Two Crystal Structures of the Isochorismate Pyruvate Lyase from Pseudomonas aeruginosa
2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês
10.1074/jbc.m605470200
ISSN1083-351X
AutoresJelena Zaitseva, Jingping Lu, Kelli L. Olechoski, Audrey L. Lamb,
Tópico(s)Protein Structure and Dynamics
ResumoEnzymatic systems that exploit pericyclic reaction mechanisms are rare. A recent addition to this class is the enzyme PchB, an 11.4-kDa isochorismate pyruvate lyase from Pseudomonas aeruginosa. The apo and pyruvate-bound structures of PchB reveal that the enzyme is a structural homologue of chorismate mutases in the AroQα class despite low sequence identity (20%). The enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites. The apo structure (2.35 Å resolution) has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure (1.95 Å resolution) has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis. Determining the structure of PchB is part of a larger effort to elucidate protein structures involved in siderophore biosynthesis, as these enzymes are crucial for bacterial iron uptake and virulence and have been identified as antimicrobial drug targets. Enzymatic systems that exploit pericyclic reaction mechanisms are rare. A recent addition to this class is the enzyme PchB, an 11.4-kDa isochorismate pyruvate lyase from Pseudomonas aeruginosa. The apo and pyruvate-bound structures of PchB reveal that the enzyme is a structural homologue of chorismate mutases in the AroQα class despite low sequence identity (20%). The enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites. The apo structure (2.35 Å resolution) has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure (1.95 Å resolution) has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis. Determining the structure of PchB is part of a larger effort to elucidate protein structures involved in siderophore biosynthesis, as these enzymes are crucial for bacterial iron uptake and virulence and have been identified as antimicrobial drug targets. Many bacteria produce low molecular mass, high affinity iron chelators called siderophores in response to an iron-limiting environment, such as that found in a host organism. Siderophores are produced in a multistep biosynthetic process, secreted into the environment where they bind available iron (frequently out-competing host organism enzymes for the iron), and are then reinternalized for iron use by the pathogen (1Sriyosachati S. Cox C.D. Infect. Immun. 1986; 52: 885-891Crossref PubMed Google Scholar, 2Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2674Crossref PubMed Scopus (897) Google Scholar, 3Visca P. Leoni L. Wilson M.J. Lamont I.L. Mol. Microbiol. 2002; 45: 1177-1190Crossref PubMed Scopus (205) Google Scholar, 4Raymond K.N. Dertz E.A. Kim S.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3584-3588Crossref PubMed Scopus (628) Google Scholar). Salicylate serves as a building block for siderophores produced by a variety of human pathogens, including pyochelin (Pseudomonas aeruginosa), yersiniabactin (Yersinia pestis and Yersinia enterocolitica), and mycobactin (Mycobacterium tuberculosis) (5Crosa J.H. Walsh C.T. Microbiol. Mol. Biol. Rev. 2002; 66: 223-249Crossref PubMed Scopus (604) Google Scholar). Recently, both salicylate biosynthesis and activation for incorporation into siderophores by these bacteria have been identified as attractive targets for antibiotic development (6Harrison A.J. Ramsay R.J. Baker E.N. Lott J.S. Acta Crystallogr Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 121-123Crossref PubMed Scopus (9) Google Scholar, 7Ferreras J.A. Ryu J.S. Di Lello F. Tan D.S. Quadri L.E. Nat. Chem. Biol. 2005; 1: 29-32Crossref PubMed Scopus (234) Google Scholar, 8Payne R.J. Kerbarh O. Miguel R.N. Abell A.D. Abell C. Org. Biomol. Chem. 2005; 3: 1825-1827Crossref PubMed Scopus (31) Google Scholar, 9Kerbarh O. Ciulli A. Howard N.I. Abell C. J. Bacteriol. 2005; 187: 5061-5066Crossref PubMed Scopus (64) Google Scholar, 10Kerbarh O. Chirgadze D.Y. Blundell T.L. Abell C. J. Mol. Biol. 2006; 357: 524-534Crossref PubMed Scopus (42) Google Scholar).P. aeruginosa synthesizes salicylate from chorismate in a two-step process (11Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (146) Google Scholar, 12Serino L. Reimmann C. Visca P. Beyeler M. Chiesa V.D. Haas D. J. Bacteriol. 1997; 179: 248-257Crossref PubMed Google Scholar, 13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 14Gaille C. Reimmann C. Haas D. J. Biol. Chem. 2003; 278: 16893-16898Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). First, chorismate is converted to isochorismate by the monofunctional enzyme PchA (14Gaille C. Reimmann C. Haas D. J. Biol. Chem. 2003; 278: 16893-16898Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Salicylate is then produced from isochorismate by a second enzyme, PchB (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). PchA is a putative structural homologue of salicylate-producing enzymes of the siderophore biosynthetic pathways from Y. entercolitica and M. tuberculosis, Irp9 and Mbt1, respectively. Irp9, a 50-kDa enzyme with a complex α/β fold, is a structural homologue of the chorismate-utilizing enzymes anthranilate synthase (TrpE subunit) and 4-amino-4-deoxychorismate synthase (PabB subunit) (10Kerbarh O. Chirgadze D.Y. Blundell T.L. Abell C. J. Mol. Biol. 2006; 357: 524-534Crossref PubMed Scopus (42) Google Scholar). MbtI is proposed to be a functional and structural homologue of Irp9 for which preliminary crystallographic information has been reported (6Harrison A.J. Ramsay R.J. Baker E.N. Lott J.S. Acta Crystallogr Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 121-123Crossref PubMed Scopus (9) Google Scholar, 15Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar). Both Irp9 and MbtI are bifunctional salicylate synthases that catalyze the production of salicylate from chorismate using a two-step process with an isochorismate intermediate (9Kerbarh O. Ciulli A. Howard N.I. Abell C. J. Bacteriol. 2005; 187: 5061-5066Crossref PubMed Scopus (64) Google Scholar). However, neither Irp9 nor MbtI shares any sequence similarity with PchB.The physiological role of PchB is as an isochorismate pyruvate lyase (IPL) 2The abbreviations used are: IPL, isochorismate pyruvate lyase; CM, chorismate mutase; EcCM, E. coli CM; TSA, transition state analogue. 2The abbreviations used are: IPL, isochorismate pyruvate lyase; CM, chorismate mutase; EcCM, E. coli CM; TSA, transition state analogue. (Fig. 1a), but PchB shows no sequence similarity to other pyruvate lyases, which proceed through a general base mechanism (16Nakai T. Mizutani H. Miyahara I. Hirotsu K. Takeda S. Jhee K.H. Yoshimura T. Esaki N. J. Biochem. (Tokyo). 2000; 128: 29-38Crossref PubMed Scopus (39) Google Scholar, 17Gallagher D.T. Mayhew M. Holden M.J. Howard A. Kim K.J. Vilker V.L. Proteins. 2001; 44: 304-311Crossref PubMed Scopus (31) Google Scholar, 18Spraggon G. Kim C. Nguyen-Huu X. Yee M.C. Yanofsky C. Mills S.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6021-6026Crossref PubMed Scopus (102) Google Scholar). Instead, PchB shares 20% sequence identity with chorismate mutases of the AroQ family α subclass, which use a Claisen rearrangement mechanism (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 19Okvist M. Dey R. Sasso S. Grahn E. Kast P. Krengel U. J. Mol. Biol. 2006; 357: 1483-1499Crossref PubMed Scopus (42) Google Scholar). Moreover, PchB has an additional catalytic activity as a chorismate mutase (CM) to produce prephenate (Fig. 1b) (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In terms of kcat/Km, the catalytic efficiency of PchB performing the CM reaction is two orders of magnitude less than that of the physiological IPL reaction (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To provide a structural frame-work for understanding the PchB catalytic mechanisms, we solved the structures of apo and pyruvate-bound PchB.EXPERIMENTAL PROCEDURESPchB Cloning and Protein ExpressionPchB-His6—The pchB gene was cloned from P. aeruginosa PAO1 genomic DNA by PCR. The forward primer (5′-TGG CGT ATC ATA TGA AAA CTC CGA A-3′) was designed to include a 5′ NdeI restriction enzyme site, and the reverse primer (5′-TTT AGA AAG CTT TGC GGC ACC CCG-3′) was designed to include a 3′ HindIII site. The PCR product was digested with NdeI/HindIII and inserted into the pET29b vector (Novagen). The resultant plasmid, which encodes the pchB gene with a C-terminal histidine tag, was then transformed into Escherichia coli BL21(DE3) cells. Protein was expressed in cells grown in Luria Bertani medium at 37 °C and 250 rpm. Protein expression was induced by the addition of 0.5 mm isopropyl-β-d-thiogalactopyranoside at an optical density at 600 nm (A600) of ∼0.7, and the cells were harvested after 3 h by centrifugation (5000 × g, 10 min, 4 °C). Cells were resuspended in ∼10 ml 25 mm Tris-HCl, pH 8, 200 mm NaCl (buffer A) per liter of harvested cells and stored at -80 °C for purification.PchB—A second overexpression system was generated to incorporate the naturally occurring stop codon and thus remove the C-terminal histidine tag. The QuikChange kit (Stratagene) and two complementary primers encoding the desired change (5′-CGG GGT GCC GCA TAA TAA AAG CTT GCG GCC-3′, 5′-GGC CGC AAG CTT TTA TTA TGC GGC ACC CCG-3′) were used according to the manufacturer's instructions.Protein PurificationPchB-His6—The cells were lysed by passage through a French Press three times. The lysate was clarified by centrifugation at 12,000 × g for 30 min at 4 °C. The cell extract was applied to a Chelating Sepharose Fast Flow (Amersham Biosciences) column charged with nickel chloride and pre-equilibrated with buffer A. The column was washed with buffer A supplemented with 5 mm imidazole. The protein was eluted with a 10-column volume 0-0.3 m imidazole gradient in buffer A. Fractions containing PchB-His6 eluted at ∼0.25 m imidazole. The collected fractions were concentrated, and the protein was further purified on a HiLoad 16/60 Superdex 75 (Amersham Biosciences) gel filtration column pre-equilibrated with 50 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm dithiothreitol, and 10% (v/v) glycerol. The purified PchB-His6 was concentrated to 28.7 mg/ml (determined by the Bradford assay) and stored at -80 °C for crystallization.PchB—The cells were lysed and the lysate clarified as described above for PchB-His6. The cell extract was applied to a Q-Sepharose Fast Flow (Amersham Biosciences) column pre-equilibrated in 25 mm Tris-HCl, pH 8.0. The protein was eluted with a 10-column volume 0-0.5 m NaCl gradient in the pre-equilibration buffer. Fractions containing PchB eluted at ∼0.1 m NaCl. The collected fractions were concentrated, and the protein was further purified using the Superdex 75 gel filtration column as described above for PchB-His6.The purified PchB was concentrated to 68 mg/ml and stored at -80 °C for crystallization.Isochorismate Pyruvate Lyase and Chorismate Mutase Activity—To ensure that purified PchB (both with and without the C-terminal histidine tag) was enzymatically active, the protein samples were subjected to assays for the known catalytic activities of PchB. Both His-tagged and untagged PchB demonstrated isochorismate pyruvate lyase activity using a coupled assay with PchA (isochorismate synthase) as previously described (data not shown) (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Both forms of PchB also demonstrated chorismate mutase activity using a previously reported protocol (data not shown) (13Gaille C. Kast P. Haas D. J. Biol. Chem. 2002; 277: 21768-21775Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Protein CrystallizationPchB-His6—Crystallization was carried out by the hanging drop method at 25 °C. Drops containing 1.5-3 μl of purified PchB-His6 protein were mixed with equal volumes of a reservoir solution composed of 0.1 m acetate buffer, pH 4.7, and 4.8-5.3 m ammonium nitrate. Large square pyramidal crystals formed after 48 h. These crystals and the structure determined from these crystals will henceforth be called "apo."PchB—Crystallization was carried out by the hanging drop method at 25 °C. Immediately prior to crystallization, the purified PchB protein was diluted to 20 mg/ml in the buffer used for gel filtration chromatography with 17 mm pyruvate and incubated on ice for 30 min. Drops containing 1 μl of this protein/pyruvate solution were mixed with a reservoir solution composed of 100 mm ADA (N-(2-acetamido)iminodiacetic acid), pH 7.0, 25% polyethylene glycol 3350, 0.15 m calcium acetate, and 10% glycerol. Large rod-shaped crystals appeared in 5-7 days and continued to grow to full size over 2-3 weeks. These crystals and the structure determined from these crystals will henceforth be called "pyruvate bound."Determination of PchB-His6 Fragment SizeCrystals of PchB-His6 dissolved in water and subjected to SDS-PAGE gave a single band corresponding to a molecular mass of 11 kDa, indicating that the crystals contained a fragment of the original PchB-His6 construct rather than the full-length protein with the C-terminal histidine tag. Further analysis of dissolved crystals by electrospray ionization mass spectrometry indicated a fragment with a mass of 11294.2 Da (experiments conducted by the University of Kansas Molecular Structures Group Mass Spectrometry Laboratory; www.msg.k-u.edu/%7Emsg/mass.html). Dissolved crystals were analyzed by N-terminal sequencing, which indicated that the N terminus was intact (experiments conducted by the Kansas State University Biotech Core Facility; www.k-state.edu/bchem/biotech/). We conclude that PchB-His6 was proteolyzed in the crystallization drop, removing amino acids 100 and 101 and the C-terminal His tag and allowing amino acids 1-99 to crystallize (Table 1). These results are consistent with the refined structure.TABLE 1Summary of the two PchB samples and structuresProtein sampleAmino acids in constructAmino acids crystallizedApo1–101 + His tag1–99Pyruvate bound1–1011–101PchB in structureOrdered amino acidsIons in active siteApoMonomer A1–41; 50–96NitrateMonomer B1–41; 49–92NitratePyruvate boundMonomer A1–41; 52–94PyruvateMonomer B2–41; 48–94PyruvateMonomer C1–982 PyruvatesMonomer D1–1002 Pyruvates Open table in a new tab Data CollectionApo Protein—For data collection, apo crystals were transferred to the reservoir solution supplemented with 14% (v/v) glycerol as a cryosolvent and flash cooled at -160 °C. Diffraction data were collected at the Protein Structure Laboratory at the University of Kansas (www.psl.ku.edu) using an RaxisIV image plate system mounted on a Rigaku RUH3R rotating anode. The exposure time per frame was 20 min with a crystal to detector distance of 150 mm. The data were processed with DENZO and SCALEPACK (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38344) Google Scholar). The crystals were initially assigned to the space group P422 with unit cell dimensions a = b = 93.2 Å and c = 60.2 Å. Data collection statistics are found in Table 2.TABLE 2Crystallographic statisticsApoPyruvate boundData collectionResolution Range (Å)28.9-2.3557.2-1.95Space GroupP43212P212121Unit Cell (Å)a = b = 93.2, c = 60.2a = 54.5, b = 74.5, c = 88.9ObservationsUnique11,52625,853Total326,890266,264Completeness (%)aValues in parentheses are for the highest resolution shell: 2.43-2.35 Å (native), 2.02-1.95Å (pyruvate bound)99.9 (100.0)95.0 (75.7)RsymbRsym = Σ|Iobs – Iavg|/ΣIobs where the summation is over all reflections0.067 (0.378)0.066 (0.405)% > 3 σ (I)84.3 (60.6)80.7 (49.4)RefinementResolution Range (Å)28.9-2.3557.2-1.95Number of reflections11,24625,825R-factorcR-factor = Σ|Fo – Fc|/ΣFo0.2240.210RfreedFor calculation of Rfree, 10% of the reflections were reserved0.2690.252Dimers/asymmetric unit12Number of atomsProtein, nonhydrogen13972984Nonprotein75218Root mean square deviationsLength (Å)0.0060.005Angles (°)1.081.08Overall B factor (Å2)42.728.6B factor for protein atoms42.528.3B factor for water oxygens58.034.9B factor for ligand44.344.3Wilson B factor (Å2)37.226.8a Values in parentheses are for the highest resolution shell: 2.43-2.35 Å (native), 2.02-1.95Å (pyruvate bound)b Rsym = Σ|Iobs – Iavg|/ΣIobs where the summation is over all reflectionsc R-factor = Σ|Fo – Fc|/ΣFod For calculation of Rfree, 10% of the reflections were reserved Open table in a new tab Pyruvate Bound—To ensure ligand binding, the drops containing pyruvate-bound crystals were supplemented with pyruvate to a final concentration of 100 mm (500 mm pyruvate in 50 mm Tris, pH 7.0, was added directly to the drop) and incubated for 5 days. For data collection, the crystals were transferred briefly to a cryosolvent composed of 30% polyethylene glycol 3350, 0.2 m calcium acetate, and 20% glycerol and then flashed cooled at -160 °C. These crystals were initially assigned to the space group P222 with unit cell dimensions a = 54.5 Å, b = 74.5 Å, c = 88.9 Å. Data collection and processing were completed as described above for the apo crystals.Structure Solution and RefinementApo—Molecular replacement calculations were performed using the program Phaser (21McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallgr. 2005; 61: 458-464Crossref PubMed Scopus (1595) Google Scholar), the data from 25 to 2.5 Å, and all possible space groups of the P422 subgroup. The E. coli chorismate mutase structure (Protein Data Bank accession code 1ECM) (22Lee A.Y. Karplus P.A. Ganem B. Clardy J. J. Am. Chem. Soc. 1995; 117: 3627-3628Crossref Scopus (195) Google Scholar) was used as a search model, yielding a clear solution in the space group P43212 with a log likelihood gain of 132.5 and no unrelated peaks higher than 75% of the difference between the top and the mean value. The map generated with this solution indicated extra density at the N terminus compared with the molecular replacement model and an area lacking density in the loop between the first and second helices. These two differences in electron density relative to the 1ECM molecular replacement model indicate a correct solution, because the map is not merely demonstrating the phase bias from the search model. After model building in the program XtalView (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar), refinement was conducting by simulated annealing and individual temperature factor refinement using CNS (24Brunger A.T. Adams P.D. LClore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). The final model contains two intertwined monomers per asymmetric unit. Monomer A comprises residues 1-41 and 50-96, whereas monomer B comprises residues 1-41 and 49-92. The model also includes 55 water molecules and 5 nitrate ions. A Ramachandran plot generated with PROCHECK (25Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26Crossref Google Scholar) shows that the model exhibits good geometry, with 97.3% of the residues in the most favored regions and 2.7% of the residues in the additionally allowed regions. Refinement statistics are in Table 2.Pyruvate Bound—Molecular replacement calculations were performed by AutoMolRep in the CCP4 program package using data from 50 to 3 Å resolution (26Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallgr. 1994; 50: 760-763Crossref PubMed Scopus (19698) Google Scholar). The apo structure (above) was used as a search model, yielding a clear solution in the space group P212121 with two dimers in the asymmetric unit. This solution had a correlation coefficient of 0.55 and an Rfactor of 0.45 (compared with the second unrelated peak with correlation coefficient of 0.27 and Rfactor of 0.57). The map generated with this solution indicated extra density in two of the monomers, corresponding to the ordering of the loop between the first and second helices. After model building in the program XtalView (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar), refinement was conducted by simulated annealing and individual temperature factor refinement using CNS (24Brunger A.T. Adams P.D. LClore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). The final model contains two dimers, each composed of two intertwined monomers. Monomer A, which is composed of residues 1-41 and 52-94, forms a dimer with Monomer B, composed of residues 2-41 and 48-94. Monomer C (residues 1-98) forms a dimer with monomer D (residues 1-100). Included in the model are 6 pyruvate molecules, 181 water molecules, and a Ca2+ ion (at a crystal contact). A Ramachandran plot generated with PROCHECK (25Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26Crossref Google Scholar) shows that the model exhibits good geometry, with 97.7% of the residues in the most favored regions and 2.2% of the residues in the additionally allowed regions.Structural Analysis and ModelingStructural Alignment—The structural alignments and calculations of root mean square deviations were all done using LSQMAN in the DEJAVU program package (27Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 277: 525-545Crossref PubMed Scopus (303) Google Scholar).Modeling—Salicylate was docked by hand into each of the pyruvate 1 sites of the pyruvate-bound PchB structure using XtalView (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar). The salicylate/pyruvate-bound model (with one salicylate and one pyruvate in each of the active sites of the closed dimer and one salicylate in each of the active sites of the open dimer) was refined using the wrong-ligand crystallographic refinement method (28Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones T.A. Structure. 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), refining the complex against the 1.95 Å pyruvate-bound PchB crystallographic data with a slow cooling simulated annealing protocol starting at 2000 K in the CNS program package (24Brunger A.T. Adams P.D. LClore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar).Figures—Figures were generated using MOLSCRIPT (29Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), PyMOL (30DeLano W.L. The PyMol Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar), and LIGPLOT (31Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4242) Google Scholar).RESULTSOverall Structure—Crystals of apo PchB, grown using ammonium nitrate as a precipitant, belong to the space group P43212. The structure was determined by molecular replacement using E. coli chorismate mutase (EcCM; Protein Data Bank accession code 1ECM) (22Lee A.Y. Karplus P.A. Ganem B. Clardy J. J. Am. Chem. Soc. 1995; 117: 3627-3628Crossref Scopus (195) Google Scholar) as a search model. The asymmetric unit contains two PchB monomers, each composed of three α-helices connected by loops to form an intertwined dimer (Fig. 2a). Several residues are disordered in both monomers, including eight to nine amino acids in the loop connecting the first and second helices and between three and seven amino acids at the C termini (Table 1).FIGURE 2Comparison of the PchB structures and the E. coli chorismate mutase structure. a, PchB apo structure. The monomers of the intertwined apo dimer are displayed in red and dark blue, and the N and C termini for each monomer are labeled. The amino acids displayed in yellow ball-and-stick represent the terminal amino acids of the disordered loop between helix 1 and helix 2. The nitrate ion bound in the active site is shown as ball-and-stick with cyan sticks. b, PchB pyruvate-bound structure. The two distinct dimers of the structure are displayed in orange and royal blue (open monomers) and purple and pink (closed monomers). In the open structure, the amino acids on either side of the disordered loop are displayed in yellow ball-and-stick. The bound pyruvate molecules are shown as ball-and-stick with cyan sticks. c, E. coli chorismate mutase structure (Protein Data Bank accession code 1ECM) (22Lee A.Y. Karplus P.A. Ganem B. Clardy J. J. Am. Chem. Soc. 1995; 117: 3627-3628Crossref Scopus (195) Google Scholar). The monomers are displayed in green and gold. The oxabicyclic acid transition state analogue is displayed in ball-and-stick with cyan sticks.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The crystals of the pyruvate-bound structure were grown using polyethylene glycol 3350 as a precipitant and belong to a different space group, P212121. The asymmetric unit for this crystal system contains two dimers of the same overall structure as the apo protein (Fig. 2b). One dimer (monomers A and B) has two open active sites as in the apo structure with disordered loops between the first and second helices and disordered C termini. In contrast, in the other dimer (monomers C and D), the corresponding loops are ordered and form a closed active site. Also, one additional turn of a C-terminal helix in each monomer is ordered. Fig. 3, a and b, displays the disorder-to-order transition for the loop between helices 1 and 2, and Table 1 includes a summary of the ordered residues in the structures.FIGURE 3Electron density maps for the apo and pyruvate-bound structures. a and b, comparison of the open and closed forms of PchB. a, the apo structure of PchB, with one monomer shown in red and the other in blue sticks. The yellow amino acids (Phe-41 and Pro-49) are the last ordered residues in the loop connecting the first and second α-helices. b, the closed, pyruvate-bound dimer with one monomer in purple and the other in pink sticks. To indicate the position of the analogous amino acids in panel a, Phe-41 and Pro-49 are again highlighted in yellow. Pyruvate 2 is depicted as green sticks with red oxygen atoms. c, stereo representation of the active site of the closed, pyruvate-bound dimer. Colors are as described in panel b. All electron density maps (cyan) are 2Fo - Fc maps contoured at 1σ. This figure was generated with PyMOL (30DeLano W.L. The PyMol Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Superposition of the apo and both pyruvate-bound dimers yields root mean square deviations ranging from 0.60 to 0.72 Å for 168-172 α-carbons, indicating that overall these three structures are very similar, as expected. The only differences are found in the loop regions leading up to the regions of disorder. The overall root mean square difference for the superposition of the PchB structures with the E. coli chorismate mutase is 1.19 Å (for all three comparisons) for 154 to 155 Cα (for the apo and open pyruvate-bound structures, respectively) and 173 Cα for the closed pyruvate-bound structure. As evident in Fig. 4, a and b, the primary area of difference between the closed pyruvate-bound PchB structure and the inhibitor-bound EcCM structure is in the loop that closes over the active site (the same loop that is disordered in the open structures) and at the termini. A structure-based sequence alignment between the pyruvate-bound closed structure and EcCM is shown in Fig. 4c.FIGURE 4Alignment of the closed pyruvate-bound PchB structure with EcCM. a, structural superposition of PchB (dark purple) and EcCM (green) displayed in the same orientation as in Fig. 2. The pyruvate molecules bound to PchB are represented in pink ball-and-stick, whereas the TSA bound to EcCM has yellow ball-and-stick. The oxygen atoms in both pyruvate and TSA are red. The N and C termini are labeled, and the red arrows identify the loop between the first a
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