Crystal structure of a phosphoribosylaminoimidazole mutase PurE (TM0446) from Thermotoga maritima at 1.77‐Å resolution
Year: 2004
Type: article
Abstract: The TM0446 gene of Thermotoga maritima encodes a phosphoribosylaminoimidazole mutase (PurE, AIRC, EC 4.1.1.21), with a molecular weight of 18,485 Da (residues 1–171) and a calculated isoelectric point of 6.3. PurE, a central enzyme in the purine biosynthesic pathway of prokaryotes, has recently been shown to be a mutase that catalyzes the sixth step of the de novo purine biosynthesis, the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide [N5-CAIR] to CAIR.1, 2 PurE's from higher eukaryotes are homologous to bacterial PurE's, but use aminoimidazole ribonucleotide (AIR) and CO2 as substrates to produce CAIR directly.3 This distinction makes prokaryotic PurEs a possible drug target for the design of antibacterial agents.4 Here, we report the crystal structure of TM0446 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).5 The structure of TM0446 [Fig. 1(A)] was determined to 1.77 Å resolution using the molecular replacement (MR) method, with a search model constructed from PurE from Escherichia coli, with a sequence identity of 49% [Protein Data Bank (PDB) code: 1QCZ].6 Data collection, model, and refinement statistics are summarized in Table I. The final model includes 2 protein molecules (residues 2–170), 2 sulphates, and 153 water molecules. No electron density was observed for residues 1 and 171. The Matthews coefficient (Vm) for TM0446 is 2.18 Å3/Da, and the estimated solvent content is 43.1%. The Ramachandran plot, produced by PROCHECK 3.4,7 shows that 92% of the residues are in the most favored regions, and 8% are in additional allowed regions. Crystal structure of TM0446. (A) Ribbon diagram of Thermotoga maritima TM0446 color coded from N-terminus (blue) to C-terminus (red), showing the domain organization and the location of the active site (arrow). The helices H1–H8 and β-strands (β1–β5) are indicated. (B) Ribbon diagram of the TM0446 octamer with one monomer shown in purple. For comparison, the PurE octamer from E. coli (PDB code: 1D7A) is shown with an aminoimidazole ribonucleotide (AIR) molecule (cpk-mode) bound to the active site. Figures 1A and 1B produced with PYMOL (DeLano Scientific LLC). (C) Diagram showing the secondary structure elements in TM0446 superimposed on its primary sequence. Figure 1C adapted from PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdsum). The final model of the TM0446 monomer consists of a single polypeptide chain of 169 amino acids composed of 5 β-strands (β1–β5), 7 α-helices (H2–H8), and one 310-helix (H1) [Fig. 1(A and C)]. The total β-strand, α-helical, and 310-helical content is 16.0%, 52.1%, and 1.2%, respectively. According to the Structural Classification of Proteins database (SCOP),8 TM0446 is a member of the “Flavodoxin-like” fold. The TM0446 monomer folds into a 3-layer α/β/α-domain with a 5-stranded (β1–β5), parallel β-sheet with 2 1 3 4 5 topology. The β-sheet is surrounded by 6 helices (H1–H6). The two C-terminal α-helices, H7 and H8, protrude away from the protein core and form a handle-like structure [Fig. 1(A)]. TM0446 forms a compact octamer with 422 symmetry and an inner channel with a diameter of 4.1 Å [Fig. 1(B)]. The structure reveals a sulphate ion coordinated by residues His39 and Arg40 in a cleft adjacent to the loop that connects strand β3 and α-helix H3. Cocrystallization experiments of PurE from E. coli (PDB code: 1D7A) have identified this region as the substrate-binding site.6 The nucleotide is bound predominantly to one subunit with conserved residues (104-MPG-106) from a second subunit making up one wall of the active site cleft [Figs. 1(B) and 2(B)]. (A) Ribbon diagram of a superposition of TM0446 (cyan) with its structural homologue PurE from E. coli (PDB code: 1D7A; white). For reference, the sulphate in TM0446 and the aminoimidazole ribonucleotide (AIR) molecule bound to PurE from E. coli are depicted in ball and stick. (B) Superposition of the active sites in TM0446 (cyan) and PurE from E. coli (white). The active site residues, the AIR molecule, and the sulphate are shown in ball and stick, with residue numbering indicated for TM0446. Pro105′ belongs to the next subunit in the octamer. The atoms are indicated as follows: carbon (cyan: TM0446; gray: PurE), nitrogen (blue), oxygen (red), phosphor (purple), and sulfur (orange). Figures produced with PYMOL (DeLano Scientific LLC). A structural similarity search, performed with the coordinates of TM0446 using the DALI server,9 indicates that the closest structural homologue is PurE from E. coli (PDB code: 1QCZ).5 The root-mean-square deviation (RMSD) between TM0446 and PurE is 1.3 Å over 159 aligned residues, with 49% sequence identity. The main differences between TM0446 and PurE from E. coli are located in the handle region at the C-terminus, where helix H7 is two turns shorter and α-helix H8 is absent in the E. coli structure [Fig. 2(A)]. All active site residues essential for catalysis are conserved [Fig. 2(B)]. According to FFAS,10 TM0446 has one paralogue, TM0965, with 28% sequence identity and one distant homologue, TM0358, with 17% sequence identity in the T. maritima proteome. In addition, human phosphoribosylaminoimidazole carboxylase (NP_006443), a 425-residue protein, contains a homologous region (residues 103–258) to TM0446, with 26% sequence identity. Models for TM0446 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm0446. The structure reported here is the first thermophilic N5-CAIR mutase (PurE) whose structure has been determined by X-ray crystallography. It reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of cocrystals of PurE from E.coli with CAIR, reveals strict conservation of the active sites. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into both the determinants for catalysis and substrate specificity of this protein family, and the thermostability of these organisms. TM0446 (PurE; TIGR: TM0446; Swissprot: Q9WYS7) was amplified by polymerase chain reaction (PCR) from T. maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM0446. The PCR product was cloned into plasmid pMH1, which encodes expression and purification tags consisting of the amino acids MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/L yeast extract, 12 g/L tryptone, 1% (v/v) glycerol, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7.6] using the E. coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) previously charged with nickel and equilibrated with Equilibration Buffer [50 mM potassium phosphate pH 7.8, 0.25 mM TCEP, 10% (v/v) glycerol, 300 mM NaCl] containing 20 mM imidazole. The resin was washed with Wash Buffer [50 mM potassium phosphate pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer Q [20 mM Tris pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) previously equilibrated with the same buffer. Protein was eluted using a linear gradient of 50–500 mM NaCl in Buffer Q, and appropriate fractions were pooled. Protein was buffer exchanged into size exclusion chromatography (SEC) buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays to 18 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method11 with standard JCSG crystallization protocols.6 The crystallization solution contained 2.0 M (NH4)2SO4, 0.2 M K/Na-tartrate, and 0.1 M TRIS (tri-sodium citrate dehydrate) at pH 5.6. The crystals were indexed in the tetragonal space group I422 (Table I). Native diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, CA) on beamline 11-1 using the BLU-ICE12 data collection environment (Table I). The data set was collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using Mosflm13 and then scaled with the program SCALA from the CCP4 suite.14 Data statistics are summarized in Table I. The structure was determined by MR using program MOLREP from the CCP4 suite.14 A homology model based on the FFAS8 alignment between TM0446 and the PurE from E. coli (PDB code: 1QCZ),6 with a sequence identity of 49%, was constructed with the modeling program WHATIF15 and used as a search model. Structure refinement was performed using REFMAC5,14 O,16 and XFIT.17 Refinement statistics are summarized in Table I. The final model includes 2 protein molecules (residues 2–170), 2 sulphates, and 153 water molecules in the asymmetric unit. No electron density was observed for residues 1, 171, and the His-tag. Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates 7 validation tools: PROCHECK 3.5.4, SFCHECK 4.0, PROVE 2.5.1, ERRAT, WASP, DDQ 2.0, and WHATCHECK. The Validation Central suite is accessible at http://www.jcsg.org. Atomic coordinates of the final model and experimental structure factors of TM0446 have been deposited with the PDB and are accessible under the code 1o4v. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).
DOI: 10.1002/prot.20023
Authors: Robert Schwarzenbacher, Lukasz Jaroszewski, Frank von Delft, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jaume M. Canaves, Jamison Cambell, Hsiu‐Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc‐André Elsliger, Said Eshagi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Alyssa Robb, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, Ian A. Wilson
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2033795664