Crystal structure of a methionine aminopeptidase (TM1478) from Thermotoga maritima at 1.9 Å resolution (2004)

The TM1478 gene of Thermotoga maritima encodes a methionine aminopeptidase (MAP; EC 3.4.11.18) with a molecular weight of 27,356 Da (250 residues) and a calculated isoelectric point of 6.6. MAP removes the ubiquitous N-terminal methionine from nascent proteins. MAP sequences are conserved from bacteria to humans, and MAP proteins usually contain a dinuclear metal center essential for activity.1 Human MAP is a molecular target for potent antiangiogenesis agents currently in clinical trials.2 Here, we report the crystal structure of TM1478 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3 The structure of TM1478 [Fig. 1(A)] was determined to 1.90 Å resolution by molecular replacement (MR) using the related MAP from Escherichia coli with a sequence identity of 42% as a search model [Protein Data Bank (PDB) code: 4MAT].4 Data collection, model, and refinement statistics are summarized in Table I. The final model includes 1 protein monomer (residues 6–254) and 195 water molecules. The Matthews coefficient (Vm) for TM1478 is 2.6 Å3/Da, and the estimated solvent content is 52.6%. The Ramachandran plot, produced by PROCHECK 3.4,5 shows that 95% of the residues are in the most favored regions, and 4.5% are in additional allowed regions. One residue, Asn77 (ϕ = 50.6°, ψ = 110.7°), is in a disallowed region. This residue is characteristic of all MAPs and is the second residue contained within a distorted type II′ β-turn (residues 76–79), observed in all structures thus far studied. The ϕ and ψ angles of Asn77 slightly deviate from the canonical values of a type II′ β turn (ϕ = 60.0°, ψ = −120.0°) by 9.4° and 10.7°, respectively. This distortion of geometry from the ϵ region of the Ramachandran plot is most likely induced by a putative sodium ion coordinated to the carboxyl oxygens of Asn77, Cys234, and Glu79. Although this ion has been observed in other MAP structures (PDB code: 2mat, 3mat, 1c21),4 it is not always assigned to be a metal, and the equivalent position has been attributed to be a water molecule (PDB code: 1mat). One cis-proline is also observed in the structure at position 184. Crystal structure of TM1478. (A) Ribbon diagram of Thermotoga maritima TM1478 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the active site (arrow). Helices (H1–H6) and β-strands (β1–β14) are indicated. (B) Diagram showing the secondary structure elements in TM1478 superimposed on its primary sequence with β-hairpins depicted in red and β-sheets labeled in red as A–D. The final model of the TM1478 monomer consists of a single polypeptide chain of 249 amino acids composed of 6 helices (H1–H6) and 14 β-strands (β1–β14). The total α-helix, 310-helix, and β-strand content is 27.3%, 2.4%, and 24.5%, respectively [Fig. 1(A and B)]. TM1478 is composed of two highly similar α/β domains characteristic of the “creatinase/aminopeptidase” fold. Domain 1 comprises residues 6–121 that contains the 3-stranded antiparallel β-sheet A (β2–β4) with 123 topology surrounded by 2 α-helices (H1, H3) and two 310-helices (H2, H4). Domain 2 comprises residues 122–254 and consists of 2 α-helices (H5, H6) followed by 3 separate antiparallel β-sheets (B–D). β-Sheet B comprises 3 β-strands (β5, β9, β12) with 123 topology, β-sheet C comprises 5 β-strands (β6–β8, β13, β14) with 21345 topology, and β-sheet D comprises 2 β-strands (β10, β11) [Fig. 1(A)]. The active site is located in the deep crevice adjacent to β-sheets A and C between the two domains [Fig. 2(A and B)]. Interestingly, no bound metal ions are found in the active site of TM1478 contrary to the expectation for this family of enzymes.4 His171 adopts a dual conformation, one in which the histidine residue resides in the canonical position ready to coordinate a divalent metal ion, whereas the other conformer points away from the active site [Fig. 2(B)]. Although not absolutely necessary for the function of the enzyme,4 it is interesting to speculate that this histidine may act as some sort of switch to stabilize metal binding. (A) Ribbon diagram of a superposition of TM1478 (cyan) and MAP from E. coli (grayish; PDB code: 1MAT). The active site residues are shown in ball and stick. (B) Close-up of the active site using superposition shown in (A). For comparison, the dinuclear cobalt center, its coordinating residues, and a bound methionine, as observed in the MAP structure from E. coli, are shown in ball and stick with carbon atoms colored gray, residue labels shown in brackets, and coordinating bonds to the metal shown in yellow dashes. The corresponding residues in TM1478 are depicted with their carbon atoms colored cyan. A structural similarity search, performed with the coordinates of TM1478 using the DALI server,6 indicates that the closest structural homologue is MAP from E. coli (PDB code: 1MAT).4 The root-mean-square deviation (RMSD) between TM1478 and MAP from E. coli was 1.2 Å over 228 aligned residues with 42% sequence identity [Fig. 2(A)]. According to FFAS,7 TM1478 has only one distant homologue in the T. maritima proteome: TM0042 (aminopeptidase P) with 26% sequence identity. Models for TM1478 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm1478. The crystal structure reported here represents a MAP from T. maritima. TM1478 is the first structure of an apo MAP without any active-site metals. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein family and the thermostability of this enzyme. Protein production and crystallization: TM1478 (TIGR: TM1478; SwissProt: Q9X1I7) 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 TM1478. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of 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 acide (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) 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 Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution Buffer [20 mM Tris, pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 300 mM imidazole). The nickel affinity eluate was buffer exchanged into crystalization buffer (20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays to 10 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method8 with standard JCSG crystallization protocols.3 The crystallization solution contained 19% isopropanol, 19% polyethylene glycol (PEG-4000), 0.095 M sodium citrate pH 5.6 and 5% glycerol. The crystals were indexed in the tetragonal space group P43212 (Table I). Data collection: Native diffraction data were collected at the Advanced Light Source (ALS, Berkeley, CA) on beamline 5.0.3. The data set was collected at 100 K using a Quantum 4 charge-coupled device (CCD) detector. Data were integrated, reduced, and scaled using the HKL2000 package9 and the CCP4 suite.10 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined by MR with AMORE10 using the E. coli (PDB code: 4MAT) MAP coordinates as a search model.4 The MR solution had a correlation coefficient of 0.41 and an initial Rcryst of 47.3%. Structure refinement was performed using the Crystallography & NMR System (CNS)11 and model building with O.12 Refinement statistics are summarized in Table I. The final model includes 1 protein molecule (residues 6–254) and 195 water molecules in the asymmetric unit. No electron density was observed for residue 255 and the N-terminal expression and purification tag. Figures 1A and 2 were produced with PYMOL (DeLano Scientific LLC). Figure 1B was produced with PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum). Validation and deposition: Analysis of the stereochemical quality of the models was accomplished using PROCHECK 3.4 and SFCHECK 4.0.5, 10 Atomic coordinates and experimental structure factors of TM1478 have been deposited with the PDB and are accessible under the code 1o0x. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), 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). Portions of this research were also carried out at The Advanced Light Source (ALS), Berkeley, CA.