Crystal structure of a PIN (PilT N‐terminus) domain (AF0591) from <i>Archaeoglobus fulgidus</i> at 1.90 Å resolution

Abstract

The AF0591 gene of Archaeoglobus fulgidus (DSM4304; gi-number: 2650039) encodes a conserved hypothetical protein, with a molecular weight of 15,335 Da (residues 1–135) and a calculated isoelectric point of 8.5. In order to extend the structural coverage of the Protein Family database (PFAM),1 we selected 331 bacterial representatives with homologues in the mouse genome from the American Type Culture Association (ATCC) proteome database (http://www.ebi.ac.uk/proteome/proteomesource.html). One of these representatives, AF0591, contains a PIN (PilT N-terminus) domain(PF01850),2 which has over 300 homologues in all kingdoms of life. The PIN domain in the PilT N-terminus is a compact domain of about 100 amino acids which polymerizes to a pilus fiber.3 In addition, the PIN domain appears to have a role in signaling given its presence in some bacterial plasmid stability proteins and in Dis3 from yeast that is implicated in mitotic control.4 Here, we report the crystal structure of AF0591 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).5 The structure of AF0591 [Fig. 1(A)] was determined to 1.90-Å resolution using the multiwavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 1 protein molecule (residues 10–134) and 115 water molecules. No electron density was observed for residues 1–9 and 135. The Matthews coefficient (Vm)6 for AF0591 is 3.04 Å3/Da, and the estimated solvent content is 59.2%. The Ramachandran plot, produced by Procheck 3.4,7 shows that 98% of the residues are in the most favored regions, and 2% are in additional allowed regions. Crystal structure of AF0591. (A) Ribbon diagram of Archaeoglobus fulgidus AF0591 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. The disulfide-bond between Cys13 and Cys100 is shown in ball and stick (orange). α-Helices H1–H6, and β-strands (β1– β5) in β-sheet A are indicated. (B) Diagram showing the secondary structure elements in AF0591 superimposed on its primary sequence. The disulfide-bond (Cys13–Cys100) is indicated, and the disordered regions are depicted by a dashed line with the corresponding sequence in brackets. Strands are labeled according to their β-sheet (A). The location of γ-turns are also indicated. Mse refers to seleno-methionine. The final model of the AF0591 monomer is composed of 5 β-strands (β1–β5) and 6 α-helices (H1–H6) [Fig. 1(A and B)]. The total β-strand and α-helical content is 16.0% and 51.2%, respectively. AF0591 folds into a α/β/α domain with a central, 5-stranded, parallel β-sheet, comprised of β-strands β1–β5, with 32145 topology and extended, 2-helical hairpin connections between β-strands β1 and β2, and β2 and β3. The β-sheet is twisted and surrounded by 6 α-helices (H1–H6). The structure contains a disulfide bond between Cys13 and Cys100 [Fig. 1(A and B)]. A structural similarity search, performed with the coordinates of AF0591 using the DALI server,8 indicates distant structural similarity for the central β-sheet of AF0591 to the catalytic domain (53EXOc) of 5′-3′ exonuclease from Bacteriophage T5 [Protein Data Bank (PDB) code: 1EXN].9 The root-mean-square deviation (RMSD) is 3.2 Å over 101 aligned residues with 14% sequence identity. Because the structural resemblance is limited only to the central β-sheet and α-helices H2, H5, and H6 [Fig. 2(B)], the structure of AF0591 can be considered a new fold. (A) Ribbon diagram of the AF0591 dimer. (B) Ribbon diagram of a superposition of AF0591 (rainbow) and the catalytic domain (residues 1–174) of 5′-3′ exonuclease from Bacteriophage T5 (gray). α-Helices and β-strands are indicated for AF0591. (C) Close-up view of the active site. Active site residues as observed in T5 5′-3′ exonuclease (labels shown in brackets) and their counterparts in AF0591 are shown in ball and stick. Colors are the same as in (B). The side-chain of E108 in AF0591 was disordered and, therefore, not modeled. For the T5 5′-3′ exonuclease, the active site was reported9 to be formed by a cluster of 7 conserved acidic residues, harboring 2 divalent metal ion binding sites. Structurally similar active sites are also present in Thermus aquaticus Pol I 5′- nuclease domain and in T4 Rnase H.10 Interestingly, when superimposed with the T5 5′-3′ exonuclease, 3 acidic AF0591 residues, D17, D106, and E108, overlap with D26, D153, and D155 of the active site with a RMSD of 0.52 Å (over all atoms, except the Cγ and carboxyl group of E108, for which no electron density was observed). The same AF0591 residues superimpose with D19, D155, and D157 of the T4 Rnase H active site with an RMSD of 0.54 Å. Another acidic AF0591 residue, D88, corresponds approximately to D130 that lies between 2 other acidic residues, E128 and D131, in the T5 5′-3′ exonuclease active site (Fig. 2C). This structural evidence allows us to speculate that the AF0591 catalytic mechanism and reaction may be similar to that of T5 5′-3′ exonuclease or T4 Rnase H. At this stage, it is difficult to specify the biologically relevant oligomerization state of the AF0591. The crystallographic packing in the AF0591 structure shows a dimer is formed through extensive interactions of the ends of the C-terminal β-strand β5 of each subunit, which intertwine into a very long 2-stranded, antiparallel β-sheet (residues 124–132). This dimer interface accounts for a buried surface area of 1174 Å2 per monomer [Fig. 2(A)]. However, this dimerization through the edge β5 strands might be a crystal-packing artifact. In the homologous T5 5′-3′ exonuclease structure, the equivalent β5 strand makes a β-turn in the middle of the corresponding sequence and then continues back as the β6-strand, antiparallel with β5, which suggests that the AF0591 β-strand may have partially unfolded in the crystal to participate in the packing interactions. Thus, further functional studies will be needed to determine the oligomerization state of AF0591. According to fold and function assignment system (FFAS),11 AF0591 has one paralogue in the T. maritima proteome: TM0495, with 23% sequence identity. Models for AF0591 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=2650039. The AF0591 structure reported here represents a new fold as judged by the Structural Classification of Proteins (SCOP) database criteria12 and the first PIN (PilT N-terminus) domain, whose structure has been determined by X-ray crystallography using the MAD method. 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 these organisms. Protein production and crystallization: AF0591 (TIGR: AF0591; SwissProt:O29664) was amplified by polymerase chain reaction (PCR) from A. fulgidus DSM 4304 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of AF0591. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer (50 mM KPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5mM imidazole, Roche ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets) with 0.5 mg/mL lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with 7 column volumes (CV) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 10 mM imidazole) and eluted with 3 CV of elute buffer (25 mM Tris 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into 10 mM Tris, pH 7.8, 150 mM NaCl and concentrated to 8 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method13 with standard JCSG crystallization protocols.5 The crystallization solution contained 30% 2-methyl 1-2,4-pentanediol (MPD) and 0.1 M 2-morpholinoethan sulfonic acid (MES) at pH 6.0. The crystals were indexed in the trigonal space group P3121 (Table I). Data collection: Anomalous diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL; Stanford, CA) on beamline 11-1 at wavelengths corresponding to the inflection point (λ1), peak (λ2), and high-energy remote (λ3) of a selenium MAD experiment, as well as a native 1.9-Å high-resolution data set (λ0), using the BLU-ICE14 data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using MOSFLM15 and then scaled with the program SCALA from the CCP4 suite.16 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined using the CCP4 suite16 and SOLVE/RESOLVE.17 Structure refinement was performed using REFMAC5,16 O18 and Xfit.19 Refinement statistics are summarized in Table I. The final model includes one protein molecule (residues 10–134) and 115 water molecules in the asymmetric unit. No electron density was observed for residues 1–9, 135, and the expression and purification tags. Figures were prepared with PYMOL (DeLano Scientific). Validation and deposition: Analysis of the stereochemical quality of the models was accomplished using Procheck 3.4 and SFcheck 4.0.7, 16 Comparison of the Asp, Asp, Glu motif from AF0591 with other Asp, Asp, Glu and Asp, Asp, Asp motifs in proteins of known structure was performed with SPASM.20 Atomic coordinates and experimental structure factors of AF0591 have been deposited with the PDB and are accessible under the code 1o4w. 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).