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• W3157089598 abstract "•Crystal structure of AdeR dimer with traceable N-terminal intrinsically disordered region.•N-terminal intrinsically disordered region AdeR is involved in proteasome proteolysis.•Crystal structure of AdeS catalytic domain demonstrates cis autophosphorylation.•AdeS can assemble into hexamer and is crucial for its full kinase activity. Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems. Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems. Two-component regulatory systems (TCSs) are ubiquitous phosphorelay signal transduction proteins, critical for sensing and responding to environmental stimuli (Groisman, 2016Groisman E.A. Feedback control of two-component regulatory systems.Annu. Rev. Microbiol. 2016; 70: 103-124https://doi.org/10.1146/annurev-micro-102215-095331Crossref PubMed Scopus (72) Google Scholar; Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215https://doi.org/10.1146/annurev.biochem.69.1.183Crossref PubMed Scopus (2311) Google Scholar). As the TCSs are abundant in bacterial genomes and respond to a wide variety of stimuli including temperature, pH, osmotic pressure, metabolic substrates, and many other signals, they are therefore important in host-pathogen interactions, bacterial pathogenesis, and antibiotic resistance. The TCSs also represent potential antimicrobial drug targets, both for broad-spectrum and precisely targeted therapy (Gotoh et al., 2010Gotoh Y. Eguchi Y. Watanabe T. Okamoto S. Doi A. Utsumi R. Two-component signal transduction as potential drug targets in pathogenic bacteria.Curr. Opin. Microbiol. 2010; 13: 232-239https://doi.org/10.1016/j.mib.2010.01.008Crossref PubMed Scopus (198) Google Scholar). Acinetobacter baumannii ranks as the top critical threat among the multidrug-resistant bacteria according to the World Health Organization, possessing an extraordinary capacity for developing multidrug resistance. The adeRS-adeABC cluster has been extensively reported to mediate multidrug resistance against many antibiotics, including aminoglycosides, fluoroquinolones, tigecycline, and chloramphenicol (Marchand et al., 2004Marchand I. Marchand I. Damier-piolle L. Damier-piolle L. Courvalin P. Courvalin P. Lambert T. Lambert T. Expression of the RND-type efflux pump AdeABC in acinetobacter baumannii is regulated by the AdeRS two-component system.Antimicrob. Agents Chemother. 2004; 48: 3298-3304https://doi.org/10.1128/AAC.48.9.3298Crossref PubMed Scopus (0) Google Scholar; Yoon et al., 2013Yoon E.J. Courvalin P. Grillot-Courvalin C. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and aders mutations.Antimicrob. Agents Chemother. 2013; 57: 2989-2995https://doi.org/10.1128/AAC.02556-12Crossref PubMed Scopus (144) Google Scholar, Yoon et al., 2015Yoon E.J. Chabane Y.N. Goussard S. Snesrud E. Courvalin P. Dé E. Grillot-Courvalin C. Contribution of resistance-nodulation-cell division efflux systems to antibiotic resistance and biofilm formation in Acinetobacter baumannii.mBio. 2015; 6: 1-13https://doi.org/10.1128/mBio.00309-15Crossref Scopus (96) Google Scholar). The A. baumannii TCS AdeRS directly reduces repression of the efflux pump AdeABC by binding to an intercistronic region between adeRS and adeABC (Wen et al., 2017Wen Y. Ouyang Z. Yu Y. Zhou X. Pei Y. Devreese B. Higgins P.G. Zheng F. Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.Nucleic Acids Res. 2017; 45: 9773-9787https://doi.org/10.1093/nar/gkx624Crossref PubMed Scopus (11) Google Scholar). A diversity of mutations in both the histidine kinase AdeS and response regulator AdeR strongly associates these proteins with antimicrobial resistance in A. baumannii (Gerson et al., 2018Gerson S. Nowak J. Zander E. Ertel J. Wen Y. Krut O. Seifert H. Higgins P.G. Diversity of mutations in regulatory genes of resistance-nodulationcell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii.J. Antimicrob. Chemother. 2018; 73: 1501-1508https://doi.org/10.1093/jac/dky083Crossref PubMed Scopus (26) Google Scholar; Nowak et al., 2016Nowak J. Schneiders T. Seifert H. Higgins P.G. The Asp20-to-Asn substitution in the response regulator AdeR leads to enhanced efflux activity of AdeB in Acinetobacter baumannii.Antimicrob. Agents Chemother. 2016; 60: 1085-1090https://doi.org/10.1128/AAC.02413-15Crossref PubMed Scopus (16) Google Scholar; Yoon et al., 2013Yoon E.J. Courvalin P. Grillot-Courvalin C. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and aders mutations.Antimicrob. Agents Chemother. 2013; 57: 2989-2995https://doi.org/10.1128/AAC.02556-12Crossref PubMed Scopus (144) Google Scholar). In the canonical TCS, the transmembrane sensor histidine kinase undergoes a conformational change upon sensing an external environment stimulus and auto-phosphorylates at a conserved histidine residue using ATP. The phosphate group can then be transferred to an aspartate residue in the cognate response regulator, which then initiates a response by modulating target gene expression (Capra and Laub, 2012Capra E.J. Laub M.T. The evolution of two-component signal transduction systems.Annu. Rev. Microbiol. 2012; 66: 325-347https://doi.org/10.1016/j.pestbp.2011.02.012Crossref PubMed Scopus (36) Google Scholar; Groisman, 2016Groisman E.A. Feedback control of two-component regulatory systems.Annu. Rev. Microbiol. 2016; 70: 103-124https://doi.org/10.1146/annurev-micro-102215-095331Crossref PubMed Scopus (72) Google Scholar; Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215https://doi.org/10.1146/annurev.biochem.69.1.183Crossref PubMed Scopus (2311) Google Scholar). Owing to the tremendous numbers of TCSs and the diverse environmental stimuli to which they respond, there have been multiple modes of activation reported both in sensor histidine kinases and response regulators (Desai and Kenney, 2017Desai S.K. Kenney L.J. To ∼P or Not to ∼P? Non-canonical activation by two-component response regulators.Mol. Microbiol. 2017; 103: 203-213https://doi.org/10.1111/mmi.13532Crossref PubMed Scopus (30) Google Scholar; Willett and Crosson, 2017Willett J.W. Crosson S. Atypical modes of bacterial histidine kinase signaling.Mol. Microbiol. 2017; 103: 197-202https://doi.org/10.1111/mmi.13525Crossref PubMed Scopus (19) Google Scholar). In this study, we characterize how conformational changes are propagated along the A. baumannii AdeRS TCS. We have found a new mechanism whereby the activity of the response regulator AdeR is dependent upon the presence of an intrinsically disordered N-terminal tail. We also determine the structure of the histidine kinase transmitter domain of AdeS using X-ray crystallography and demonstrate, via negative stain electron microscopy and small-angle X-ray scattering (SAXS), that the full cytoplasmic domain of AdeS forms a hexamer in solution necessary for its histidine kinase activity. The present investigation sheds light on new structural mechanisms whereby signals are transduced and regulated along the TCS AdeRS in A. baumannii. Structure-based sequence alignment of A. baumannii response regulator AdeR with other response regulator family indicates that it contains an extended N-terminal sequence, “FDHSFSFDCQD,” which consists of three hydrophobic F and three acidic D residues (Figure S1). The electron density of this tail could not be observed in the 1.4 Å resolution crystal structure of the AdeR receiver domain (Wen et al., 2017Wen Y. Ouyang Z. Yu Y. Zhou X. Pei Y. Devreese B. Higgins P.G. Zheng F. Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.Nucleic Acids Res. 2017; 45: 9773-9787https://doi.org/10.1093/nar/gkx624Crossref PubMed Scopus (11) Google Scholar), indicating that it is disordered and able to adopt multiple conformations. Its disordered nature was further validated by SAXS (Wen et al., 2017Wen Y. Ouyang Z. Yu Y. Zhou X. Pei Y. Devreese B. Higgins P.G. Zheng F. Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.Nucleic Acids Res. 2017; 45: 9773-9787https://doi.org/10.1093/nar/gkx624Crossref PubMed Scopus (11) Google Scholar). In the current study, crystallization and structure determination of an AdeR K112A mutant interestingly demonstrate a completely traceable N-terminal chain at 1.8 Å, running along a groove across the dimer interface formed by helices α4 and α5 (Figures 1A and S2A, Table S1). The binding of the ordered N-terminal region to the other AdeR monomer coincides with a rearrangement of the α4-β5-α5 dimerization interface, characterized by a 15 Å shift of residue R103 in the α4 helix, as indicated by an alignment of the structures of AdeRWT and AdeRK112A (Figure 1A) and a coloring of this alignment by root-mean-square deviation (Figure S2B). Between the two AdeR monomers in the crystal asymmetric unit, the exact conformation of the N-terminal tail varies, which is a further indication of its structural plasticity. However, a common feature in both observed conformations is the electrostatic interaction of the side chains of F6 and R128 with the C-terminal end of helix α4 across the dimer interface. The structure reveals a multitude of dipoles with their negative ends oriented toward F6 and R128, including the non-hydrogen-bonded carbonyl oxygens of V99, M100, and A101 at the C-terminal end of helix α4, as well as the carbonyl oxygens of L102 and R103 (Figures 1B and S2A). These interactions re-orient and stabilize helix α4, lengthening it relative to the wild-type structure so that its N-terminal end extends further back to D93. In the crystal structure of wild-type AdeR, this arrangement is prevented by a steric clash between the β4-α4 loop and the side chain of K112, which is held in place by a salt bridge with D63. Hence, it appears that the K112A mutation is coupled to a re-orientation and increased structuring of both the α4 helix and the disordered N-terminal tail. These structural changes further lead to a rearrangement of the α4-β5-α5 dimerization interface, causing a small shift in the relative orientation of the monomers within the homodimer (Figures 1A and S2B). To verify whether the intrinsically disordered N-terminal region is involved in the regulation of the efflux pump AdeABC, we tested transcription of the adeB gene and antimicrobial susceptibility upon truncation of the AdeR N-terminal intrinsically disordered region. Quantitative RT-PCR showed that truncation of the N-terminal tail results in decreased adeB expression (Figure 1C) and an increase in susceptibility to gentamicin and streptomycin (Figure 1D, Table S2). This suggests that the N-terminal tail is needed to fully activate adeB expression. The K112A mutation also decreased adeB expression and increased antibiotic susceptibility. The explanation for this result is that the K112 side chain hydrogen bonds to the phosphate group of phosphoaspartate in homologous response regulator proteins like PhoP. We thus propose that the observed structure of the K112A mutant of AdeR represents a conformation that is partially activated by disruption of the K112-D63 hydrogen bond and structuring of the N-terminal disordered tail, but full activation requires phosphorylation of D63 and its subsequent interaction with K112. The N terminus is extensively reported to be an important determinant of protein half-life in vivo via the N-end rule, which highlights the preference of the proteasome for certain N-terminal degradation signals (N-degrons) (Bachmair et al., 1986Bachmair A. Finley D. Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue.Science. 1986; 234: 179-186https://doi.org/10.1126/science.3018930Crossref PubMed Scopus (1300) Google Scholar; Kim et al., 2014Kim H.K. Kim R.R. Oh J.H. Cho H. Varshavsky A. Hwang C.S. The N-terminal methionine of cellular proteins as a degradation signal.Cell. 2014; 156: 158-169https://doi.org/10.1016/j.cell.2013.11.031Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar; Varshavsky, 2011Varshavsky A. The N-end rule pathway and regulation by proteolysis.Protein Sci. 2011; 20: 1298-1345https://doi.org/10.1002/pro.666Crossref PubMed Scopus (419) Google Scholar). An N-terminal D, E, F, H, Q, or Y residue confers a short half-life to a protein, being readily recognized by the proteasome (Bachmair et al., 1986Bachmair A. Finley D. Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue.Science. 1986; 234: 179-186https://doi.org/10.1126/science.3018930Crossref PubMed Scopus (1300) Google Scholar; Kim et al., 2014Kim H.K. Kim R.R. Oh J.H. Cho H. Varshavsky A. Hwang C.S. The N-terminal methionine of cellular proteins as a degradation signal.Cell. 2014; 156: 158-169https://doi.org/10.1016/j.cell.2013.11.031Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar; Varshavsky, 2011Varshavsky A. The N-end rule pathway and regulation by proteolysis.Protein Sci. 2011; 20: 1298-1345https://doi.org/10.1002/pro.666Crossref PubMed Scopus (419) Google Scholar). The intrinsically disordered N-terminal region of AdeR “FDHSFSFDCQD” has shown high frequency of D, F, H, and Q residues, so proteolytic processing would produce a new N-terminal tail that would be highly susceptible to proteasomal degradation. We performed an in vivo proteolysis assay of AdeR with and without the intrinsically disordered N-terminal region in A. baumannii. Owing to the lack of a commercially available anti-AdeR antibody, we used an anti-His tag antibody to detect the C-terminal His-tagged AdeR expressed from our plasmid. The proteolysis assay demonstrates that AdeR is degraded in vivo with a half-life around 70 min, whereas truncation of the N-terminal region significantly slowed the rate of degradation (Figures 2A and 2B ). Lactacystin and its derivative β-lactone are covalent inhibitors of the prokaryotic proteasome (Craiu et al., 1997Craiu A. Gaczynska M. Akopian T. Gramm C.F. Fenteany G. Goldberg A.L. Rock K.L. Lactacystin and clasto-lactacystin β-lactone modify multiple proteasome β-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation.J. Biol. Chem. 1997; 272: 13437-13445https://doi.org/10.1074/jbc.272.20.13437Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar; Ōmura and Crump, 2019Ōmura S. Crump A. Lactacystin: first-in-class proteasome inhibitor still excelling and an exemplar for future antibiotic research.J. Antibiot. (Tokyo). 2019; 72: 189-201https://doi.org/10.1038/s41429-019-0141-8Crossref PubMed Scopus (12) Google Scholar), and addition of the β-lactone reduced the proteolysis rate of AdeR in vivo (Figures 2A and 2B). Thus, although the intrinsically disordered N-terminal region of AdeR is needed for maximal activation, its presence also renders it susceptible to proteasomal degradation. We further tested whether truncation of the disordered N-terminal region of AdeR would modify its DNA-binding activity. Isothermal titration calorimetery demonstrated that N-terminally truncated AdeR interacts with its target DNA with similar binding affinity (Kd = 20 nM) and thermodynamic properties (Figure 2C) (Wen et al., 2017Wen Y. Ouyang Z. Yu Y. Zhou X. Pei Y. Devreese B. Higgins P.G. Zheng F. Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.Nucleic Acids Res. 2017; 45: 9773-9787https://doi.org/10.1093/nar/gkx624Crossref PubMed Scopus (11) Google Scholar). Thus, although the N-terminal tail of AdeR is required for its maximal activation, it is not needed for high-affinity DNA binding. Instead, it could be acting through any of the following potential mechanisms: stabilization of an AdeR dimer configuration that distorts and activates bound DNA, recruitment of an unknown factor to the AdeR-DNA complex, or facilitation of AdeS-mediated phosphorylation of AdeR. The sensor histidine kinase AdeS consists of two transmembrane N-terminal helices linked by a short extracellular sensor domain (residue 34−61), a HAMP domain (residue 84−138), a DHp (dimerization histidine phosphotransfer) domain (residue 146−204), and a C-terminal catalytic ATP (CA)-binding domain (residue 204−357) (Figure 3A). To investigate the kinetic properties of AdeS, we purified different recombinant constructs consisting of the AdeS full cytoplasmic domain (HAMP-DHp-CA), HAMP-DHp domain, and histidine kinase transmitter domain (DHp-CA), confirmed by SDS-PAGE (Figure 3B). The size exclusion chromatography profile indicated that the AdeS DHp-CA domain elutes as dimer with a calculated molecular weight of 47 kDa, whereas the cytoplasmic domain elutes with a calculated molecular weight around 190 kDa (Figure 3B), suggestive of a hexameric assembly. The HAMP-DHp domain elutes as a broader peak, which may imply a non-globular shape or the presence of mixed oligomers (Figure 3B). RT-qPCR experiments of adeB expression indicated that the AdeS constructs lacking the transmembrane sensor domain still retain kinase activity in vivo. However, truncation of the HAMP domain or mutation of the catalytic histidine H149 totally abolishes its activity (Figure 3C). RT-qPCR detection of adeB expression also correlated with antibiotic minimum inhibitory concentration values determined for different AdeS construct strains (Table S3). We further measured the kinase activity of the AdeS cytoplasmic domain and the DHp-CA domain (Figures 3D and 3E). The cytoplasmic domain has a Km of 5.25 ± 0.20 μM for ATP and Vmax of 1.1 μmol/mg⋅min−1 for autophosphorylation, whereas the DHp-CA domain alone displays no phosphoryl transfer activity (Figures 3D and 3E). To further elucidate the molecular mechanism and assembly of AdeS, we carried out a combined X-ray crystallography and electron microscopy study of recombinant AdeS. The crystal structure of apo AdeS DHp-CA was solved by molecular replacement using a trimmed version of the catalytic domain of histidine kinase CpxA (PDB ID; 5FLK) (Mechaly et al., 2017Mechaly A.E. Soto Diaz S. Sassoon N. Buschiazzo A. Betton J.-M. Alzari P.M. Structural coupling between autokinase and phosphotransferase reactions in a bacterial histidine kinase.Structure. 2017; 25: 939-944https://doi.org/10.1016/j.str.2017.04.011Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), which has an overall sequence identity of 25% with AdeS (Figure S3), and an I-TASSER model of the DHp domain of AdeS as search templates. The AdeS DHp-CA domain consists of a symmetric homodimer, in which the α1-α2 DHp domain forms a central four helix bundle with a large parallel dimerization interface, which is flanked by CA domains on each side (Figure 4A). The conserved phosphorylation site histidine 149 is located at the N-terminal end of the α1 helix, whereas residues 138−147 were present in our DHp-CA construct but not visible in the crystal structure. This contrasts with CpxA, in which the α1 helix extends further N-terminally and is continuous with the HAMP C-terminal helix (Figure S3). The α1 helix of AdeS extends from H149 to D167, followed by a short linker from G168-D173, leading to a long α2 helix from E174 to N202. The short interhelical linker in AdeS travels in a different direction and is longer than the corresponding linker in CpxA, so that the α2 helices are “swapped” when the homodimer of AdeS is compared with CpxA. This is why the CA domain in AdeS appears positioned to catalyze a cis autophosphorylation of His149 (within the same monomer), whereas CpxA appears to catalyze a trans-phosphorylation (Figure 4B). A flexible linker between Q203 and N208 imparts a high degree of mobility to the CA domains when the structure of AdeS is compared with CpxA and other histidine kinases. In our AdeS structure, there are some hydrophobic contacts between the α1 helix (L197 and V200) of the DHp domain and the CA domain (V326 and A329), fixing the positioning of the CA domains. These must be transient for the CA domain to be able to move into the correct position to autophosphorylate H149 (Figures 4A and 4B). All the residues involved in the ATP binding and autophosphorylation are identical in AdeS and CpxA (Figure S3). Co-crystallization attempts for the AdeS full cytoplasmic domain and for DHp-CA domain with ATP, ADP, and ATP analogues were unsuccessful. We therefore turned to direct visualization of the full cytoplasmic domain with negative stain electron microscopy, using a highly monodisperse preparation of purified cytoplasmic AdeS (HAMP-DHp-CA), both in the apo state and in the presence of ATP and Mg2+ (Figures 4C and S4). In total 619 and 319 micrographs were collected for the apo and ATP-bound states, respectively. After contrast transfer function estimation, 566 and 299 micrographs were screened out for the particle-picking procedure. About 37,000 particles for each individual dataset were used to do the 2D classification. Comparing the two class averages, more compact density distribution within each blade was observed in the ATP-bound state (Figure 4C). The gold-standard Fourier shell correlation curves at 0.143 criterion indicate a final resolution of 25 Å (apo state) and 22 Å (ATP-bound state), respectively (Figure S4). The projections obtained from certain Euler angles of the final model matched well with the experimental 2D class averages, with the three-blade propeller-like orientation corresponding to the top view of the 3D model (Figure 4C). We then manually docked our crystal structure of DHp-CA dimer into the two electron microscopic (EM) models by using the Fit-in-map routine of UCSF Chimera software. The crystal structures occupied the three blade-like densities outside well with the central joint-density vacant. It demonstrates that the DHp-CA dimers assemble further into a trimer-of-dimers mediated by the centrally located N-terminal HAMP domain. The arrangement requires greater flexibility between the HAMP domains and DHp domains than was observed in CpxA, and this may have contributed to the difficulty in crystallizing the full-length cytoplasmic AdeS construct. Comparison of the coordinates of the docked crystal structures shows a slightly clockwise rotation of the CA domains in the ATP-bound model relative to the apo model. Taken together, negative stain EM maps of the apo and ATP-incubated cytoplasmic AdeS both revealed an assembly into a hexamer, although the ATP-incubated cytoplasmic AdeS displayed better-defined CA domains, suggesting that there was a greater conformational heterogeneity in the apo state, with binding of ATP stabilizing a more uniform activated state (Figures 4D and S4). To further explore the physiological assembly of AdeS, we carried out SAXS and an in-cell cross-linking study. Plots of the Guinier region provide evidence of the overall high quality of the SAXS data, and corresponding distance distributions indicated the overall size of the measured samples (Figures 5A and 5B ). The SAXS data confirm the dimeric assembly of the AdeS DHp-CA domain and the hexameric assembly of the AdeS full cytoplasmic domain, as judged by the molecular weight calculated by MoW2 and the Porod volume approach, which is consistent with our results with size exclusion chromatography (Table S4). The AdeS DHp-CA domain SAXS experimental data strongly confirm its dimeric crystal structure with a Χ2 value of 1.8. Furthermore, the AdeS full cytoplasmic domain model was generated by fitting three copies of the AdeS DHp-CA dimer and a hexameric HAMP domain model generated from I-TASSER in the negative stain EM map. The validation of the AdeS full cytoplasmic domain model with the SAXS experimental data using AllosMod-FoXS shows good agreement with the hexameric assembly in solution (Figure 5C) (Yang and Zhang, 2015Yang J. Zhang Y. I-TASSER server: new development for protein structure and function predictions.Nucleic Acids Res. 2015; 43: W174-W181https://doi.org/10.1093/nar/gkv342Crossref PubMed Scopus (817) Google Scholar). To further verify the oligomeric state of full-length AdeS in vivo, we carried out an in-cell formaldehyde cross-linking experiment employing a low-copy-number full-length AdeS expression plasmid with His-tag. A. baumannii cells treated with and without formaldehyde were disrupted; the AdeS full-length protein was purified and detected by western blotting using an anti-His-tag antibody (Figure 5D). The results indicated that cross-linked AdeS behaved as an oligomer with a molecular weight higher than 170 kDa, whereas the sample without cross-linking behaved as a monomer on SDS-PAGE. Both samples showed a light band corresponding to a dimer population, which could have resulted from incomplete denaturation in SDS (Figure 5D). Taken together, our combined X-ray crystallographic/SAXS/negative stain EM study determines the physiological assembly of AdeS to be a trimer-of-dimers and allows us to propose a structural model for the full cytoplasmic domain of AdeS. The mechanism by which signals are transduced along the ubiquitous TCSs remains a profound mystery because of a continuous chain of conformational transitions that must occur within each component. Perhaps the most easily understood part of the signal transduction is the transfer of a phosphate group from the histidine kinase sensor to the response regulator. Downstream of this transfer, phosphorylation of the aspartate causes long-range conformational changes throughout the receiver domain that shift its equilibrium from a monomer to dimer. Classically, the phosphorylation of the aspartate on the β3 strand has been described as causing a conformational switch in a β4 strand threonine, which is passed on to a β5 strand tyrosine (so-called “Y-T” coupling), which is in turn transmitted to the α4 and α5 helices that constitute the dimerization interface (Bachhawat and Stock, 2007Bachhawat P. Stock A.M. Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofiuoride.J. Bacteriol. 2007; 189: 5987-5995https://doi.org/10.1128/JB.00049-07Crossref PubMed Scopus (58) Google Scholar; Bourret, 2010Bourret R.B. Receiver domain structure and function in response regulator proteins.Curr. Opin. Microbiol. 2010; 13: 142-149https://doi.org/10.1016/j.mib.2010.01.015Crossref PubMed Scopus (154) Google Scholar). Although “Y-T” coupling has been challenged (Campagne et al., 2016Campagne S. Dintner S. Gottschlich L. Thibault M. Bortfeld-Miller M. Kaczmarczyk A. Francez-Charlot A. Allain F.H.T. Vorholt J.A. Role of the PFXFATG[G/Y] motif in the activation of SdrG, a response regulator involved in the alphaproteobacterial general stress response.Structure. 2016; 24: 1237-1247https://doi.org/10.1016/j.str.2016.05.015Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar; Ouyang et al., 2019Ouyang Z. Zheng F. Chew J.Y. Pei Y. Zhou J. Wen K. Han M. Lemieux M.J. Hwang P.M. Wen Y. Deciphering the activation and recognition mechanisms of Staphylococcus aureus response regulator ArlR.Nucleic Acids Res. 2019; 47: 11418-11429https://doi.org/10.1093/nar/gkz891Crossref PubMed Scopus (4) Google Scholar), it is clear that at least some response regulator receiver domains employ a different mechanism of signal transduction (Desai and Kenney, 2017Desai S.K. Kenney L.J. To ∼P or Not to ∼P? Non-canonical activation by two-component response regulators.Mol. Microbiol. 2017; 103: 203-213https://doi.org/10.1111/mmi.13532Crossref PubMed Scopus (30) Google Scholar). Despite the h" @default.
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• W3157089598 title "Proteolysis and multimerization regulate signaling along the two-component regulatory system AdeRS" @default.
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