in solution (65). Additionally, alternative dimers involving α1-α5 (6, 71) or

330 other surfaces (13) have also been discovered for unphosphorylated RRs but the physiological

331 relevance of these dimers awaits further studies.

332 The NtrC subfamily of RRs displays two major modes of dimerization, with each involving

333 different subsets of the α4-β5-α5 face (Figure 5c). The α4-β5 dimer interface is often seen in

334 phosphorylated RRs (Table 1) or unphosphorylated RRs with meta-active conformations (32, 37,

335 84). Another β5-α5 dimer is associated with inactive conformations (36, 84), as indicated by the

336 outward orientation of the Phe residue at the Y position. Dimer interaction centers around the β5-α5

337 face, sometimes with a slightly tilted α5 helix in contact with both β5 and α4. Formation of the

338 inactive β5-α5 dimer is believed to inhibit the AAA,+ ATPase output domain from oligomerization

16 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 339 and transcription initiation (10, 84). For the LytTR subfamily, structural information is limited and a

340 dimer also involving the α4-β5 subset of the α4-β5-α5 face has been observed for ComE (16).

341 Diverse dimerization modes have also been observed in the NarL subfamily. An α4-β5 dimer

342 interface and the phosphorylation-dependent orientation of the Phe residue have been reported for

343 the REC domain of FixJ (14). However, most RRs from the NarL subfamily show an α1-α5 dimer

344 interface (Table 1) distinct from the widely used α4-β5-α5 surface (7, 20, 29, 60, 82, 106). The

345 dimer interface mainly involves the α1 helix, the β1-α1 and β5-α5 loops (Figure 5d).

346 Correspondingly, significant structural rearrangements spanning to the α1 helix have been observed

- 347 when apo- and BeF3 -bound structures are compared. As discussed earlier, many RRs of the NarL

348 subfamily display a constitutive inward orientation of the residue at the Y position, thus this residue

349 is unlikely to be involved in allosteric regulation. Instead, different switching mechanisms involving

350 the T residue and other residues at α1, the β1-α1, β4-α4 and β5-α5 loops have been proposed for

351 different RRs, such as VraR, DesR and RcsB (20, 60, 106). In addition to the α1-α5 dimer interface,

352 the α4-β5-α5 surface is also remodeled to alter the positioning of the accessory β6 and α6 that are

353 directly linked to the effector domain. Another dimer mode involving β6 and α6 is common (Figure

354 5d, right) and several RRs, such as VraR (60), DesR (106), Spr1814 (82) and LiaR (29), show both

355 α1-α5 and β6-α6 dimer modes within a single crystal. It has been suggested that the β6-α6 dimer

356 may help RRs to form higher order oligomers on DNA to bind arrays of DNA-binding sites (106).

357 DNA-bound structures of RcsB again indicate an asymmetric dimer with different rotation symmetry

358 axes for the REC and DBDs (38). Different relative positions of the REC and DBDs are frequently

359 observed for different full length RRs. Asymmetry is believed to result from either different REC-

360 DBD interactions (20, 38) or a flexible linker that enables different disposition or even domain-swap

361 of individual domains trapped in crystals (29, 60).

17 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 362

363 5. RESPONSE REGULATOR – HISTIDINE KINASE INTERACTIONS

364 HK Conformational States

365 HKs have a modular architecture with a large variety of sensory domains (extracellular,

366 transmembrane or cytoplasmic) linked to a conserved catalytic domain by one or more signal

367 transducing domains (12, 69) (Figure 1a). The cytoplasmic enzymatic core of HKs consists of a

368 dimerization histidine phosphotransfer (DHp) domain and a catalytic ATP-binding (CA) domain.

369 The DHp domain contains the conserved phosphorylatable His residue and mediates dimerization of

370 the HK to form a 4-helix bundle that is essential for its activity (Figure 6). The DHp dimer is

371 flanked by the two α/β catalytic CA domains containing the kinase active sites that catalyze

372 phosphoryl transfer from ATP to the His residues. Autophosphorylation of the HK provides a high-

373 energy phorphoryl group for subsequent phosphotransfer to the RR. Most HKs also mediate

374 dephosphorylation of their cognate RRs in order to modulate the output response.

375 Large conformational changes in HKs occur during transitions between kinase,

376 phosphoryltranfer and phosphatase states. In the HisKA_3 subfamily of HKs (e.g. DesK of Bacillus

377 subtilis), the switch between states involves a large rotation of the DHp domains, a mechanism that

378 is less pronounced in the HisKA subfamily. Specific regulatory mechanisms have been described for

379 individual HKs, including the stabilization of the phosphatase state by ligand binding (e.g. c-di-GMP

380 binding on CckA) and the inhibition of the phosphatase state by a pH-dependent conformational

381 switch (33, 62). In contrast to the different conformations of HKs, structures of HK-REC domain

382 complexes determined to date indicate that REC domains bound to either phosphotransfer or

383 phosphatase states of HKs have similar conformations, with the REC domain displaying a meta-

384 active conformation, as defined in the previous section.

18 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 385 Partner Recognition and Specificity

386 Specificity of HK-RR pairs is crucial for the integrity of signaling pathways, given the large number

387 of different TCSs that typically exist in a single cell. Specificity within a pair is mediated by

388 recognition residues that participate in HK-RR binding. These residues are located in helix α1 of the

389 REC domain and in the two α helices of the HK DHp domain that contribute to the binding interface.

390 Signaling pathways have been successfully rewired by substituting as few as three co-evolved

391 specificity residues (88, 96). Additional strategies are employed to further ensure specificity of the

392 pairs. For example, the phosphatase activity of HKs eliminates non-specific phosphorylation by non-

393 cognate kinases or small molecule phosphodonors such as AcP. The low-abundance of HKs relative

394 to RRs also minimizes cross-phosphorylation between non-cognate pairs (57).

395 In an HK-RR complex, the REC domain inserts its α1 helix and β5-α5 loop between the two

396 helices of the DHp domain, primarily contacting the DHp α1 helix that contains the

397 phosphorylatable His residue. This conserved mode of binding buries the active site of the REC

398 domain at the HK-RR interface (Figure 6). Thus, conserved residues of both the HK and RR form a

399 substantial subset of the interface residues, with specificity being determined by a relatively small

400 set of variable residues. In addition to these specificity residues in α1 of the REC domain and the

401 two helices of the DHp domain, additional variable contacts can involve the β2-α2, β3-α3 and β4-

402 α4 loops of the REC domain interacting with the CA domain, the C-terminal region of the DHp and

403 the DHp-CA linker of the other protomer of the dimer (Figure 6) (21, 72, 74, 88, 107, 108, 114, 118,

404 122). Other contacts can occur between the REC domain and additional domains within the HK,

405 increasing the specificity and/or stability of the pair (e.g. contacts between the PAS domain of ThkA

406 and the RR TrrA) (118). These elements suggest that beyond the conserved REC-DHp interface that

407 buries the active site, the binding interface can vary greatly in different HK-RR pairs.

19 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 408 A model based on structures of HK FixL and full-length RR FixJ suggests that the RR

409 effector domain is free to move and does not participate in binding to the HK (116). However, a lack

410 of structures of full-length RRs bound to HKs leaves open the question as to how effector domains

411 might be positioned relative to REC domains and domains of the HK. Given the great diversity in

412 REC-effector domain arrangements in different inactive RRs, it is possible that diversity exists in the

413 ways effector domains interact in different HK-RR complexes. It is easy to envision how an effector

414 domain could participate in the stability of the complex and/or the efficiency of catalysis. Indeed, the

415 meta-active conformation of the REC domain observed in HK-REC domain complexes might be a

416 consequence of isolated REC domains being unhindered by REC-effector interactions that could bias

417 conformational states.

418 Phosphotransfer State

419 Structural and biochemical studies of DesK-DesR complexes suggest that one HK dimer binds to

420 one RR molecule in an asymmetric conformation. The DesK-DesR structure and a docking model of

421 CpxA-CpxR in a putative phosphotransfer state show asymmetry within the HK dimer, with the CA

422 domain of one monomer bound to the DHp domain of the other monomer, leaving the second CA

423 domain untethered with enough space for RR binding to the DHp. Structural data from the DesK-

424 DesR complex and from CpxA-CpxR model suggest that this highly dynamic state is coupled with

425 autokinase activity, involves back-and-forth movements of the DHp and CA domains, and is

426 modulated by REC domain binding (72, 107).

427 Phosphatase State

428 In contrast to the asymmetrical domain arrangements associated with phosphotransfer, HK-RR

429 complexes in putative dephosphorylation states have symmetrical arrangements of the HK DHp and

430 CA domains. The REC domains are also arranged symmetrically, each interacting with a DHp

20 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 431 domain with a 2:2 HK:RR stoichiometry (62, 107). The HK in its phosphatase state is more rigid

432 than in the phosphotransfer state, with the N-terminal coiled-coil of the DHp domains being highly

433 stable.

434 Despite the large number of structures recently determined, the mechanistic details of the

435 enzymatic reactions involving HKs and RRs are not fully understood. Discrimination between the

436 phosphotransfer and phosphatase states that have been trapped in crystal structures still remains

437 challenging. The situation is further complicated by distinct phosphatase mechanisms used by HKs

438 of different subtypes. A major limitation of current studies is the use of truncated proteins, most

439 notably the cytoplasmic domains of transmembrane HKs lacking sensor and transmembrane regions

440 that in intact proteins control the signaling states of HKs, and REC domains of RRs in the absence of

441 effector domains that are known to influence their conformational equilibria. While conserved

442 features of HK-RR interactions are beginning to be elucidated, variations on the theme are

443 anticipated. Similar to many other aspects of TCS structure and function, it is likely that the great

444 variety of domain architectures in TCS proteins enable different modes of HK-RR interactions and

445 regulatory mechanisms that are adapted to individual HK-RR pairs.

446

447 6. NON-CANONICAL MODES OF RR REGULATION

448 The canonical mechanism for regulation of RR activity involves phosphorylation at a conserved Asp

449 in the REC domain, stabilizing an active conformation that enables effector domain function.

450 Regulation of RR activity, in addition to or in place of Asp phosphorylation, potentially can be

451 achieved in many different ways including post-translational modifications at other sites that bias the

452 conformational equilibrium of the REC domain or directly alter effector domain function,

453 interactions that lower the energetic barrier for transition to an active conformation, ligand binding

21 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 454 to the RR, and regulation of expression to control RR levels and thus dimerization or

455 oligomerization. Indeed, each of these strategies has been observed and a few examples are

456 described below.

457 Reversible protein acetylation is known to regulate metabolic enzymes and transcription in

458 bacteria (49) and was documented in CheY more than 20 years ago (90). Enzyme catalyzed

459 acetylation of CheY at K91 and K106 increases in response to acetate and promotes clockwise

460 flagellar rotation, with acetylation at K91 proposed to alter conformational dynamics of the β4α4

461 loop (39). Autoacetylation with AcCoA as acetyl donor at other sites in CheY is thought to link

462 chemotaxis to the metabolic state of the cell (120). Another well-characterized example of

463 acetylation occurs in E. coli transcription factor RcsB. Acetylation of K154, a residue in the helix-

464 turn-helix motif that interacts with a phosphate in the DNA backbone, disrupts DNA binding, down-

465 regulates expression of the acid stress response genes, and compromises survival in acidic conditions

466 (22, 51). It should be noted that the stoichiometry of acetylation has not been determined, thus

467 physiological effects observed with mutations that mimic fully unmodified or acetylated states might

468 overestimate regulation that occurs in wild-type cells. A proteomics study in E. coli documented

469 acetylation of seven RRs: ArcA, CheY, CpxR, EvgA, NarL, OmpR and RcsB (22). The E. coli

470 genome encodes 26 putative Gcn5 N-acetyltransferases (GNATs) and a single known deacetylase

471 (CobB) (49). While the best-characterized acetyltransferase, PatZ, is proposed to mediate a global

472 strategy linking regulation to metabolic state, the large number of acetyltransferases raises the

473 possibility of substrate specificity that might enable system-specific signaling mechanisms.

474 Signal-induced, HK-dependent, phosphorylation-independent monomer to dimer activation

475 has been observed for several RR transcription factors. In the extensively studied B. subtilis system,

476 binding of DesR to DesK promotes an active conformation of DesR. At sufficiently high RR

22 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 477 concentrations and with a slow active to inactive state transition, dimerization can occur upon

478 release of the RR from the HK with subsequent stabilization of the dimer upon DNA binding (106).

479 Similar phosphorylation-independent activation of OmpR by HK EnvZ has been observed in acidic

480 conditions (23). Cyanobacterial transcription factor NblR contains a conserved Asp, but lacks other

481 residues necessary for phosphorylation. HK NblS is required for phosphorylation-independent

482 activation of NblR. However, no NblS-NblR interactions have been detected and the activating

483 monomer to dimer transition is postulated to be promoted by another protein partner (93).

484 Multiple strategies have been identified for regulation of orphan RRs that lack a conserved

485 Asp and/or other conserved residues necessary for phosphorylation. In streptomycetes, two atypical

486 RR transcription factors that lack residues necessary for phosphorylation, JadR1 and RedZ, are

487 regulated by the end products of the antibiotic biosynthetic pathways they control. The antibiotic

488 JdB binds directly to the JadR1 REC domain, disrupting DNA binding (112). A different strategy is

489 used by Helicobacter pylori HP1043, which exists as a constitutively active dimer in vitro with a

490 crystal structure similar to that of other activated OmpR subfamily members (50). Levels of HP1043

491 are regulated both transcriptionally and post-transcriptionally leading to speculation that control of

492 expression of this constitutively active RR may be the sole mechanism for regulating HP1403

493 activity (75).

494 Combinations of these mechanisms create even more strategies. Streptomyces coelicolor

495 GlnR, which regulates genes for nitrogen assimilation, is an orphan OmpR subfamily RR that lacks

496 residues for Asp phosphorylation and forms a constitutive α4-β5-α5 dimer (61). GlnR is

497 phosphorylated at 6 Ser/Thr sites in the DNA-binding domain under N-rich conditions, disrupting

498 DNA binding. GlnR is also acetylated at multiple Lys residues in the DNA-binding domain, with

499 acetylation enhancing DNA binding (4).

23 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 500

501 7. CONCLUDING REMARKS

502 When the structure of an RR REC domain was first reported thirty years ago, a central question was

503 how this single conserved domain could regulate responses as diverse as flagellar rotation,

504 transcription, and enzyme activity. The answer that emerged defined a mechanism that was both

505 simple and versatile. The small α/β REC domain exists in equilibrium between two primary

506 conformations with phosphorylation stabilizing an active conformation. This phosphorylation-

507 regulated switch enables regulatory strategies via any type of activating or inhibitory

508 macromolecular interactions that discriminate between the two states. Hundreds of structures of RRs

509 have provided descriptions of the conformations of REC domains in inactive and active states,

510 interactions with effector domains, DNA, HKs and auxiliary proteins. These structures provide a

511 foundation for identifying conserved features as well as specific variations in individual RRs.

512 Beyond the universally conserved enzymatic mechanisms facilitated by configurations at the active

513 site, other features such as Y-T coupling, regions of conformational perturbations, domain

514 arrangements and modes of DNA binding show distinct trends among RRs within specific

515 subfamilies.

516 Numerous variations on most every feature of RRs have been observed and undoubtedly,

517 many more remain to be discovered. The plasticity of the REC domain and versatility of RR design

518 allow an almost unlimited array of adaptations to fit the needs of individual signaling systems.

519 Defining details of how specific structural features impact function is important for interpreting the

520 nuances of RR behavior within specific TCSs as well as for pursuit of applied projects such as

521 development of antimicrobial therapeutics or the engineering of synthetic biosensing pathways.

24 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 522 Our current understanding of RR structure is largely informed by compiling information

523 derived from many partial structural descriptions of different RRs. Very few structures are available

524 for full-length multi-domain RRs in both inactive and active states. The crystal structures that do

525 exist are constrained by the limitations of the methodology, specifically the capture of single discrete

526 states that do not reflect conformational distributions and the potential promotion of inter- and/or

527 intra-molecular interactions that are influenced by the high concentrations of proteins used in

528 crystallization and/or stabilization of conformations necessary for crystallization and lattice

529 interactions. While NMR studies have provided information about dynamics and conformational

530 distributions in solution, size limitations have mostly precluded studies of full-length RRs, dimers,

531 oligomers and complexes of RRs with HKs or other macromolecular partners. Thus, while structures

532 unambiguously define allowable states, it is important to keep in mind the conformationally dynamic

533 nature of RRs and the potential influence of associated domains and macromolecular partners when

534 interpreting structures determined by methodologies with technical limitations. The emergence of

535 new structural methods such as high-resolution cryo-electron microscopy promise to provide

536 solutions to some of these challenges.

537

538 DISCLOSURE STATEMENT

539 The authors are not aware of any affiliations, memberships, funding, or financial holdings that might

540 be perceived as affecting the objectivity of this review.

541

542 ACKNOWLEDGMENTS

25 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 543 Research in the Stock laboratory is supported by grant R01 GM47958 from the National Institutes of

544 Health. S.B. was supported in part by the Foundation pour la Recherche Médicale (FRM) Grant

545 SPE201803005070.

546

547

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701 95:723-37

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770 component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical

771 potential sensor LytS. F1000Res. 4:79

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775 87. Pazy Y, Wollish AC, Thomas SA, Miller PJ, Collins EJ, et al. 2009. Matching biochemical

776 reaction kinetics to the timescales of life: structural determinants that influence the

777 autodephosphorylation rate of response regulator proteins. J. Mol. Biol. 392:1205-20

778 88. Podgornaia AI, Laub MT. 2013. Determinants of specificity in two-component signal

779 transduction. Curr. Opin. Microbiol. 16:156-62

780 89. Pontiggia F, Pachov DV, Clarkson MW, Villali J, Hagan MF, et al. 2015. Free energy

781 landscape of activation in a signalling protein at atomic resolution. Nat. Commun. 6:7284

782 90. Ramakrishnan R, Schuster M, Bourret RB. 1998. Acetylation at Lys-92 enhances signaling by

783 the chemotaxis response regulator protein CheY. Proc. Natl. Acad. Sci. USA 95:4918-23

784 91. Robinson VL, Wu T, Stock AM. 2003. Structural analysis of the domain interface in DrrB, a

785 response regulator of the OmpR/PhoB subfamily. J. Bacteriol. 185:4186-94

786 92. Romling U, Gomelsky M, Galperin MY. 2005. C-di-GMP: the dawning of a novel bacterial

787 signalling system. Mol. Microbiol. 57:629-39

788 93. Ruiz D, Salinas P, Lopez-Redondo ML, Cayuela ML, Marina A, Contreras A. 2008.

789 Phosphorylation-independent activation of the atypical response regulator NblR. Microbiology

790 154:3002-15

791 94. Sidote DJ, Barbieri CM, Wu T, Stock AM. 2008. Structure of the Staphylococcus aureus AgrA

792 LytTR domain bound to DNA reveals a beta fold with an unusual mode of binding. Structure

793 16:727-35

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795 structure of wild-type apoCheY. J. Biol. Chem. 276:28637-40

796 96. Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, et al. 2008. Rewiring the

797 specificity of two-component signal transduction systems. Cell 133:1043-54

798 97. Sourjik V, Schmitt R. 1996. Different roles of CheY1 and CheY2 in the chemotaxis of

799 Rhizobium meliloti. Mol. Microbiol. 22:427-36

800 98. Sourjik V, Schmitt R. 1998. Phosphotransfer between CheA, CheY1, and CheY2 in the

801 chemotaxis signal transduction chain of Rhizobium meliloti. Biochemistry 37:2327-35

802 99. Stock AM, Robinson VL, Goudreau PN. 2000. Two-component signal transduction. Annu. Rev.

803 Biochem. 69:183-215

804 100. Surette MG, Levit M, Liu Y, Lukat G, Ninfa EG, et al. 1996. Dimerization is required for the

805 activity of the protein histidine kinase CheA that mediates signal transduction in bacterial

806 chemotaxis. J. Biol. Chem. 271:939-45

807 101. Thomas SA, Brewster JA, Bourret RB. 2008. Two variable active site residues modulate

808 response regulator phosphoryl group stability. Mol. Microbiol. 69:453-65

809 102. Thomas SA, Immormino RM, Bourret RB, Silversmith RE. 2013. Nonconserved active site

810 residues modulate CheY autophosphorylation kinetics and phosphodonor preference.

811 Biochemistry 52:2262-73

812 103. Tindall MJ, Porter SL, Maini PK, Armitage JP. 2010. Modeling chemotaxis reveals the role of

813 reversed phosphotransfer and a bi-functional kinase-phosphatase. PLoS Comput. Biol. 6

814 104. Toro-Roman A, Mack TR, Stock AM. 2005. Structural analysis and solution studies of the

815 activated regulatory domain of the response regulator ArcA: a symmetric dimer mediated by

816 the α4-β5-α5 face. J. Mol. Biol. 349:11-26

38 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 817 105. Toro-Roman A, Wu T, Stock AM. 2005. A common dimerization interface in bacterial

818 response regulators KdpE and TorR. Protein Sci. 14:3077-388

819 106. Trajtenberg F, Albanesi D, Ruetalo N, Botti H, Mechaly AE, et al. 2014. Allosteric activation

820 of bacterial response regulators: the role of the cognate histidine kinase beyond

821 phosphorylation. MBio 5:e02105

822 107. Trajtenberg F, Imelio JA, Machado MR, Larrieux N, Marti MA, et al. 2016. Regulation of

823 signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife

824 5:e21422

825 108. Varughese KI. 2005. Conformational changes of Spo0F along the phosphotransfer pathway. J.

826 Bacteriol. 187:8221-27

827 109. Villali J, Pontiggia F, Clarkson MW, Hagan MF, Kern D. 2014. Evidence against the "Y-T

828 coupling" mechanism of activation in the response regulator NtrC. J. Mol. Biol. 426:1554-67

829 110. Volkman BF, Lipson D, Wemmer DE, Kern D. 2001. Two-state allosteric behavior in a single

830 domain signaling protein. Science 291:2429-33

831 111. Volz K. 1993. Structural conservation in the CheY superfamily. Biochemistry 32:11741-53

832 112. Wang L, Tian X, Wang J, Yang H, Fan K, et al. 2009. Autoregulation of antibiotic biosynthesis

833 by binding of the end product to an atypical response regulator. Proc. Natl. Acad. Sci. USA

834 106:8617-22

835 113. Wheeler TJ, Clements J, Finn RD. 2014. Skylign: a tool for creating informative, interactive

836 logos representing sequence alignments and profile hidden Markov models. BMC

837 Bioinformatics 15:7

39 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 838 114. Willett JW, Herrou J, Briegel A, Rotskoff G, Crosson S. 2015. Structural asymmetry in a

839 conserved signaling system that regulates division, replication, and virulence of an intracellular

840 pathogen. Proc .Natl. Acad. Sci. USA 112:E3709-18

841 115. Wolfe AJ. 2010. Physiologically relevant small phosphodonors link metabolism to signal

842 transduction. Curr. Opin. Microbiol. 13:204-9

843 116. Wright GSA, Saeki A, Hikima T, Nishizono Y, Hisano T, et al. 2018. Architecture of the

844 complete oxygen-sensing FixL-FixJ two-component signal transduction system. Sci. Signal. 11

845 117. Wu J, Ohta N, Newton A. 1998. An essential, multicomponent signal transduction pathway

846 required for cell cycle regulation in Caulobacter. Proc. Natl. Acad. Sci. USA 95:1443-48

847 118. Yamada S, Sugimoto H, Kobayashi M, Ohno A, Nakamura H, Shiro Y. 2009. Structure of

848 PAS-linked histidine kinase and the response regulator complex. Structure 17:1333-44

849 119. Yan D, Cho HS, Hastings CA, Igo MM, Lee SY, et al. 1999. Beryllofluoride mimics

850 phosphorylation of NtrC and other bacterial response regulators. Proc. Natl. Acad. Sci. USA

851 96:14789-94

852 120. Yan J, Barak R, Liarzi O, Shainskaya A, Eisenbach M. 2008. In vivo acetylation of CheY, a

853 response regulator in chemotaxis of Escherichia coli. J. Mol. Biol. 376:1260-71

854 121. Zapf J, Madhusudan M, Grimshaw CE, Hoch JA, Varughese KI, Whiteley JM. 1998. A source

855 of response regulator autophosphatase activity: the critical role of a residue adjacent to the

856 Spo0F autophosphorylation active site. Biochemistry 37:7725-32

857 122. Zapf J, Sen U, Madhusudan, Hoch JA, Varughese KI. 2000. A transient interaction between

858 two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular

859 recognition and phosphotransfer in signal transduction. Structure 8:851-62

40 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 860 123. Zhao R, Collins EJ, Bourret RB, Silversmith RE. 2002. Structure and catalytic mechanism of

861 the E. coli chemotaxis phosphatase CheZ. Nat. Struct. Biol. 9:570-75

862 124. Zhu X, Rebello J, Matsumura P, Volz K. 1997. Crystal structures of CheY mutants Y106W

863 and T87I/Y106W: CheY activation correlates with movement of residue 106. J. Biol. Chem.

864 272:5000-06

865 125. Zhu Y, Qin L, Yoshida T, Inouye M. 2000. Phosphatase activity of histidine kinase EnvZ

866 without kinase catalytic domain. Proc. Natl. Acad. Sci. USA 97:7808-13

867

41 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 868

869 TERMS AND DEFINITIONS 870 871 AcP acetyl phosphate

- 872 BeF3 beryllofluoride

873 CA catalytic/ATP-binding (domain)

874 DBD DNA-binding domain

875 DHp dimerization/histidine phosphotransfer (domain)

876 ECF extracytoplasmic function

877 HK histidine kinase

878 HPt histidine-containing phosphotransfer (domain)

879 PDB Protein Data Bank

880 REC receiver (domain of response regulator protein)

881 RR response regulator protein

882 SDRR single-domain response regulator

883 TCS two-component system

884

42 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 885 FIGURE CAPTIONS

886 Figure 1. Common features of RRs. (a) Schematic diagram of the prototypical TCS pathway. (b)

887 The conserved (βα)5 fold of REC domains. Phosphorylation site residues and the phosphoryl group

- 888 mimic, beryllofluoride (BeF3 ), are shown in sticks. (c) Sequence conservation of RECs. The

889 profiled hidden Markov model (HMM) for the REC protein family (Pfam PF00072) is shown as

890 sequence logos (113) with the secondary structure elements illustrated. Heights of individual stacked

891 letters at each position correspond to information contents, reflecting the probability of observing the

892 particular amino acids at each position. Phosphorylation site residues (stars), named after the most

893 conserved amino acids (DD, D, T and K), are among the most conserved residues in REC domains.

894

895 Figure 2. Classification of RRs by their effector domains. The percentile distribution is indicated for

896 RR effector functional classes and RR subfamilies. RR subfamilies are defined by effector domain

897 folds identified in Pfam. Representative proteins are traditionally used to name the OmpR, NarL and

898 NtrC subfamilies with effector domains named Trans_reg_C, GerE and Sigma54_activa in Pfam.

899 Representative structures of each subfamily are shown with REC domains colored in grey and

900 effector domains in colors (PDB ids: KdpE, 4KNY; RcsB, 5W43; LuxO, 5EP0; AgrA, 3BS1; AmiR,

901 1QO0; WspR, 3BRE; CheB, 1A2O).

902

903 Figure 3. Phosphorylation site of the REC domain. The phosphoryl group is positioned by a network

904 of hydrogen bonds (dashed lines) with side chains of the highly conserved residues (orange) as well

905 as backbone atoms of non-conserved residues (light pink). Non-conserved active site residues are

906 labeled by their relative sequence positions to the nearest conserved residues, such as T+1, indicating

43 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 907 one residue C-terminal to the conserved Thr/Ser residue. (a) Phosphorylation site of the archetype

908 RR CheY in the active conformation (PDB id: 1FQW) with residues that are differently positioned

909 in the inactive conformation (2CHE) shown in cyan. (b) The putative trigonal bipyramidal transition

910 state for both phosphorylation and dephosphorylation. X represents the leaving group of the

911 phosphodonor for phosphorylation or the attacking water for dephosphorylation. (c) Surface view of

912 the active site in the phosphatase-REC complex (3HZH).

913

914 Figure 4. RR regulatory strategies. (a) Distinct inactive and active RR conformations exemplified

- 915 by full-length RR VraR (PDB ids: 4GVP, 4IF4). BeF3 is shown in red spheres. (b) Schematic

916 diagrams of RR regulatory mechanisms. Functional sites of effector domains, such as enzyme sites

917 or DNA recognition regions, are shown as pink dots. These sites can be buried or exposed in a wide

918 variety of inactive RR conformations and their activity depends on different interactions between the

919 REC and effector domains. Phosphorylation of the RR can relieve REC domain inhibition, promote

920 effector function or both. Representative RRs that utilize these strategies are indicated.

921

922 Figure 5. Phosphorylation-induced conformational changes in the REC domain. RR structures with

- 923 or without BeF3 were aligned using the conserved strands β1, β3, β4, and β5 to compute the average

924 backbone RMSD per residue (a, right). RMSD values above the median + 2x MAD (median

925 absolute deviation) are considered as significant conformational changes and the corresponding

- 926 residues are colored blue. Non-REC structural elements are colored cyan. BeF3 is shown as red

927 spheres and residues involved in the potential Y-T coupling are shown as green or gray sticks.

928 Representative protein structures shown for the (a) Stand-alone, (b) OmpR, (c) NtrC and (d) NarL

44 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 929 RR subfamilies are CheY (1F4V), DrrB (3NNS, 1P2F), NtrC1 (1ZY2, 1NY5) and VraR (4IF4). Y/T

930 residues from the inactive CheY structure (2CHE) are differently positioned from the active

931 structure. For the OmpR, NtrC and NarL subfamilies, two proteins from each subfamily were used

932 for RMSD analyses and both showed similar regions of conformational changes. Substantial changes

933 in α1 are also observed in one protein from the NtrC subfamily (pink).

934

935 Figure 6. Structure of the HK-RR complex. Ribbon (a) and surface (b) views of the HK856-RR468

936 complex (PDB id, 3DGE). Residues that determine the HK-RR interaction specificity (19, 96) are

937 highlighted in light orange and cyan in the HK and RR, respectively. HK-RR contacts also involve

938 other surface regions (grey), including both active sites of the HK (red) and the RR (pink).

45 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 28 Abstract

29 Response regulators function as the output components of two-component systems, which couple

30 the sensing of environmental stimuli to adaptive responses. Response regulators typically

31 contain conserved receiver (REC) domains that function as phosphorylation-regulated switches

32 to control the activities of effector domains that elicit output responses. This modular design is

33 extremely versatile, enabling different regulatory strategies tuned to the needs of individual

34 signaling systems. This review summarizes functional features that underlie response regulator

35 function. An abundance of atomic resolution structures and complementary biochemical data

36 have defined the mechanisms for response regulator enzymatic activities, revealed trends in

37 regulatory strategies utilized by response regulators of different subfamilies and provided

38 insights into interactions of response regulators with their cognate histidine kinases. Among the

39 hundreds of thousands of response regulators identified, variations abound. This article provides

40 a framework for understanding structural features that enable function of canonical response

41 regulators and a basis for distinguishing non-canonical configurations.

42

3 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 43 1. INTRODUCTION

44 Two-component systems (TCSs) are the predominant multi-step signaling pathways in bacteria.

45 Two conserved proteins, a histidine protein kinase (HK) and response regulator (RR), constitute

46 the core system (Figure 1a). Autophosphorylation of the HK at a conserved His provides a high-

47 energy phosphoryl group that is transferred to a conserved Asp in the RR, resulting in RR

48 activation (99). Stimuli, sensed either directly or indirectly by the HK, regulate opposing

49 autophosphorylation/phosphotransfer and RR phosphatase activities of the HK, thus determining

50 the level of phosphorylation of the RR and the output response. A large variety of input domains

51 in HKs and output domains in RRs allows the coupling of an almost limitless array of chemical

52 or physical stimuli to diverse output responses. The conserved proteins and the systems

53 themselves are extremely versatile. An enormous range of variations in protein activities, domain

54 architectures and system configurations allows adaptation of TCSs to the needs of specific

55 signaling systems.

56 TCSs are found in bacteria, archaea (41) and eukaryotes such as slime molds, yeast, and

57 plants (2). While great diversity exists among all TCSs it should be noted that some specific

58 trends in system configurations are found in different organisms. The Pfam database (35) lists

59 >342,000 entries of proteins containing the conserved receiver (REC, named Response_reg in

60 Pfam) domain that characterizes RRs and >650 structures of such proteins in the Protein Data

61 Bank (PDB) (18). In addition to their presence in RRs, REC domains are also found in hybrid

62 HKs that function within phospho-relay systems involving multiple phosphotransfer steps,

63 accounting for ~10% of REC domain-containing proteins. The scope of this review will focus on

64 bacterial RRs that mediate output of TCSs. Among these RRs, variations abound. The plasticity

65 of the REC domain and the versatility of TCS architecture has driven the evolution of RRs that

4 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 66 are uniquely adapted and fine-tuned for function in specific pathways. Out of necessity, this

67 review will focus on canonical RRs with full understanding, but lack of specific

68 acknowledgement of individual cases, that for every feature noted, exceptions exist.

69 This review provides an overview of the structure of RRs and features that provide

70 conserved enzymatic activities in REC domains. Beyond the core domain structures and

71 common enzymatic mechanisms, RRs display great variation in domain arrangements that

72 provide a variety of mechanistically distinct regulatory mechanisms. However, as structures have

73 accumulated, trends in regulatory strategies used by different subfamilies of RRs have begun to

74 emerge and these will be explored in this review.

75

76 2. RESPONSE REGULATOR ARCHITECTURE

77 REC Domains

78 RRs are defined by the presence of a conserved REC domain. The REC domain fold is

79 composed of five α helices surrounding a central five-stranded parallel β-sheet with a 21345

80 topology (Figure 1b). Sequence identity between REC domains is usually 20-30%, showing

81 great variations (Figure 1c). The central hydrophobic β-strands, β1, β3, β4 and β5, are more

82 conserved than the peripheral helices and loops. The most conserved residues include the site of

83 phosphorylation, an Asp at the C-terminus of β3, and several other residues in the β-α loops,

84 which constitute the active site. The highly conserved phosphorylation site and the variable

85 peripheral sequences allow RRs to couple phosphorylation to diverse effector functions.

86 Effector Domains

87 RRs typically function as the output components of signaling pathways. Regulatory REC

88 domains can be linked either covalently or non-covalently to a great diversity of effectors and

5 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 89 thus control numerous diverse responses. Figure 2 summarizes the distribution of the major RR

90 effector domains identified in the Pfam database. Approximately one fourth of RRs (23%)

91 consist solely of a REC domain. These single-domain RRs (SDRRs) regulate intermolecular

92 effectors such as the chemotaxis system regulator CheY, which binds to the flagellar motor

93 component FliM controlling flagellar rotation. CheY-like proteins found in chemotaxis-like or

94 chemosensory systems mediate responses other than chemotaxis, for example biofilm formation

95 regulated by Cle proteins (77). Other functions for SDRRs include acting as intermediates in

96 phosphorelays (e.g. the general stress response proteins SdrG and MrrA, and the sporulation

97 protein Spo0F) (45, 63), as phosphate sinks to control phosphate flux within phosphorelays (e.g.

98 Rhizobium meliloti CheY1) (97), as allosteric regulators of HKs (e.g. Caulobacter crescentus

99 DivK) (24, 117) or as protease adapters (e.g. C. crescentus CpdR) (56).

100 The largest class of RRs (64.5%) is composed of RRs containing a DNA-binding domain

101 (DBD) with subfamilies defined by different DBD folds. The OmpR subfamily (29% of RRs)

102 contains a winged-helix effector domain (68), the NarL subfamily (19%) a four-helix DNA-

103 binding HTH domain (73), the NtrC subfamily (7%) a Fis-type HTH domain fused to an AAA+

104 ATPase domain and the LytTR (5.5%) a predominantly β fold (94). The abundance of RRs in

105 this class likely reflects the importance of transcriptional regulation as a response to

106 environmental change. A small class of RRs (1%) harbors an RNA-binding domain belonging to

107 the ANTAR subfamily of anti-termination factors such as AmiR (79).

108 RRs with enzymatic domains account for ~8% of RRs. A major group within this class (3%

109 of RRs) is the regulators of cyclic di-GMP including cyclases (GGDEF) and phosphodiesterases

110 (EAL, HD-GYP) (92). A second major group (2%) is the chemotaxis methylesterase CheB

111 proteins (31). A variety of different enzymatic domains have been identified in other RRs (3%)

6 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 112 such as the PP2C phosphatase domain of RsbY and the hybrid kinase domain of FrzE (30, 54).

113 RRs with protein binding domains account for ~1% of RRs. Examples in this class include

114 CheV, with a CheW-like domain that connects chemoreceptors to the chemotaxis histidine

115 kinase CheA; RssB, which regulates turnover of the stress response sigma factor RpoS; and

116 PhyR, a regulator of the general stress response that contains an extracytoplasmic function (ECF)

117 sigma factor-like domain (1, 11, 66). While RRs often contain a simple REC-effector domain

118 architecture they can have complex domain organizations with additional signaling domains

119 including PAS, GAF, HisKa, HATPase, etc. The large variety of RRs identified to date

120 emphasizes the versatility of the REC domain with no apparent limits on the types of effector

121 domains that can be controlled by this phosphorylation-activated switch.

122

123 3. ENZYMATIC ACTIVITIES OF RRs

124 Conserved Phosphorylation Site

125 One of the defining characteristics of the REC domain is its highly conserved phosphorylation

126 site. Due to the lability of the high-energy acyl phosphate, structural characterization of the

- 127 active site often relies on beryllofluoride (BeF3 ) that noncovalently binds to the phosphorylation

- 128 site Asp and serves as a mimic of the phosphoryl group (119). Different BeF3 -bound RR

129 structures reveal a conserved active site with a network of hydrogen bonds (Figure 3a). The

130 carboxylate side chains of the acidic residue duo (DD) at the β1-α1 loop participate in

131 coordinating a Mg2+ required for catalysis. Sidechains of two additional residues, a Thr/Ser (T) at

132 the β4-α4 loop and a Lys (K) at the β5-α5 loop, together with backbone atoms of non-conserved

133 active site residues, coordinate phosphate oxygens in the phosphorylated REC domain.

7 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 134 RR phosphorylation level, the ultimate determining factor of TCS output for canonical

135 RRs, is regulated by multiple enzyme activities, including phosphotransfer from HKs,

136 dephosphorylation by auxiliary phosphatases or bifunctional HKs that also possess RR

137 phosphatase activity, autophosphorylation by small-molecule phosphodonors such as

138 phosphoramidate and acetyl phosphate (AcP) (67, 115), and autodephosphorylation activities.

139 Similar phosphorylation sites with almost identical positioning of active site residues have been

140 observed in numerous RR or RR complex structures (21, 81, 86, 107). A pentavalent phosphorus

141 intermediate is believed to be a common transition state for different activities of the REC

142 domain (Figure 3b). Both phosphorylation and dephosphorylation reactions can proceed through

143 either an associative or a dissociative mechanism depending on how tight or extended the

144 transition state is. A phosphodonor or water molecule needs to be in line with the acyl phosphate

145 bond that is to be formed or broken. The phosphotransferase or phosphatase helps position these

146 molecules to further enhance the reaction rates. In all cases, the majority of residues involved in

147 coordinating the phosphorus intermediate are from the REC domain. Thus, most RRs are

148 catalytically competent of autophosphorylation and autodephosphorylation in the absence of any

149 enzymatic protein partner and in vitro analyses of these reactions often provide insights into RR

150 regulatory mechanisms.

151 Autodephosphorylation and Autophosphorylation

152 Despite the highly conserved active site geometry, RRs show large variations in rates of

153 autophosphorylation and autodephosphorylation (87, 101, 102). For example,

154 autodephosphorylation rate constants of the REC domain range over six orders of magnitude,

155 giving phosphorylation half-lives of seconds to hours and even days (101). Such great diversity

156 is partly attributed to several variable residues surrounding the active site, such as positions D+2

8 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 157 (two residues C-terminal to the conserved D), T+1 and T+2 (one and two residues C-terminal to

158 T) (Figure 3b and 3c). Backbone atoms of D+2 and T+1 directly form hydrogen bonds with the

159 phosphoryl group while the charge, size and hydrophobicity of sidechains at these three positions

160 may affect the energy barrier of the transition state, facilitate the positioning or block the in-line

161 path of the phosphodonor or attacking water (53, 80, 102, 121). In more than half of RR

162 sequences, a limited number of amino acid combinations are preferred at these positions and the

163 distribution of preferred amino acids correlates with RR effector subfamilies (53, 80). REC

164 enzyme activities are therefore suggested to co-evolve with effector domain regulation and these

165 residues represent functional sites for modulating the stability of RR phosphorylation to match

166 the timescale of individual TCS output responses.

167 Phosphorylation by small molecules, particularly AcP, has long been suggested to be

168 physiologically relevant in some RRs to couple TCS output to global conditions (115). RR

169 phosphorylation, often in the absence of the cognate HK, can be influenced by cellular AcP

170 levels that are sensitive to the metabolic state of cell. Because the phosphorylation rate by AcP is

171 usually much slower than the rate of phosphotransfer by the cognate HK and often offset by the

172 phosphatase activity of bifunctional HKs or other auxiliary phosphatases (43, 57), the

173 contribution of phosphorylation by AcP to TCS output is typically minimal in wild-type cells.

174 However, given the great diversity of TCSs, AcP can play a significant role in some systems,

175 specifically those with a fast RR autophosphorylation rate and/or a slow phosphatase rate (59,

176 85).

177 HK-mediated Activities

178 RR phosphatase activities mediated by HKs or auxiliary phosphatases are believed to function

179 through positioning a water molecule and stimulating the intrinsic RR autophosphatase activity

9 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 180 (17). Structure of CheY3 complexed with the phosphatase CheX (86) reveals an amide side

181 chain inserting into the RR active site and forming a hydrogen bond with the attacking water

182 molecule (Figure 3c). Similar positioning of the amide from a Gln or Asn residue has been

183 observed in different phosphatases with distinct structures, such as CheX (86), CheZ (123) and

184 RapH (81), as well as the phosphatase state of DesK, a bifunctional HK from the second largest

185 HK subfamily, HisKA_3 (107). The Gln-containing sequence motif, DXXXQ, is located

186 immediately after the phospho-accepting His residue in HKs from the HisKA_3 subfamily. The

187 His residue does not appear to be required for RR dephosphorylation despite its close proximity

188 to the phosphatase-essential Gln (52) . For the largest HK subfamily, HisKA, the His residue

189 may play a role in RR dephosphorylation (62, 125) but the exact phosphatase mechanism is less

190 clear. An EXXN/T motif similar to CheX-like phosphatases has been identified and the

191 conserved Asn/Thr residue is suggested to be the catalytic residue (52). However, among the

192 available structures of RR complexes with HKs of the HisKA subfamily, the side chain of the

193 Asn/Thr residue is not at a similar position as the Asn/Gln in other phosphatases and is distant

194 from the active site Asp. A dual engagement model has been suggested involving both His and

195 Asn/Thr residues positioning the catalytic water molecule (62). Roles of the two residues may

196 differ for individual HisKA proteins depending on structural details and structures unequivocally

197 capturing the phosphatase state are needed to elucidate the mechanism.

198 Loss of RR phosphorylation can result from autodephosphorylation, dephosphorylation

199 by the HK and back-transfer to the cognate HK (34, 98), i.e. the reverse reaction of

200 phosphotransfer. The back-transferred histidyl phosphoryl group can be further transferred to

201 ADP (46, 100) or to other RRs that function as phosphate sinks to modulate phosphorylation

202 levels (3, 98, 103). Back-transfer is suggested to accompany an associative transfer mechanism

10 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 203 that has a tight RR phosphorylation transition state and appears to be evolved for bi-directional

204 transfer common in CheA or other HPt-containing phosphorelay proteins (107, 122).

205 Unidirectional phosphotransfer with little back-transfer, observed in several canonical HKs, is

206 linked to a dissociative mechanism and the asymmetry of the Mg2+ position in an extended

207 transition state (107). The dissociative mechanism can be distinguished by a long distance

208 between the conserved Asp and His residues in an HK-RR complex structure. It remains to be

209 investigated whether there is any domain preference or sequence signature that distinguishes the

210 two mechanisms and determines the relative rates of forward- and back-transfer. Residues

211 affecting the transition state stability, such as non-conserved residues at D+2, T+1 and T+2

212 positions, also modulate the HK-catalyzed phosphotransfer rate (53, 107). Due to multiple

213 phosphorylation and dephosphorylation reactions present simultaneously, quantitation of

214 individual activities is often complicated by interference from other activities. Furthermore, RR

215 activities measured in vitro with truncated cytoplasmic fragments of transmembrane HKs require

216 careful examination and may differ greatly from their full-length counterparts in the cellular

217 environment (43).

218

219 4. REGULATORY MECHANISMS IN RR SUBFAMILIES

220 As a phosphorylation-activated switch between inactive and active conformations, the REC

221 domain mediates effector functions through intramolecular and/or intermolecular interactions.

222 Structures of individual REC and effector domains usually undergo subtle changes upon

223 phosphorylation, but the overall structures may vary greatly because of different domain

224 arrangements. A typical RR regulatory mechanism is exemplified by Staphylococcus aureus

225 VraR (Figure 4a), one of a few RRs with full-length protein structures available in both

11 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 - 226 phosphorylated (or BeF3 -bound) and unphosphorylated states (60). The monomeric

227 unphosphorylated VraR adopts a closed conformation with extensive contacts between the REC

228 and DBD effector domains, holding the DNA-recognition helix at a position unfavorable for

229 dimerization on DNA. Phosphorylation results in an extended conformation with a flexible linker

230 between the two domains and an altered REC surface that promotes RR dimerization for DNA

231 binding.

232 Negative regulation in the inactive state and positive regulation in the active state, as

233 shown for VraR, are two common mechanisms mediated by the REC domain (Figure 4b). For

234 positive regulation, the REC domain facilitates the effector domain function, as shown for many

235 DBD-containing RRs in which dimerization of the phosphorylated REC domains is thought to

236 promote DNA binding and transcription regulation. Effector domains of some RRs, such as

237 NtrC1 (58) and CheB (31), are catalytically competent when alone, but are inhibited by the REC

238 domain in the inactive state. The two mechanisms are not exclusive and many RRs use both.

239 Regulatory details for individual RRs show great variations that may have been evolved to adapt

240 each protein to its unique structure and function. For example, in inactive states, interactions

241 between REC and effector domains can differ dramatically (Figure 4b), even within the same

242 subfamily of RRs (42). Functional sites, such as the DNA-recognition helix for DBDs and the

243 active site for enzymatic effector domains, can be buried within the REC-effector interface or

244 exposed but held at unfavorable positions by tight interactions. Extended conformations with few

245 interdomain contacts have also been observed for a few RRs that are believed to employ a

246 positive regulatory mechanism.

247 Inactive and Active Conformational States

12 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 248 The REC interaction surface that mediates effector domain function is usually distant from the

249 phosphorylation site. A dynamic allosteric mechanism allows conformational changes at the

250 phosphorylation site to propagate to the distal interaction surface. The REC domain samples

251 different allosteric conformations and exists in equilibrium between inactive and active

252 conformations, with phosphorylation shifting the equilibrium. A “Y-T coupling” allosteric

253 mechanism was initially described for CheY (25, 124) and several other RRs (14, 47).

254 Rearrangement of the conserved T at the phosphorylation site (Figure 3a) is believed to correlate

255 with the rotameric conformation of a conserved Tyr/Phe (Y) residue in the β5 strand. In the

256 active state, the aromatic side chain is oriented toward the interior of the REC domain, distinct

257 from the outward position in the inactive state (Figure 5a), resulting in alteration of the α4-β5-α5

258 face, a surface widely used by many RRs for interdomain interactions. Because of the readily

259 recognizable position of the two residues, they are often used to classify structures as inactive or

260 active, with the caveat that the conformational change involves a broad surface of the REC

261 domain and Y-T coupling is not the only allosteric mechanism (20, 109).

262 X-ray crystallography has been central to understanding RR regulatory mechanisms

263 although conformations trapped in crystals represent only static snapshots of RR conformational

264 dynamics and can be influenced by experimental conditions, crystal lattice contacts and high

265 protein concentrations used in crystallization. A particular interaction interface or

266 dimerization/oligomerization mode observed in crystal structures, such as domain-swapped

267 dimers observed in several RRs (17, 26, 55), may not be physiologically relevant and requires

268 complementary experiments for validation. Nevertheless, an increasing number of RR structure

269 snapshots start to reveal different states of conformational trajectories and trends of prevalent

270 regulatory strategies in RR subfamilies.

13 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 271 DBD-containing RRs, the largest class of RRs, have the greatest number of X-ray

272 structures available, making it possible to analyze the allosteric conformational features within

- 273 individual RR subfamilies (Table 1). All structures crystalized with phosphorylated or BeF3 -

274 bound RRs display inward orientations of the side chain at the conserved Y position while

275 conformations of unphosphorylated RRs are diverse. For unphosphorylated RRs in the OmpR

276 and NtrC subfamilies, both outward and inward orientations of Y residues have been observed,

277 with the inward orientation more readily observed for the REC domain alone than for multi-

278 domain proteins (Table 1). This is consistent with NMR studies suggesting that

279 unphosphorylated RRs exist in equilibrium between inactive and active conformations (110)

280 while interactions with the effector domain shift the equilibrium to the inactive state (27). The

281 inward orientation of Y is predominant for most NarL subfamily members irrespective of

282 phosphorylation status, thus is unlikely indicative of the active state, but rather a result of

283 packing the α4-β5-α5 face with an accessory β6 strand, a structural feature found in many RRs of

284 the NarL subfamily (20, 60, 83, 106). Therefore, the Y-T coupling mechanism is not universal in

285 all RRs. Even for NtrC in which orientation switching has been observed, interconversion of the

286 aromatic side chain has been suggested not to be involved in allosteric regulation because its rate

287 of conversion is faster than the rate of active/inactive state conversion (109). RRs appear to be

288 highly plastic for allosteric regulation with diverse mechanisms matching their sequence and

289 structural features.

290 The Y and T residues are two of many residues that may participate in allosteric

291 regulation. As discussed earlier, the phosphorylation site involves an intricate hydrogen-bond

292 network with several residues from the β1-α1, β3-α3, β4-α4 and β5-α5 loops. It has been

293 recognized that interactions, such as salt bridges and van der Waals contacts, of residues at D+1,

14 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 294 T+1, T+2, K and other positions can impact loop conformations and propagate the

295 conformational changes to different areas of the REC domain (5, 14, 20, 44, 70, 106).

296 Conformations with only subsets of active site residues at proper hydrogen-bonding positions

297 have been discovered for many RRs (37, 95, 106) and are often referred to as meta-active states.

298 A single RR can have multiple meta-active states with different combinations of loop

299 conformations, as shown for NtrX (37). Unphosphorylated RRs can exist in equilibrium between

300 the active state and multiple inactive or meta-active conformations and there are multiple

301 pathways for transition to the active state (37, 44, 89). NMR relaxation data also support a model

302 of segmental motions of multiple allosteric residues for activation of CheY, instead of a strict

303 two-state switching model (70).

304 Just as phosphorylation shifts the equilibrium for effector domain regulation, the reverse

305 scenario, with output domains affecting phosphorylation, has also been observed (8, 37). It has

306 been suggested that adoption of an active conformation precedes phosphorylation (44, 53).

307 Phosphorylation can be accelerated by any effect that increases the population of active

308 conformation, such as relieving inhibition from the output domain by truncation, DNA binding

309 in DBD-containing RRs, dimerization at high protein concentrations and HK-RR interaction (8,

310 9, 27, 28, 37, 66). Surface changes caused by these different interactions can propagate through

311 the connecting βα loops to the phosphorylation site and other regions of the REC domain,

312 increasing the active state population. One extraordinary example is DesR in which the HK-RR

313 interaction can stabilize the active state of DesR and promote dimerization (66). Besides the

314 phosphorylation-activated switch, interaction with the HK also functions as an allosteric switch

315 to increase the population of active conformation for transcription activation. The same principle

316 may be the basis for many phosphorylation-independent regulatory mechanisms.

15 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 317 Dimerization Modes and Corresponding Conformational Changes

318 Phosphorylation-promoted conformational changes are routinely identified by comparing X-ray

- 319 or NMR structures obtained for unphosphorylated and BeF3 -bound RRs. As shown in Figure 5a,

320 for the stand-alone RR CheY, significant differences in positions of backbone atoms occur at the

321 β4-α4, β5-α5 loops and part of the α4-β5-α5 surface where CheY binds the FliM effector protein

322 to regulate flagellar rotation. It is unsurprising that structural elements of the REC domain

323 undergoing the largest rearrangements upon phosphorylation often correlate with protein-protein

324 interaction surfaces, particularly, the dimer interface for DBD-containing RRs. Certain

325 dimerization modes are popular for specific RR subfamilies with corresponding conformational

326 changes.

327 A dimerization mode using the complete α4-β5-α5 surface is predominant in the OmpR

328 subfamily (Figure 5b). Dimer interaction involves salt bridges between pairs of highly

329 conserved charged residues within the α4-β5-α5 region (104, 105). For the DrrB protein shown

330 in Figure 5b, conformational changes span the entire dimer surface. A Tyr residue is prevalent at

331 the Y position (111) and switching of Tyr orientations has been observed. In several RRs (40, 78,

332 91), the hydroxyl group of Tyr is in close proximity to a polar residue from the DBD (Figure 5b

333 right), suggesting a potential role in effector inhibition. This is unlikely to be a common

334 mechanism given the wide diversity of domain arrangements within the subfamily (8). RRs from

335 the OmpR subfamily often recognize tandem DNA sites and two DBDs bind DNA in a head-to-

336 tail manner (15, 48, 64, 76). The translational symmetry of DBDs coupled with the rotational

337 symmetry of the REC domain predicts a flexible linker or different REC-DBD interfaces for

338 individual RR monomers within the RR-DNA complex. Such asymmetry of REC-DBD

339 interfaces has been observed for KdpE and PmrA (65, 76). However, NMR studies suggest that

16 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 340 the REC-DBD interaction observed for PmrA in the crystal structure is transient in solution (65).

341 Additionally, alternative dimers involving α1-α5 (6, 71) or other surfaces (13) have also been

342 discovered for unphosphorylated RRs but the physiological relevance of these dimers awaits

343 further studies.

344 The NtrC subfamily of RRs displays two major modes of dimerization, with each

345 involving different subsets of the α4-β5-α5 face (Figure 5c). The α4-β5 dimer interface is often

346 seen in phosphorylated RRs (Table 1) or unphosphorylated RRs with meta-active conformations

347 (32, 37, 84). Another β5-α5 dimer is associated with inactive conformations (36, 84), as

348 indicated by the outward orientation of the Phe residue at the Y position. Dimer interaction

349 centers around the β5-α5 face, sometimes with a slightly tilted α5 helix in contact with both β5

350 and α4. Formation of the inactive β5-α5 dimer is believed to inhibit the AAA,+ ATPase output

351 domain from oligomerization and transcription initiation (10, 84). For the LytTR subfamily,

352 structural information is limited and a dimer also involving the α4-β5 subset of the α4-β5-α5 face

353 has been observed for ComE (16).

354 Diverse dimerization modes have also been observed in the NarL subfamily. An α4-β5

355 dimer interface and the phosphorylation-dependent orientation of the Phe residue have been

356 reported for the REC domain of FixJ (14). However, most RRs from the NarL subfamily show

357 an α1-α5 dimer interface (Table 1) distinct from the widely used α4-β5-α5 surface (7, 20, 29, 60,

358 82, 106). The dimer interface mainly involves the α1 helix, the β1-α1 and β5-α5 loops (Figure

359 5d). Correspondingly, significant structural rearrangements spanning to the α1 helix have been

- 360 observed when apo- and BeF3 -bound structures are compared. As discussed earlier, many RRs

361 of the NarL subfamily display a constitutive inward orientation of the residue at the Y position,

362 thus this residue is unlikely to be involved in allosteric regulation. Instead, different switching

17 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 363 mechanisms involving the T residue and other residues at α1, the β1-α1, β4-α4 and β5-α5 loops

364 have been proposed for different RRs, such as VraR, DesR and RcsB (20, 60, 106). In addition to

365 the α1-α5 dimer interface, the α4-β5-α5 surface is also remodeled to alter the positioning of the

366 accessory β6 and α6 that are directly linked to the effector domain. Another dimer mode

367 involving β6 and α6 is common (Figure 5d, right) and several RRs, such as VraR (60), DesR

368 (106), Spr1814 (82) and LiaR (29), show both α1-α5 and β6-α6 dimer modes within a single

369 crystal. It has been suggested that the β6-α6 dimer may help RRs to form higher order oligomers

370 on DNA to bind arrays of DNA-binding sites (106). DNA-bound structures of RcsB again

371 indicate an asymmetric dimer with different rotation symmetry axes for the REC and DBDs (38).

372 Different relative positions of the REC and DBDs are frequently observed for different full

373 length RRs. Asymmetry is believed to result from either different REC-DBD interactions (20,

374 38) or a flexible linker that enables different disposition or even domain-swap of individual

375 domains trapped in crystals (29, 60).

376

377 5. RESPONSE REGULATOR – HISTIDINE KINASE INTERACTIONS

378 HK Conformational States

379 HKs have a modular architecture with a large variety of sensory domains (extracellular,

380 transmembrane or cytoplasmic) linked to a conserved catalytic domain by one or more signal

381 transducing domains (12, 69) (Figure 1a). The cytoplasmic enzymatic core of HKs consists of a

382 dimerization histidine phosphotransfer (DHp) domain and a catalytic ATP-binding (CA) domain.

383 The DHp domain contains the conserved phosphorylatable His residue and mediates

384 dimerization of the HK to form a 4-helix bundle that is essential for its activity (Figure 6). The

385 DHp dimer is flanked by the two α/β catalytic CA domains containing the kinase active sites that

18 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 386 catalyze phosphoryl transfer from ATP to the His residues. Autophosphorylation of the HK

387 provides a high-energy phorphoryl group for subsequent phosphotransfer to the RR. Most HKs

388 also mediate dephosphorylation of their cognate RRs in order to modulate the output response.

389 Large conformational changes in HKs occur during transitions between kinase,

390 phosphoryltranfer and phosphatase states. In the HisKA_3 subfamily of HKs (e.g. DesK of

391 Bacillus subtilis), the switch between states involves a large rotation of the DHp domains, a

392 mechanism that is less pronounced in the HisKA subfamily. Specific regulatory mechanisms

393 have been described for individual HKs, including the stabilization of the phosphatase state by

394 ligand binding (e.g. c-di-GMP binding on CckA) and the inhibition of the phosphatase state by a

395 pH-dependent conformational switch (33, 62). In contrast to the different conformations of HKs,

396 structures of HK-REC domain complexes determined to date indicate that REC domains bound

397 to either phosphotransfer or phosphatase states of HKs have similar conformations, with the REC

398 domain displaying a meta-active conformation, as defined in the previous section.

399 Partner Recognition and Specificity

400 Specificity of HK-RR pairs is crucial for the integrity of signaling pathways, given the large

401 number of different TCSs that typically exist in a single cell. Specificity within a pair is mediated

402 by recognition residues that participate in HK-RR binding. These residues are located in helix α1

403 of the REC domain and in the two α helices of the HK DHp domain that contribute to the

404 binding interface. Signaling pathways have been successfully rewired by substituting as few as

405 three co-evolved specificity residues (88, 96). Additional strategies are employed to further

406 ensure specificity of the pairs. For example, the phosphatase activity of HKs eliminates non-

407 specific phosphorylation by non-cognate kinases or small molecule phosphodonors such as AcP.

19 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 408 The low-abundance of HKs relative to RRs also minimizes cross-phosphorylation between non-

409 cognate pairs (57).

410 In an HK-RR complex, the REC domain inserts its α1 helix and β5-α5 loop between the

411 two helices of the DHp domain, primarily contacting the DHp α1 helix that contains the

412 phosphorylatable His residue. This conserved mode of binding buries the active site of the REC

413 domain at the HK-RR interface (Figure 6). Thus, conserved residues of both the HK and RR

414 form a substantial subset of the interface residues, with specificity being determined by a

415 relatively small set of variable residues. In addition to these specificity residues in α1 of the REC

416 domain and the two helices of the DHp domain, additional variable contacts can involve the β2-

417 α2, β3-α3 and β4-α4 loops of the REC domain interacting with the CA domain, the C-terminal

418 region of the DHp and the DHp-CA linker of the other protomer of the dimer (Figure 6) (21, 72,

419 74, 88, 107, 108, 114, 118, 122). Other contacts can occur between the REC domain and

420 additional domains within the HK, increasing the specificity and/or stability of the pair (e.g.

421 contacts between the PAS domain of ThkA and the RR TrrA) (118). These elements suggest that

422 beyond the conserved REC-DHp interface that buries the active site, the binding interface can

423 vary greatly in different HK-RR pairs.

424 A model based on structures of HK FixL and full-length RR FixJ suggests that the RR

425 effector domain is free to move and does not participate in binding to the HK (116). However, a

426 lack of structures of full-length RRs bound to HKs leaves open the question as to how effector

427 domains might be positioned relative to REC domains and domains of the HK. Given the great

428 diversity in REC-effector domain arrangements in different inactive RRs, it is possible that

429 diversity exists in the ways effector domains interact in different HK-RR complexes. It is easy to

430 envision how an effector domain could participate in the stability of the complex and/or the

20 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 431 efficiency of catalysis. Indeed, the meta-active conformation of the REC domain observed in

432 HK-REC domain complexes might be a consequence of isolated REC domains being unhindered

433 by REC-effector interactions that could bias conformational states.

434 Phosphotransfer State

435 Structural and biochemical studies of DesK-DesR complexes suggest that one HK dimer binds to

436 one RR molecule in an asymmetric conformation. The DesK-DesR structure and a docking

437 model of CpxA-CpxR in a putative phosphotransfer state show asymmetry within the HK dimer,

438 with the CA domain of one monomer bound to the DHp domain of the other monomer, leaving

439 the second CA domain untethered with enough space for RR binding to the DHp. Structural data

440 from the DesK-DesR complex and from CpxA-CpxR model suggest that this highly dynamic

441 state is coupled with autokinase activity, involves back-and-forth movements of the DHp and CA

442 domains, and is modulated by REC domain binding (72, 107).

443 Phosphatase State

444 In contrast to the asymmetrical domain arrangements associated with phosphotransfer, HK-RR

445 complexes in putative dephosphorylation states have symmetrical arrangements of the HK DHp

446 and CA domains. The REC domains are also arranged symmetrically, each interacting with a

447 DHp domain with a 2:2 HK:RR stoichiometry (62, 107). The HK in its phosphatase state is more

448 rigid than in the phosphotransfer state, with the N-terminal coiled-coil of the DHp domains being

449 highly stable.

450 Despite the large number of structures recently determined, the mechanistic details of the

451 enzymatic reactions involving HKs and RRs are not fully understood. Discrimination between

452 the phosphotransfer and phosphatase states that have been trapped in crystal structures still

453 remains challenging. The situation is further complicated by distinct phosphatase mechanisms

21 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 454 used by HKs of different subtypes. A major limitation of current studies is the use of truncated

455 proteins, most notably the cytoplasmic domains of transmembrane HKs lacking sensor and

456 transmembrane regions that in intact proteins control the signaling states of HKs, and REC

457 domains of RRs in the absence of effector domains that are known to influence their

458 conformational equilibria. While conserved features of HK-RR interactions are beginning to be

459 elucidated, variations on the theme are anticipated. Similar to many other aspects of TCS

460 structure and function, it is likely that the great variety of domain architectures in TCS proteins

461 enable different modes of HK-RR interactions and regulatory mechanisms that are adapted to

462 individual HK-RR pairs.

463

464 6. NON-CANONICAL MODES OF RR REGULATION

465 The canonical mechanism for regulation of RR activity involves phosphorylation at a conserved

466 Asp in the REC domain, stabilizing an active conformation that enables effector domain function.

467 Regulation of RR activity, in addition to or in place of Asp phosphorylation, potentially can be

468 achieved in many different ways including post-translational modifications at other sites that bias

469 the conformational equilibrium of the REC domain or directly alter effector domain function,

470 interactions that lower the energetic barrier for transition to an active conformation, ligand

471 binding to the RR, and regulation of expression to control RR levels and thus dimerization or

472 oligomerization. Indeed, each of these strategies has been observed and a few examples are

473 described below.

474 Reversible protein acetylation is known to regulate metabolic enzymes and transcription

475 in bacteria (49) and was documented in CheY more than 20 years ago (90). Enzyme catalyzed

476 acetylation of CheY at K91 and K106 increases in response to acetate and promotes clockwise

22 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 477 flagellar rotation, with acetylation at K91 proposed to alter conformational dynamics of the β4α4

478 loop (39). Autoacetylation with AcCoA as acetyl donor at other sites in CheY is thought to link

479 chemotaxis to the metabolic state of the cell (120). Another well-characterized example of

480 acetylation occurs in E. coli transcription factor RcsB. Acetylation of K154, a residue in the

481 helix-turn-helix motif that interacts with a phosphate in the DNA backbone, disrupts DNA

482 binding, down-regulates expression of the acid stress response genes, and compromises survival

483 in acidic conditions (22, 51). It should be noted that the stoichiometry of acetylation has not been

484 determined, thus physiological effects observed with mutations that mimic fully unmodified or

485 acetylated states might overestimate regulation that occurs in wild-type cells. A proteomics study

486 in E. coli documented acetylation of seven RRs: ArcA, CheY, CpxR, EvgA, NarL, OmpR and

487 RcsB (22). The E. coli genome encodes 26 putative Gcn5 N-acetyltransferases (GNATs) and a

488 single known deacetylase (CobB) (49). While the best-characterized acetyltransferase, PatZ, is

489 proposed to mediate a global strategy linking regulation to metabolic state, the large number of

490 acetyltransferases raises the possibility of substrate specificity that might enable system-specific

491 signaling mechanisms.

492 Signal-induced, HK-dependent, phosphorylation-independent monomer to dimer

493 activation has been observed for several RR transcription factors. In the extensively studied B.

494 subtilis system, binding of DesR to DesK promotes an active conformation of DesR. At

495 sufficiently high RR concentrations and with a slow active to inactive state transition,

496 dimerization can occur upon release of the RR from the HK with subsequent stabilization of the

497 dimer upon DNA binding (106). Similar phosphorylation-independent activation of OmpR by

498 HK EnvZ has been observed in acidic conditions (23). Cyanobacterial transcription factor NblR

499 contains a conserved Asp, but lacks other residues necessary for phosphorylation. HK NblS is

23 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 500 required for phosphorylation-independent activation of NblR. However, no NblS-NblR

501 interactions have been detected and the activating monomer to dimer transition is postulated to

502 be promoted by another protein partner (93).

503 Multiple strategies have been identified for regulation of orphan RRs that lack a

504 conserved Asp and/or other conserved residues necessary for phosphorylation. In streptomycetes,

505 two atypical RR transcription factors that lack residues necessary for phosphorylation, JadR1 and

506 RedZ, are regulated by the end products of the antibiotic biosynthetic pathways they control. The

507 antibiotic JdB binds directly to the JadR1 REC domain, disrupting DNA binding (112). A

508 different strategy is used by Helicobacter pylori HP1043, which exists as a constitutively active

509 dimer in vitro with a crystal structure similar to that of other activated OmpR subfamily

510 members (50). Levels of HP1043 are regulated both transcriptionally and post-transcriptionally

511 leading to speculation that control of expression of this constitutively active RR may be the sole

512 mechanism for regulating HP1403 activity (75).

513 Combinations of these mechanisms create even more strategies. Streptomyces coelicolor

514 GlnR, which regulates genes for nitrogen assimilation, is an orphan OmpR subfamily RR that

515 lacks residues for Asp phosphorylation and forms a constitutive α4-β5-α5 dimer (61). GlnR is

516 phosphorylated at 6 Ser/Thr sites in the DNA-binding domain under N-rich conditions,

517 disrupting DNA binding. GlnR is also acetylated at multiple Lys residues in the DNA-binding

518 domain, with acetylation enhancing DNA binding (4).

519

520 7. CONCLUDING REMARKS

521 When the structure of an RR REC domain was first reported thirty years ago, a central question

522 was how this single conserved domain could regulate responses as diverse as flagellar rotation,

24 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 523 transcription, and enzyme activity. The answer that emerged defined a mechanism that was both

524 simple and versatile. The small α/β REC domain exists in equilibrium between two primary

525 conformations with phosphorylation stabilizing an active conformation. This phosphorylation-

526 regulated switch enables regulatory strategies via any type of activating or inhibitory

527 macromolecular interactions that discriminate between the two states. Hundreds of structures of

528 RRs have provided descriptions of the conformations of REC domains in inactive and active

529 states, interactions with effector domains, DNA, HKs and auxiliary proteins. These structures

530 provide a foundation for identifying conserved features as well as specific variations in

531 individual RRs. Beyond the universally conserved enzymatic mechanisms facilitated by

532 configurations at the active site, other features such as Y-T coupling, regions of conformational

533 perturbations, domain arrangements and modes of DNA binding show distinct trends among RRs

534 within specific subfamilies.

535 Numerous variations on most every feature of RRs have been observed and undoubtedly,

536 many more remain to be discovered. The plasticity of the REC domain and versatility of RR

537 design allow an almost unlimited array of adaptations to fit the needs of individual signaling

538 systems. Defining details of how specific structural features impact function is important for

539 interpreting the nuances of RR behavior within specific TCSs as well as for pursuit of applied

540 projects such as development of antimicrobial therapeutics or the engineering of synthetic

541 biosensing pathways.

542 Our current understanding of RR structure is largely informed by compiling information

543 derived from many partial structural descriptions of different RRs. Very few structures are

544 available for full-length multi-domain RRs in both inactive and active states. The crystal

545 structures that do exist are constrained by the limitations of the methodology, specifically the

25 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 546 capture of single discrete states that do not reflect conformational distributions and the potential

547 promotion of inter- and/or intra-molecular interactions that are influenced by the high

548 concentrations of proteins used in crystallization and/or stabilization of conformations necessary

549 for crystallization and lattice interactions. While NMR studies have provided information about

550 dynamics and conformational distributions in solution, size limitations have mostly precluded

551 studies of full-length RRs, dimers, oligomers and complexes of RRs with HKs or other

552 macromolecular partners. Thus, while structures unambiguously define allowable states, it is

553 important to keep in mind the conformationally dynamic nature of RRs and the potential

554 influence of associated domains and macromolecular partners when interpreting structures

555 determined by methodologies with technical limitations. The emergence of new structural

556 methods such as high-resolution cryo-electron microscopy promise to provide solutions to some

557 of these challenges.

558

559 DISCLOSURE STATEMENT

560 The authors are not aware of any affiliations, memberships, funding, or financial holdings that

561 might be perceived as affecting the objectivity of this review.

562

563 ACKNOWLEDGMENTS

564 Research in the Stock laboratory is supported by grant R01 GM47958 from the National

565 Institutes of Health. S.B. was supported in part by the Foundation pour la Recherche Médicale

566 (FRM) Grant SPE201803005070.

567

568

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805 88. Podgornaia AI, Laub MT. 2013. Determinants of specificity in two-component signal

806 transduction. Curr. Opin. Microbiol. 16:156-62

807 89. Pontiggia F, Pachov DV, Clarkson MW, Villali J, Hagan MF, et al. 2015. Free energy

808 landscape of activation in a signalling protein at atomic resolution. Nat. Commun. 6:7284

809 90. Ramakrishnan R, Schuster M, Bourret RB. 1998. Acetylation at Lys-92 enhances signaling

810 by the chemotaxis response regulator protein CheY. Proc. Natl. Acad. Sci. USA 95:4918-23

811 91. Robinson VL, Wu T, Stock AM. 2003. Structural analysis of the domain interface in DrrB,

812 a response regulator of the OmpR/PhoB subfamily. J. Bacteriol. 185:4186-94

813 92. Romling U, Gomelsky M, Galperin MY. 2005. C-di-GMP: the dawning of a novel bacterial

814 signalling system. Mol. Microbiol. 57:629-39

37 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 815 93. Ruiz D, Salinas P, Lopez-Redondo ML, Cayuela ML, Marina A, Contreras A. 2008.

816 Phosphorylation-independent activation of the atypical response regulator NblR.

817 Microbiology 154:3002-15

818 94. Sidote DJ, Barbieri CM, Wu T, Stock AM. 2008. Structure of the Staphylococcus aureus

819 AgrA LytTR domain bound to DNA reveals a beta fold with an unusual mode of binding.

820 Structure 16:727-35

821 95. Simonovic M, Volz K. 2001. A distinct meta-active conformation in the 1.1-Å resolution

822 structure of wild-type apoCheY. J. Biol. Chem. 276:28637-40

823 96. Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, et al. 2008. Rewiring the

824 specificity of two-component signal transduction systems. Cell 133:1043-54

825 97. Sourjik V, Schmitt R. 1996. Different roles of CheY1 and CheY2 in the chemotaxis of

826 Rhizobium meliloti. Mol. Microbiol. 22:427-36

827 98. Sourjik V, Schmitt R. 1998. Phosphotransfer between CheA, CheY1, and CheY2 in the

828 chemotaxis signal transduction chain of Rhizobium meliloti. Biochemistry 37:2327-35

829 99. Stock AM, Robinson VL, Goudreau PN. 2000. Two-component signal transduction. Annu.

830 Rev. Biochem. 69:183-215

831 100. Surette MG, Levit M, Liu Y, Lukat G, Ninfa EG, et al. 1996. Dimerization is required for

832 the activity of the protein histidine kinase CheA that mediates signal transduction in

833 bacterial chemotaxis. J. Biol. Chem. 271:939-45

834 101. Thomas SA, Brewster JA, Bourret RB. 2008. Two variable active site residues modulate

835 response regulator phosphoryl group stability. Mol. Microbiol. 69:453-65

38 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 836 102. Thomas SA, Immormino RM, Bourret RB, Silversmith RE. 2013. Nonconserved active site

837 residues modulate CheY autophosphorylation kinetics and phosphodonor preference.

838 Biochemistry 52:2262-73

839 103. Tindall MJ, Porter SL, Maini PK, Armitage JP. 2010. Modeling chemotaxis reveals the role

840 of reversed phosphotransfer and a bi-functional kinase-phosphatase. PLoS Comput. Biol. 6

841 104. Toro-Roman A, Mack TR, Stock AM. 2005. Structural analysis and solution studies of the

842 activated regulatory domain of the response regulator ArcA: a symmetric dimer mediated

843 by the α4-β5-α5 face. J. Mol. Biol. 349:11-26

844 105. Toro-Roman A, Wu T, Stock AM. 2005. A common dimerization interface in bacterial

845 response regulators KdpE and TorR. Protein Sci. 14:3077-388

846 106. Trajtenberg F, Albanesi D, Ruetalo N, Botti H, Mechaly AE, et al. 2014. Allosteric

847 activation of bacterial response regulators: the role of the cognate histidine kinase beyond

848 phosphorylation. MBio 5:e02105

849 107. Trajtenberg F, Imelio JA, Machado MR, Larrieux N, Marti MA, et al. 2016. Regulation of

850 signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action.

851 Elife 5:e21422

852 108. Varughese KI. 2005. Conformational changes of Spo0F along the phosphotransfer pathway.

853 J. Bacteriol. 187:8221-27

854 109. Villali J, Pontiggia F, Clarkson MW, Hagan MF, Kern D. 2014. Evidence against the "Y-T

855 coupling" mechanism of activation in the response regulator NtrC. J. Mol. Biol. 426:1554-

856 67

857 110. Volkman BF, Lipson D, Wemmer DE, Kern D. 2001. Two-state allosteric behavior in a

858 single domain signaling protein. Science 291:2429-33

39 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 859 111. Volz K. 1993. Structural conservation in the CheY superfamily. Biochemistry 32:11741-53

860 112. Wang L, Tian X, Wang J, Yang H, Fan K, et al. 2009. Autoregulation of antibiotic

861 biosynthesis by binding of the end product to an atypical response regulator. Proc. Natl.

862 Acad. Sci. USA 106:8617-22

863 113. Wheeler TJ, Clements J, Finn RD. 2014. Skylign: a tool for creating informative,

864 interactive logos representing sequence alignments and profile hidden Markov models.

865 BMC Bioinformatics 15:7

866 114. Willett JW, Herrou J, Briegel A, Rotskoff G, Crosson S. 2015. Structural asymmetry in a

867 conserved signaling system that regulates division, replication, and virulence of an

868 intracellular pathogen. Proc .Natl. Acad. Sci. USA 112:E3709-18

869 115. Wolfe AJ. 2010. Physiologically relevant small phosphodonors link metabolism to signal

870 transduction. Curr. Opin. Microbiol. 13:204-9

871 116. Wright GSA, Saeki A, Hikima T, Nishizono Y, Hisano T, et al. 2018. Architecture of the

872 complete oxygen-sensing FixL-FixJ two-component signal transduction system. Sci. Signal.

873 11

874 117. Wu J, Ohta N, Newton A. 1998. An essential, multicomponent signal transduction pathway

875 required for cell cycle regulation in Caulobacter. Proc. Natl. Acad. Sci. USA 95:1443-48

876 118. Yamada S, Sugimoto H, Kobayashi M, Ohno A, Nakamura H, Shiro Y. 2009. Structure of

877 PAS-linked histidine kinase and the response regulator complex. Structure 17:1333-44

878 119. Yan D, Cho HS, Hastings CA, Igo MM, Lee SY, et al. 1999. Beryllofluoride mimics

879 phosphorylation of NtrC and other bacterial response regulators. Proc. Natl. Acad. Sci.

880 USA 96:14789-94

40 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 881 120. Yan J, Barak R, Liarzi O, Shainskaya A, Eisenbach M. 2008. In vivo acetylation of CheY, a

882 response regulator in chemotaxis of Escherichia coli. J. Mol. Biol. 376:1260-71

883 121. Zapf J, Madhusudan M, Grimshaw CE, Hoch JA, Varughese KI, Whiteley JM. 1998. A

884 source of response regulator autophosphatase activity: the critical role of a residue adjacent

885 to the Spo0F autophosphorylation active site. Biochemistry 37:7725-32

886 122. Zapf J, Sen U, Madhusudan, Hoch JA, Varughese KI. 2000. A transient interaction

887 between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of

888 molecular recognition and phosphotransfer in signal transduction. Structure 8:851-62

889 123. Zhao R, Collins EJ, Bourret RB, Silversmith RE. 2002. Structure and catalytic mechanism

890 of the E. coli chemotaxis phosphatase CheZ. Nat. Struct. Biol. 9:570-75

891 124. Zhu X, Rebello J, Matsumura P, Volz K. 1997. Crystal structures of CheY mutants Y106W

892 and T87I/Y106W: CheY activation correlates with movement of residue 106. J. Biol. Chem.

893 272:5000-06

894 125. Zhu Y, Qin L, Yoshida T, Inouye M. 2000. Phosphatase activity of histidine kinase EnvZ

895 without kinase catalytic domain. Proc. Natl. Acad. Sci. USA 97:7808-13

896

41 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 897

898 TERMS AND DEFINITIONS 899 900 AcP acetyl phosphate

- 901 BeF3 beryllofluoride

902 CA catalytic/ATP-binding (domain)

903 DBD DNA-binding domain

904 DHp dimerization/histidine phosphotransfer (domain)

905 ECF extracytoplasmic function

906 HK histidine kinase

907 HPt histidine-containing phosphotransfer (domain)

908 PDB Protein Data Bank

909 REC receiver (domain of response regulator protein)

910 RR response regulator protein

911 SDRR single-domain response regulator

912 TCS two-component system

913

42 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 914 FIGURE CAPTIONS

915 Figure 1. Common features of RRs. (a) Schematic diagram of the prototypical TCS pathway. (b)

916 The conserved (βα)5 fold of REC domains. Phosphorylation site residues and the phosphoryl

- 917 group mimic, beryllofluoride (BeF3 ), are shown in sticks. (c) Sequence conservation of RECs.

918 The profiled hidden Markov model (HMM) for the REC protein family (Pfam PF00072) is

919 shown as sequence logos (113) with the secondary structure elements illustrated. Heights of

920 individual stacked letters at each position correspond to information contents, reflecting the

921 probability of observing the particular amino acids at each position. Phosphorylation site

922 residues (stars), named after the most conserved amino acids (DD, D, T and K), are among the

923 most conserved residues in REC domains.

924

925 Figure 2. Classification of RRs by their effector domains. The percentile distribution is indicated

926 for RR effector functional classes and RR subfamilies. RR subfamilies are defined by effector

927 domain folds identified in Pfam. Representative proteins are traditionally used to name the

928 OmpR, NarL and NtrC subfamilies with effector domains named Trans_reg_C, GerE and

929 Sigma54_activa in Pfam. Representative structures of each subfamily are shown with REC

930 domains colored in grey and effector domains in colors (PDB ids: KdpE, 4KNY; RcsB, 5W43;

931 LuxO, 5EP0; AgrA, 3BS1; AmiR, 1QO0; WspR, 3BRE; CheB, 1A2O).

932

933 Figure 3. Phosphorylation site of the REC domain. The phosphoryl group is positioned by a

934 network of hydrogen bonds (dashed lines) with side chains of the highly conserved residues

935 (orange) as well as backbone atoms of non-conserved residues (light pink). Non-conserved

43 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 936 active site residues are labeled by their relative sequence positions to the nearest conserved

937 residues, such as T+1, indicating one residue C-terminal to the conserved Thr/Ser residue. (a)

938 Phosphorylation site of the archetype RR CheY in the active conformation (PDB id: 1FQW) with

939 residues that are differently positioned in the inactive conformation (2CHE) shown in cyan. (b)

940 The putative trigonal bipyramidal transition state for both phosphorylation and

941 dephosphorylation. X represents the leaving group of the phosphodonor for phosphorylation or

942 the attacking water for dephosphorylation. (c) Surface view of the active site in the phosphatase-

943 REC complex (3HZH).

944

945 Figure 4. RR regulatory strategies. (a) Distinct inactive and active RR conformations

- 946 exemplified by full-length RR VraR (PDB ids: 4GVP, 4IF4). BeF3 is shown in red spheres. (b)

947 Schematic diagrams of RR regulatory mechanisms. Functional sites of effector domains, such as

948 enzyme sites or DNA recognition regions, are shown as pink dots. These sites can be buried or

949 exposed in a wide variety of inactive RR conformations and their activity depends on different

950 interactions between the REC and effector domains. Phosphorylation of the RR can relieve REC

951 domain inhibition, promote effector function or both. Representative RRs that utilize these

952 strategies are indicated.

953

954 Figure 5. Phosphorylation-induced conformational changes in the REC domain. RR structures

- 955 with or without BeF3 were aligned using the conserved strands β1, β3, β4, and β5 to compute the

956 average backbone RMSD per residue (a, right). RMSD values above the median + 2x MAD

957 (median absolute deviation) are considered as significant conformational changes and the

44 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 - 958 corresponding residues are colored blue. Non-REC structural elements are colored cyan. BeF3 is

959 shown as red spheres and residues involved in the potential Y-T coupling are shown as green or

960 gray sticks. Representative protein structures shown for the (a) Stand-alone, (b) OmpR, (c) NtrC

961 and (d) NarL RR subfamilies are CheY (1F4V), DrrB (3NNS, 1P2F), NtrC1 (1ZY2, 1NY5) and

962 VraR (4IF4). Y/T residues from the inactive CheY structure (2CHE) are differently positioned

963 from the active structure. For the OmpR, NtrC and NarL subfamilies, two proteins from each

964 subfamily were used for RMSD analyses and both showed similar regions of conformational

965 changes. Substantial changes in α1 are also observed in one protein from the NtrC subfamily

966 (pink).

967

968 Figure 6. Structure of the HK-RR complex. Ribbon (a) and surface (b) views of the HK856-

969 RR468 complex (PDB id, 3DGE). Residues that determine the HK-RR interaction specificity (19,

970 96) are highlighted in light orange and cyan in the HK and RR, respectively. HK-RR contacts

971 also involve other surface regions (grey), including both active sites of the HK (red) and the RR

972 (pink).

45 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Table 1. Dimerization modes and orientations of the conserved aromatic residue in RR subfamilies.

Position of Y/F Family P Active Dimer Interface Alt. Dimer Interfaces (inward)

α4-β5-α5 (10/10) OmpR + 10/10a N.A. (1zes, 1xhf, 4uhk, 4s04, 6br7,…)

3/9 (MD) α4-β5-α5 (MD 3/9, Rec 16/18) α1-α5 (2/27) (1b00, 4uhs) - 9+3b/18(Rec) (1xhe, 2hqr, 3nhz, 3r0j, 4kny, 4uhs,…) Other (1mvo)

α4-β5 (4/5) NtrC + 6/6 β5-α5 (1/5) (2vui) (1l5y, 1zy2, 2jrl, 4d6y)

0/4 (MD) α4-β5 (MD 0/4, Rec 2/7) β5-α5 (5/9) - 2/7(Rec) (3cfy, 4d6x) (1l5z, 2jk1, 3dzd, 4i4u, 5m7n)

α1-α5 (7/8) β6-α6 (6/8) (4if4, 4ldz, 4zmr, 5hev…) NarL + 8/8 (4if4, 4ldz, 4zmr, 5hev, 5o8z, 4e7p, 4le0) α4-β5 (1/8) (1d5w)

6/8 (MD) α1-α5 (MD 4/8, Rec 5/8) β6-α6 (7/16) (4hye, 4le1, 2qsj, 5f64…) - 7/8(Rec) (1rnl, 5vxn, 4hye, 4le1, 3eul, 3b2n…) α4-β5 (1/8) (1dbw)

1/1 (MD) LytTR +/-c α4-loop-β5 (4cbv, 4ml3) N.A. 1/1(Rec) a: Numbers are for individual protein fragments. The REC domain or multi-domain (MD) fragments of the same protein are individually counted. b: Mixed positioning. Protein chains contain both inward and outward orientations of Y within a single crystal symmetry unit. c: Phosphorylation status is mimicked with D to E or D to A mutations.

46

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v2 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 1

a Phosphoryl transfer b α1 P α2 β2 Stimuli His Asp Responses β1 Input DHp CA REC Output β4 α3 β3

Histidine Kinase Response Regulator α α5 4 β5 c β1 α1 β2 α2 β3 α3 β4 α4 β5 α5 DD D T K 3.3 ** * * * 1.7 M IC (bits) V I V L I G L D I S T F E I I ED V E E L L V D Y Y I P NE D DN L G I V E I L MD V V GEA D P V QT I L V L N K L L V D N R G I L Q I S M I L I L V A SH N T L L P H G D F I I I A N P V I E A E E VK P V Q K I S E L K KE L K E E K S R E V L L L K L E L Q VL SK R R L G F A V S M G V E R V I S D K D T I SQ F K K E S AA I A E M N L E I E K T L A D RA I E I E K L E L G A M S E E K K E Q N E I I V K V E V N A A N N VK E L K Q A Q E I AQ F A KK Q L E I E DV T DE Q K I Q E K MV D K VK I E R R F V A K VQ S V E DDM E L E A KVMT V MA I V R S S N K T E L K R F V A L L L S KA I A M KV R E KG D AD R K A E Q A T F R R A ED T F NSA E A K T QQ SR AR F A C AE A K E KE MSK F A VM A A D H A E I V QR PD A C T K S E G D N A A K A AQ V NAVQ AA F AAKS I R D Q A T R T Y SK S E S S D A M G L S AV I MT R R AQ S A S S S L F T NS T L VK T NR A Q AKK T I MFM E RA A I S R A C GAS S AA Q AR M G N I R F AA SQ Q D E K T EKG A A Y F F R F AAQ N L KK N ML A A Q F S K R A C I P C N YQ L A M T M G S QA T S S DK T A C A L SAR F SQAA S T S V T NEQ T F A DNA E QF Y A S T GN KDV D YA QF DRS T T AN RQG T K T A F N C C T RRD M T M QD R A QQP S T R Y T T S S A T Q M HSAQA P GQD L T A KE F T S L N S Q T D Q Y E I T S MN T RE T P K S S L S A S Q L T R M D G S T Y T R T QM F T R L S T AKG S S T RVQN T F C L R DRS A T T QAD DYKS T T DK T S T S T YN T S NE Q T L I F H T L NS T S T S AAHQ T T S NP A R Q I S EN L H Q N D C F F H GN S SAS M AQ R T TMRGKRG F EN W SH AR F A F L GG M S C V T SQQ KR T E L N Y F HE S K L GM N VDS Y T T RD T S F T Y Q T N MT S Y M K L V F T D VAY C L R C DDGK L Y PH I D G Y P N L KS C Y A Q DH MM N L L NN GG V GG I V YQ T VR K L R L L H L G T I NE Q SH S P P I Y R VMS R N T S M R D VQ H D A D R S R NY T V V R T T Y C KG Y L L VH SK T VNGDQS SN T V P GQERV C NP E T I SWT S GDDQS P YRS F L R T T HAK L P SNY GN T T HGDGNV S G EWGC D L F T G PA T HMRV I VQKY GAAP VQR G L KR L VMT YRRKV VD I L L T ENGC D L E Y G T NHNM I QSKGG L T L L KKH L HHGGGMV P G Y Y VNG Y I H F V E P L G Y GDY G L T R L R F LQQY S C KVRV ENEKV VNS G S PNAV E VT V VHY P P P VRKR T S S 0 D G K

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v247 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 2

AgrA LuxO RcsB DNA Binding 66% NarL WspR (GGDEF) 19% NtrC 7% LytTR 6% Misc. OmpR 5% AmiR 29% KdpE RNA Binding 1% Protein Binding 1% Other 1% Enzymatic 8% GGDEF/EAL 3% CheB 2% Misc. 3% Stand alone 23% CheB (Methylesterase)

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v248 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 3

a b X = HK or small donor (phosphorylation) c Phosphatase β4-α4 X = OH2 (dephosphorylation) loop D+2 X T+1 T+1 T BeF3- T+2 N D+2 T O OH H2O β α O 2 5- 5 O O D+2 loop 2+ P O - Mg Mg O D T+1 N DD2 O OH2 D - O DD2 K O O K N - DD2 K O Active DD1 D DD1 DD1 Inactive β1-α1 loop

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v249 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 4

a Inactive Active

Rec P

VraR

DNA binding domain Linker

Recognition helix

b Inactive Active

CheB E NarL E R E E E PrrA E ... MtrA Inhibition RitR Relief E E E Enzyme R DrrB P DctD, NtrC1, LuxO, ... CheB E VraR EvgA PhoPMtb ... P P P P R R R R Positive ... R Regulation E E DrrD E E ChrA E FixJ Enzyme

KdpE, PmrA, RcsB, ... PleD

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v250 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 5

CheY a β1 α1 β2 α2 β3 α3 β4 α4 β5 α5

4 α5 T 2

Y 0 FliM Phos. Dependent Backbone RMSD (Å) 0 20 40 60 80 100 120 α4 Residue Number

b α5 α4’ OmpR subfamily α5 DNA-binding domain

β β5 P 5 β5’

α4 α5’ α4 4-5-5 Dimer c NtrC subfamily α5 α4’

β5 β5’ β5 β5’ P α4 α5’

4-5 Dimer 5-5 Dimer NarL subfamily d α1 α1’ 1-5 surface

β6’ α α 1’ 5 α5’ α6 β6’ α α1 6’ Linker β6

α4 α4’ 1-5 surface

1-5 Dimer Alternative Dimer

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v251 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019 Figure 6

a HK b HK’

His HK

specificity HK-RR region Interface RR’ RR active site RR α1

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27554v252 | CC BY 4.0 Open Access | rec: 21 Mar 2019, publ: 21 Mar 2019