Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m700556200
ISSN1083-351X
AutoresMatthew J. Bottomley, Ester Muraglia, Renzo Bazzo, Andrea Carfı́,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoMany Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C12-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an α-β-α fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-Å resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors. Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C12-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an α-β-α fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-Å resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors. Evolution has endowed bacteria with a wide variety of diffusible chemical signals that are used for communication both within and between species. The most common form of intercellular communication in Gram-negative bacteria is “quorum sensing,” so named because the bacteria initiate coordinated activities only when a “quorum” population density is reached (1Fuqua W.C. Winans S.C. Greenberg E.P. J. Bacteriol. 1994; 176: 269-275Crossref PubMed Scopus (2194) Google Scholar). This synchronization of individual behavior into cooperative group activity can have many different benefits, e.g. to produce virulence factors only when the bacterial population is sufficiently dense to survive host immune responses (2Winans S.C. Bassler B.L. J. Bacteriol. 2002; 184: 873-883Crossref PubMed Scopus (126) Google Scholar, 3Parsek M.R. Greenberg E.P. Trends Microbiol. 2005; 13: 27-33Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 4Bjarnsholt T. Givskov M. Anal. Bioanal. Chem. 2007; 387: 409-414Crossref PubMed Scopus (98) Google Scholar). The first quorum sensing system described was that of Vibrio fischeri, a symbiont of the Hawaiian bobtail squid (5Ruby E.G. Annu. Rev. Microbiol. 1996; 50: 591-624Crossref PubMed Scopus (242) Google Scholar, 6Nealson K.H. Platt T. Hastings J.W. J. Bacteriol. 1970; 104: 313-322Crossref PubMed Google Scholar). The squid has a “light organ” wherein it provides nutrition for V. fischeri. Upon reaching high population density the bacteria produce bioluminescence enabling the squid to cancel its own shadow and thereby avoid predation (7Visick K.L. McFall-Ngai M.J. J. Bacteriol. 2000; 182: 1779-1787Crossref PubMed Scopus (133) Google Scholar). The quorum sensing mechanism triggering bioluminescence depends on the bacterial synthase LuxI, which constitutively produces a signal molecule, an acylhomoserine lactone (acyl-HSL or AHL), 2The abbreviations used are: AHL, acylhomoserine lactone; QSI, quorum sensing inhibitor; HSL, homoserine lactone; LBD, ligand-binding domain. the autoinducer. The AHL diffuses through the bacterial envelope, and, upon reaching a threshold concentration, the AHL binds and activates its receptor LuxR, a transcriptional activator of the luciferase operon (1Fuqua W.C. Winans S.C. Greenberg E.P. J. Bacteriol. 1994; 176: 269-275Crossref PubMed Scopus (2194) Google Scholar). Many different Gram-negative bacteria have now been shown to use quorum sensing systems composed of LuxI/LuxR homologues and AHL autoinducers that elicit diverse responses (8Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 685-695Crossref PubMed Scopus (836) Google Scholar). The LuxI homologues show moderate amino acid conservation (28–35% identity), whereas the LuxR homologues display only 10–25% identity. Of the many quorum sensing systems known, that of Pseudomonas aeruginosa is the most intensively studied due to the potentially fatal effects of its infections, which kill several thousand immunocompromised individuals each year (9Costerton J.W. Trends Microbiol. 2001; 9: 50-52Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). P. aeruginosa is an opportunistic pathogen causing death in the majority of cystic fibrosis sufferers and in AIDS patients, burn victims, and neutropenic cancer patients (10Lyczak J.B. Cannon C.L. Pier G.B. Microbes Infect. 2000; 2: 1051-1060Crossref PubMed Scopus (1001) Google Scholar). It is the most common Gram-negative bacterium found in hospital-acquired infections, being responsible for nosocomial pneumonia, urinary tract infections, and surgical wound or bloodstream infections (11Van Delden C. Iglewski B.H. Emerg. Infect. Dis. 1998; 4: 551-560Crossref PubMed Scopus (614) Google Scholar). Current therapies lack efficacy, partly because P. aeruginosa creates and inhabits surface-associated biofilms conferring increased resistance to antibiotics and host immune responses (12Anwar H. Dasgupta M.K. Costerton J.W. Antimicrob. Agents Chemother. 1990; 34: 2043-2046Crossref PubMed Scopus (269) Google Scholar). P. aeruginosa can also adapt to overcome the selective pressures imposed by growth-restricting antibiotics. Therefore, there is an urgent requirement for novel antibacterial treatments to counter P. aeruginosa. It is thus of interest that quorum sensing systems control many aspects of P. aeruginosa pathogenesis, e.g. the expression of virulence factors and a greater resistance of biofilms to antibiotics and the immune system (13Van Delden C. Pesci E.C. Pearson J.P. Iglewski B.H. Infect. Immun. 1998; 66: 4499-4502Crossref PubMed Google Scholar, 14Winzer K. Williams P. Int. J. Med. Microbiol. 2001; 291: 131-143Crossref PubMed Scopus (194) Google Scholar, 15Juhas M. Eberl L. Tummler B. Environ. Microbiol. 2005; 7: 459-471Crossref PubMed Scopus (296) Google Scholar, 16Rasmussen T.B. Givskov M. Int. J. Med. Microbiol. 2006; 296: 149-161Crossref PubMed Scopus (617) Google Scholar). This presents opportunities for the design of quorum sensing inhibitors (QSIs), which reduce virulence, pathogenicity, and resistance rather than directly inhibiting growth, with the important ramifications that they would be unlikely to exert selective pressures leading to the emergence of drug-resistant bacteria and would not kill the beneficial gut flora (17Hentzer M. Wu H. Andersen J.B. Riedel K. Rasmussen T.B. Bagge N. Kumar N. Schembri M.A. Song Z. Kristoffersen P. Manefield M. Costerton J.W. Molin S. Eberl L. Steinberg P. Kjelleberg S. Hoiby N. Givskov M. EMBO J. 2003; 22: 3803-3815Crossref PubMed Scopus (1133) Google Scholar). In P. aeruginosa, quorum sensing of high population density induces the production of virulence factors potentially fatal to the infected patient (18Gambello M.J. Kaye S. Iglewski B.H. Infect. Immun. 1993; 61: 1180-1184Crossref PubMed Google Scholar, 19Smith R.S. Iglewski B.H. Curr. Opin. Microbiol. 2003; 6: 56-60Crossref PubMed Scopus (513) Google Scholar). The LasI synthase of P. aeruginosa constitutively produces the signal 3-oxo-C12-HSL (N-3-oxododecanoyl-l-homoserine lactone), which accumulates with population growth and activates LasR, a transcriptional regulator (R protein) homologous to LuxR. When activated by 3-oxo-C12-HSL, LasR dimers bind target gene promoters and activate the transcription of many toxic virulence factors, including exoproteases, exotoxins, and secondary metabolites (18Gambello M.J. Kaye S. Iglewski B.H. Infect. Immun. 1993; 61: 1180-1184Crossref PubMed Google Scholar, 19Smith R.S. Iglewski B.H. Curr. Opin. Microbiol. 2003; 6: 56-60Crossref PubMed Scopus (513) Google Scholar, 20Winson M.K. Camara M. Latifi A. Foglino M. Chhabra S.R. Daykin M. Bally M. Chapon V. Salmond G.P. Bycroft B.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9427-9431Crossref PubMed Scopus (426) Google Scholar, 21Kiratisin P. Tucker K.D. Passador L. J. Bacteriol. 2002; 184: 4912-4919Crossref PubMed Scopus (143) Google Scholar, 22Schuster M. Urbanowski M.L. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15833-15839Crossref PubMed Scopus (222) Google Scholar). Activated LasR also participates in the maturation of biofilms, which commonly result in persistent pathogenic infections (3Parsek M.R. Greenberg E.P. Trends Microbiol. 2005; 13: 27-33Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 23Davies D.G. Parsek M.R. Pearson J.P. Iglewski B.H. Costerton J.W. Greenberg E.P. Science. 1998; 280: 295-298Crossref PubMed Scopus (2590) Google Scholar). Overall, quorum sensing in P. aeruginosa probably controls over 350 genes, of which ∼30% encode virulence factors (16Rasmussen T.B. Givskov M. Int. J. Med. Microbiol. 2006; 296: 149-161Crossref PubMed Scopus (617) Google Scholar, 24Schuster M. Lostroh C.P. Ogi T. Greenberg E.P. J. Bacteriol. 2003; 185: 2066-2079Crossref PubMed Scopus (873) Google Scholar). Evidence now shows that QSIs targeting LasR can attenuate the pathogenicity of P. aeruginosa (17Hentzer M. Wu H. Andersen J.B. Riedel K. Rasmussen T.B. Bagge N. Kumar N. Schembri M.A. Song Z. Kristoffersen P. Manefield M. Costerton J.W. Molin S. Eberl L. Steinberg P. Kjelleberg S. Hoiby N. Givskov M. EMBO J. 2003; 22: 3803-3815Crossref PubMed Scopus (1133) Google Scholar, 25Hentzer M. Riedel K. Rasmussen T.B. Heydorn A. Andersen J.B. Parsek M.R. Rice S.A. Eberl L. Molin S. Hoiby N. Kjelleberg S. Givskov M. Microbiology. 2002; 148: 87-102Crossref PubMed Scopus (823) Google Scholar, 26Persson T. Givskov M. Nielsen J. Curr. Med. Chem. 2005; 12: 3103-3115Crossref PubMed Scopus (81) Google Scholar). QSI compounds have been obtained from natural sources or by synthesis of AHL analogues or by screening random libraries (reviewed in Ref. 16Rasmussen T.B. Givskov M. Int. J. Med. Microbiol. 2006; 296: 149-161Crossref PubMed Scopus (617) Google Scholar), but so far none of these are suitable for medical application. Therefore, we sought to aid the rational development and/or optimization of QSIs by generating molecular information on the uppermost protein in the hierarchy of quorum sensing regulators in P. aeruginosa, namely LasR (2Winans S.C. Bassler B.L. J. Bacteriol. 2002; 184: 873-883Crossref PubMed Scopus (126) Google Scholar, 17Hentzer M. Wu H. Andersen J.B. Riedel K. Rasmussen T.B. Bagge N. Kumar N. Schembri M.A. Song Z. Kristoffersen P. Manefield M. Costerton J.W. Molin S. Eberl L. Steinberg P. Kjelleberg S. Hoiby N. Givskov M. EMBO J. 2003; 22: 3803-3815Crossref PubMed Scopus (1133) Google Scholar, 19Smith R.S. Iglewski B.H. Curr. Opin. Microbiol. 2003; 6: 56-60Crossref PubMed Scopus (513) Google Scholar, 27Pesci E.C. Pearson J.P. Seed P.C. Iglewski B.H. J. Bacteriol. 1997; 179: 3127-3132Crossref PubMed Scopus (679) Google Scholar, 28Latifi A. Foglino M. Tanaka K. Williams P. Lazdunski A. Mol. Microbiol. 1996; 21: 1137-1146Crossref PubMed Scopus (552) Google Scholar, 29Lequette Y. Lee J.H. Ledgham F. Lazdunski A. Greenberg E.P. J. Bacteriol. 2006; 188: 3365-3370Crossref PubMed Scopus (163) Google Scholar, 30Fuqua C. J. Bacteriol. 2006; 188: 3169-3171Crossref PubMed Scopus (103) Google Scholar). Here we present the structure of LasR in complex with its autoinducer and interpret this structure in the context of the activation and inhibition of quorum sensing. Sample Preparation—Using a P. aeruginosa cosmid from the Pseudomonas Genetic Stock Center (Greenville, NC), full-length LasR (GenBank™ D30813) was PCR-cloned into a pETM-11 vector (EMBL Heidelberg). When expressed in Escherichia coli, either in the presence or absence of 3-oxo-C12-HSL, full-length LasR (239 residues) was largely insoluble and was resistant to refolding. Subsequently, numerous shorter LasR constructs were cloned and tested for expression. A construct spanning Met-1 to Lys-173, the predicted ligand-binding domain (LBD), was highly soluble in the presence of 3-oxo-C12-HSL. The TraR LBD (Met-1 to Thr-165) was cloned from an existing in-house plasmid (31Vannini A. Volpari C. Gargioli C. Muraglia E. Cortese R. De Francesco R. Neddermann P. Di Marco S. EMBO J. 2002; 21: 4393-4401Crossref PubMed Scopus (288) Google Scholar). In 1 liter of rich LB medium with induction by 0.4 mm isopropyl 1-thio-β-d-galactopyranoside at 23 °C for 18 h, LasR-LBD (Met-1 to Lys-173) expression yielded ∼100 mg of His6-tagged protein. Protein solubility was highly dependent on the presence in the growth medium of 3-oxo-C12-HSL, synthesized in-house from S-(-)-homoserine lactone hydrobromide and the acyl Meldrum’s acid intermediate of Oikawa, as described (32Zhang L. Murphy P.J. Kerr A. Tate M.E. Nature. 1993; 362: 446-448Crossref PubMed Scopus (362) Google Scholar). Native protein was produced in E. coli BL21 cells, and selenomethionine derivative protein was produced in E. coli strain B834. The B834 cells were grown in M9 medium supplemented with all natural amino acids except methionine, plus 50 mg/liter selenomethionine. Proteins were first purified by Ni2+ affinity chromatography, as described previously (33Bottomley M.J. Collard M.W. Huggenvik J.I. Liu Z. Gibson T.J. Sattler M. Nat. Struct. Biol. 2001; 8: 626-633Crossref PubMed Scopus (176) Google Scholar). The His6 tag was removed by incubation at 23 °C with tobacco etch virus protease. The sample was ultimately purified on a Superdex-75 gel filtration column equilibrated in 40 mm Tris/HCl, pH 7.5, 200 mm sodium chloride, 3 mm dithiothreitol, and 50 μm 3-oxo-C12-HSL. The molecular mass of the purified LasR-LBD protein (19,432 Da per monomer) and the presence of 3-oxo-C12-HSL (C16H27NO4, 298 Da) in a buffer-exchanged sample lacking excess AHL were confirmed by mass spectrometry. Impact of AHLs and QSIs on Protein Solubility—LasR-LBD and TraR-LBD proteins were produced in E. coli from the tightly regulated pETM-11 vector, in the presence or absence of 10 μm AHLs or QSIs. Expression was induced at 21 °C at an optical density (A600 nm) of 0.4 unit using 0.2 mm isopropyl 1-thio-β-d-galactopyranoside. To account for any variable effects of the AHLs or QSIs on overall cell growth and/or protein synthesis, cell cultures were harvested not after a predetermined time but rather only when the A600 nm had reached 1.4 units, typically 4–6 h. Cells were disrupted by microfluidization in lysis buffer: 40 mm Tris-HCl, pH 8.0, 200 mm NaCl, 5 mm imidazole, 5 mm 2-mercaptoethanol, 0.5% (w/v) glycerol, 0.01% (w/v) Nonidet P-40 detergent, and a protease inhibitor mixture. The lysate was centrifuged at 12,000 rpm for 30 min. The soluble supernatants were purified by Ni2+ affinity chromatography. The insoluble pellets were resuspended in lysis buffer plus 2% SDS detergent. Normalized sample volumes were examined by SDS-PAGE. Crystallization and X-ray Data Collection of LasR-LBD in Complex with 3-Oxo-C12-HSL—Single LasR-LBD crystals were obtained from 10 mg/ml stocks of native or selenomethionine derivative protein using the hanging-drop vapor diffusion method. Crystals grew after 1–2 days equilibration at 18 °C against a reservoir of 20% w/v polyethylene glycol 4000, 80 mm calcium acetate, 40 mm Hepes, pH 7.3, 5 mm dithiothreitol, and 50 μm 3-oxo-C12-HSL. X-ray diffraction data were collected from flash-frozen crystals in a 100 K nitrogen cryostream. Three cycles of crystal annealing by the flash-annealing method (34Heras B. Martin J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 1173-1180Crossref PubMed Scopus (185) Google Scholar) improved the diffraction quality. Structure Determination and Refinement—We were unable to solve the x-ray data from native LasR-LBD crystals by molecular replacement using the known TraR structure as a search model (it has only 16% sequence identity). Thus, a single anomalous dispersion experiment was performed with the x-ray wavelength tuned to the selenium peak (0.980 Å) determined from an x-ray fluorescence scan (beamline BW7A, DESY, EMBL Hamburg). Diffraction images were indexed and integrated in the P21 space group using MOSFLM. The data were consistent with a unit cell containing two LasR-LBD dimers and 42% solvent. Each native LasR-LBD monomer contains four methionine residues (excluding the N-terminal methionine), and consequently 16 selenomethionine sites were sought using CNX (Accelrys Software Inc.). The Patterson maps obtained revealed peaks for all 16 selenomethionine sites, which were refined and used for phase calculation and density modification, yielding a clearly interpretable electron density map. Further density modification, solvent flattening, and phase extension to 1.8 Å provided a map that was auto-traced to 80% completeness by ARP/wARP (35Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (461) Google Scholar). Model building was performed using O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refinement using REFMAC (37Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar), defining one TLS group per protein chain. Structure Analysis and Modeling of Interactions with QSIs—The structural quality was analyzed using the Molprobity server (38Davis I.W. Murray L.W. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2004; 32: W615-W619Crossref PubMed Scopus (820) Google Scholar). Structural alignments of LasR, TraR, and SdiA were performed using DALI (39Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar). Protein-ligand images were prepared with PyMOL. 3W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA. For modeling the LasR·QSI interactions, hydrogen atoms were added to LasR to simulate a pH of 7.4. The positions of these hydrogens and of the protein side chains were optimized by energy minimization (5000 steepest descent steps), using the Merck molecular force field (MMFF-S) as implemented in Macromodel version 9.0 (Schrödinger LLC software), keeping first the protein backbone and then the AHL structure rigid. Subsequently, the AHL geometry was allowed to relax while keeping the protein rigid. Each QSI, except TP-1 and TP-5, was manually docked into the binding site by superimposing the key atomic fragments onto the AHL lactone ring and amide group. Because the binding pocket is buried, it was not possible to perform automated surface docking. The LasR protein structure was restrained as a rigid body, and the position of the QSI was subjected to conjugate gradient energy minimization until convergence was reached. In the case of TP-1 and TP-5, 500 conformers, generated by Monte Carlo type conformational analysis, were docked into LasR and submitted to energy minimization as above. The structures shown represent the LasR·QSI complexes with the lowest energy values. Purification of Soluble LasR—Despite the medical significance of LasR, there are very few reports describing its biochemical or biophysical characterization, presumably due to the noted difficulty of purifying soluble LasR (41Smith K.M. Bu Y. Suga H. Chem. Biol. 2003; 10: 81-89Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and the need to synthesize its AHL. Therefore, we cloned differently sized LasR proteins and screened for useful expression constructs. We found one C-terminally truncated construct that enabled the production of large amounts of soluble LasR protein. Indicative of functional integrity, this LasR protein was soluble and stable only if produced in the presence of its cognate AHL ligand, 3-oxo-C12-HSL. The dependence on AHL for solubility is similar to that reported for full-length TraR (42Zhu J. Winans S.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1507-1512Crossref PubMed Scopus (279) Google Scholar), and was also observed herein for a ligand-binding domain construct of TraR (Fig. 1). Decreasing amounts of the LasR protein were soluble if produced in the presence of one of the following AHLs: 3-oxo-C8-HSL, 3-oxo-C6-HSL, and C4-HSL, the autoinducers of TraR, LuxR, and RhlR, respectively (Fig. 1). Despite the apparent similarities of these quorum sensing systems, none of the non-cognate AHLs formed stable complexes with LasR, i.e. they had all precipitated 1–2 h post-purification. This lack of promiscuity by LasR agrees with previous findings that the autoinducers of LasR and LuxR show virtually no cross-functionality (43Pearson J.P. Gray K.M. Passador L. Tucker K.D. Eberhard A. Iglewski B.H. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 197-201Crossref PubMed Scopus (809) Google Scholar). The soluble LasR construct (hereafter termed LasR-LBD) spanned the predicted LBD and oligomerization domain and lacked the C-terminal 60 residues of the DNA-binding domain. Structure Determination of the LasR·AHL Complex—The soluble LasR-LBD·3-oxo-C12-HSL complex was purified to homogeneity and crystallized. We solved the structure via the single anomalous dispersion method up to 1.8-Å resolution (Table 1). The crystallographic asymmetric unit contains four LasR-LBD·AHL complexes, and electron density was clear for the four AHLs and for all residues of the four near-identical protein monomers (average pairwise Cα r.m.s.d. values 0.43 Å for 162 residues), with the exception of the terminal residues Met-1 to Asp-5 and His-169 to Lys-173. All residues lie within the favored regions of the Ramachandran plot (93.7% in the most favorable region).TABLE 1Data collection and refinement statistics for LasR-LBD complexed with 3-oxo-C12-HSLData collection statisticsSpace groupP21Cell dimensions (/)a = 53.82, b = 85.33, c = 75.51Cell anglesα = γ = 90.0°, β = 95.8°Protein chains per unit cell4Solvent content in unit cell45%Resolution range (/)40.0-1.80Number of unique reflectionsaValues for the outermost resolution shell spanning 1.9-1.8 Å are in parentheses61,495 (8,825)MultiplicityaValues for the outermost resolution shell spanning 1.9-1.8 Å are in parentheses8.1 (7.5)CompletenessaValues for the outermost resolution shell spanning 1.9-1.8 Å are in parentheses97.8 (96.9)Mean I/S.D.aValues for the outermost resolution shell spanning 1.9-1.8 Å are in parentheses26.4 (3.1)RsymaValues for the outermost resolution shell spanning 1.9-1.8 Å are in parentheses9.0 (58.3)Refinement statisticsRfree (5% of total reflections)25.4Rwork20.9No. reflections in Rfree set3,112 (218)Protein residuesbThe purified LasR-LBD was 175 residues, but for each chain some of the following residues were not visible in the electron density maps: the N-terminal Gly-Ala tag, Met-1 to Asp-5, and His-169 to Lys-173660Protein atoms (non-hydrogen)5,140Ligand atoms (non-hydrogen)84Water moleculescOnly includes water molecules visible above 1.1σ in the 2Fo – Fc electron density map542Quality of structurer.m.s.d. bond lengths (/)0.007r.m.s.d. bond angles (degrees)1.10B protein main chaindAverage B-factor for specified set after TLS refinement3.63B protein side chains4.65B ligand2.82B water molecules15.48a Values for the outermost resolution shell spanning 1.9-1.8 Å are in parenthesesb The purified LasR-LBD was 175 residues, but for each chain some of the following residues were not visible in the electron density maps: the N-terminal Gly-Ala tag, Met-1 to Asp-5, and His-169 to Lys-173c Only includes water molecules visible above 1.1σ in the 2Fo – Fc electron density mapd Average B-factor for specified set after TLS refinement Open table in a new tab The biologically relevant complex in the unit cell is a symmetrical dimer of LasR-LBDs, with each monomer containing one deeply buried ligand (Fig. 2). The monomer fold is an α-β-α sandwich with three α-helices packed on both sides of a five-stranded anti-parallel β-sheet. The 3-oxo-C12-HSL lies parallel to the β-sheet and is buried from the solvent in a pocket formed between the β-sheet and helices α3, α4, and α5. On the other side of the β-sheet, helix α6 makes the majority of the intermolecular H-bonds and hydrophobic contacts contributing to the formation of a large dimer interface burying ∼1900 Å2 of surface area. According to dynamic light scattering, analytical gel filtration, and NMR spectroscopy (data not shown), the LasR-LBD is a constitutive dimer in solution, presumably enabling the binding in vivo of full-length LasR to promoters with two DNA target motifs (19Smith R.S. Iglewski B.H. Curr. Opin. Microbiol. 2003; 6: 56-60Crossref PubMed Scopus (513) Google Scholar, 21Kiratisin P. Tucker K.D. Passador L. J. Bacteriol. 2002; 184: 4912-4919Crossref PubMed Scopus (143) Google Scholar, 22Schuster M. Urbanowski M.L. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15833-15839Crossref PubMed Scopus (222) Google Scholar). Analysis of the AHL Binding Site in LasR—The LasR-LBD structure reveals the atomic interactions between LasR and its autoinducer. All the polar groups of 3-oxo-C12-HSL, except the lactone ring oxygen, make H-bonds with the LasR-LBD. A total of six intermolecular H-bonds were observed: five direct and one water-mediated, involving Tyr-56, Trp-60, Arg-61, Asp-73, Thr-75, and Ser-129 (Fig. 3). In particular Tyr-56, Trp-60, Asp-73, and Ser-129 are strongly conserved in LuxR homologues, reflecting the shared activation mechanism by AHLs with identical HSL headgroups (Fig. 1B). In contrast with this conservation, the long acyl chain extends into a cavity lined with hydrophobic residues, some of which are seen only in LasR and not in other LuxR homologues, e.g. Leu-40, Tyr-47, Cys-79, and Thr-80 (Fig. 4C). These non-conserved residues contact the extreme methylene/methyl groups of the acyl chain, which are absent from the shorter AHL ligands of most LuxR homologues. The tight encapsulation of the AHL by LasR means that its function can be abolished even by relatively conservative point mutations in the AHL binding pocket, e.g. Y64H, A70E, D73E, P74L, and W88Y (21Kiratisin P. Tucker K.D. Passador L. J. Bacteriol. 2002; 184: 4912-4919Crossref PubMed Scopus (143) Google Scholar, 44Cabrol S. Olliver A. Pier G.B. Andremont A. Ruimy R. J. Bacteriol. 2003; 185: 7222-7230Crossref PubMed Scopus (82) Google Scholar). These structural features presumably provide high ligand specificity and minimize cross-talk between different bacterial species.FIGURE 4Overall fold comparisons of LasR, TraR, and SdiA. A, ribbon diagrams of superposed LasR (blue) and SdiA (pink) monomers (SdiA coordinates from PDB entry 2AVX). The orientation of LasR is similar to that in Fig. 2. B, ribbon representations of superposed LasR and TraR. The LBD monomers on the left (blue, LasR; green, TraR) have been superimposed, clearly revealing the structural similarity of their folds (Cα r.m.s.d. 2.4 Å for 150 residues). However, the overall structural differences of the dimers are revealed by the monomers on the right (cyan, LasR; red, TraR), due to their different dimer interfaces, they are rotated ∼90° with respect to each other such that their β-sheets are roughly perpendicular. C, a structure-based sequence alignment of LasR, TraR, and SdiA. The major secondary structure elements of LasR and TraR are shown above and below the alignment, respectively; they are also conserved in SdiA. Asterisks indicate the residues making H-bonds to the AHLs; hashes indicate additional residues forming the ligand-binding pockets of LasR (above) and TraR (below). Numbering is for LasR.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structural Comparisons of LasR: the Overall Fold—Despite low amino acid sequence identities (10–16%), the LasR-LBD monomer shows considerable structural similarity to the quorum sens
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