Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold (2006)

The hyperthermophilic bacterium Thermotoga maritima has been the target of choice at the Joint Center for Structural Genomics (JCSG) for pipeline development and genome-wide structural annotation.1 In order to increase the fold coverage for this bacterium, the structure determination of the TM1367 gene product, which encodes a hypothetical protein with a molecular weight of 13,791 Da (residues 1–124) and a calculated isoelectric point of 4.86, was undertaken. This protein is a representative of the PFam2 domain of unknown function 369 (DUF369). Currently, no sequence-based functional annotation has been made for this protein, since profile-based sequence similarity methods, such as PSI-BLAST3 and Meta-BASIC,4 found similarities only to other hypothetical proteins, mainly of archaeal and bacterial origin. Here, we report the crystal structure of TM1367, which was determined using the semiautomated, high-throughput pipeline of JCSG,1 and propose that TM1367 is a novel member of the cyclophilin (peptidylprolyl isomerase) fold. The crystal structure of TM1367 [Fig. 1(A)] was determined to 1.90 Å resolution using the multi-wavelength anomalous diffraction (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final refined structure includes three TM1367 monomers (residues 1–124 for chains A and B, and residues 1–123 for chain C), three His-tag residues for chains A and B, five His-tag residues for chain C, three PEG-200 ligands, one Ni2+ ion, and 243 water molecules in the asymmetric unit. The Matthews' coefficient (Vm)5 for TM1367 is 2.31 Å3/Da, and the estimated solvent content is 43.4%. The Ramachandran plot, produced by MolProbity,6 shows that 98.4 and 100.0% of the residues are in favored and allowed regions, respectively. A: Crystal structure of TM1367. Stereo ribbon diagram of TM1367 monomer color-coded from N-terminus (blue) to C-terminus (red). Helices (H1 and H2) and β-strands (β1–β8) are labeled. The structure includes His residues from the purification tag (three in chains A and B and five in chain C). B: Diagram showing the secondary structural elements in TM1367 superimposed on its primary sequence. The helices, β-strands of sheet A, and β-bulges are indicated. The β-hairpins are depicted as red loops. TM1367 is composed of eight β-strands (β1–β8), one α-helix (H1), and one 310 helix (H2) [Fig. 1(A,B)]. The total β-strand, α-helical, and 310-helical content is 41.9, 7.3, and 4.8%, respectively. The TM1367 monomer is comprised of a single structural domain (1–124) and belongs to the cyclophilin (peptidylprolyl isomerase) fold of the SCOP database.7 SCOP defines the core of this fold as an eight-stranded, antiparallel, closed β-barrel [Fig. 1(A)]. In addition to the β-strands, TM1367 and other members of the cyclophilin (peptidylprolyl isomerase) fold contain an α-helix (H1), which is located between β-strands β2 and β3, and a 310 helix (H2) between β-strands β7 and β8. Unlike other enzymes based on a β-barrel architecture, which generally have their active sites inside the β-barrel, members of this fold have both ends of the β-barrel closed due to the presence of the helices, and the active site is located on the exterior of the β-barrel. The interior of the closed β-barrel of TM1367 is extremely hydrophobic, similar to other members of the cyclophilin (peptidylprolyl isomerase) fold. A DALI8 structural similarity search, using the TM1367 structure as a query, found similarities to human cyclophilin A (PDB: 2cpl, Z = 10.8) and the C-terminal domain of a protein of unknown function from Bacillus cereus (PDB: 1x7f, Z = 8.3), whose structure was determined by the Midwest Center for Structural Genomics. The structural alignment of TM1367 with human cyclophilin A superimposes 115 Cα atoms with an RMSD of 2.7 Å (sequence identity 10%) [Fig. 2(A,B)], while the C-terminal domain of the protein of unknown function from B. cereus can be aligned over 100 Cα atoms with an RMSD of 2.9 Å and a sequence identity of 13%. A: Structural superposition of TM1367 (blue) and the human cyclophilin A (PDB: 2cpl, gray). Residues corresponding to the calcineurin binding site of human cyclophilin A are shown in ball-and-stick representation. B: Residues corresponding to PPlase active site of cyclophilin A are shown in ball-and-stick representation. TM1367 residues are shown in brackets. Cyclophilin A (CyPA) belongs to a group of cytosolic enzymes called peptidylprolyl cis-trans isomerases or rotamases (PPIase, E.C. 5.2.1.8). These enzymes mediate the conversion of Xaa-Pro peptide bonds between trans and cis conformations in Pro-containing polypeptides and have been proposed to play an important role in catalyzing the refolding of partly-denatured proteins in vivo. Based on structural and biochemical evidence, it has been suggested that the mechanism of the PPIase activity of CyPA depends on its ability to recognize a specific conformation of the peptide rather than the sequence.9-11 Cyclosporin A (CsA) is a fungally produced “11-residue” cyclic peptide that can bind CyPA with a very high affinity and inhibit its enzymatic function. It is a highly effective immunosuppressant whose main clinical use is in the prevention of organ-transplant rejection and in the treatment of autoimmune disorders mediated by T cells. Apart from the PPIase activity of CyPA, the CyPA-CsA binary complex binds to and inhibits the Ca2+-activated Ser/Thr phosphatase calcineurin. Binding of the CyPA-CsA complex to calcineurin, which plays an essential role in the antigen-induced proliferation of T cells, prevents it from dephosphorylating NFATp (nuclear factor of activated T cells), which suppresses T cell proliferation. It has been argued that the presence of the CyPA-CsA binary complex, rather than the inhibition of the rotamase activity, interferes with T cell proliferation.12, 13 The calcineurin binding site of human CyPA is comprised of Arg69, Asn71, Glu81, Lys82, Glu84, Pro105, Ser147, and Arg1489, 14 [Fig. 2(A)]. This site is not conserved in TM1367, and most of the residues of the calcineurin binding site correspond to insertions in human CyPA, as compared to TM1367 [Fig. 2(A)]. However, this is not totally unexpected since calcineurin inhibition by the CyPA-CsA complex has so far been reported only in mammals.15 CyPA contains a large number of isozymes, including cyclophilins B and C, that vary in their organism/tissue distribution.16 For example, while CyPA is found in the cytosol, CyPB and CyPC are found in the endoplasmic reticulum (ER) and mitochondrial matrices, respectively. The sequences at the ligand binding site of these different forms of cyclophilin-type PPIases are highly conserved.9 In human CyPA, the CsA binding site (also the rotamase active site) is comprised of Arg55, Phe60, Gln63, Thr73, Asn102, Phe113, Trp121, and His126 [Fig. 2(B)]. The corresponding side-chain residues in TM1367 (shown in brackets) are Asn37, Glu41, Tyr44, Pro70, Cys76, and Val95. Thr73 and Trp121 of CyPA are present in insert regions and have no corresponding residues in TM1367 [Fig. 2(B)]. It has been proposed that Arg55 of human CyPA, which is crucial for enzymatic function, hydrogen bonds with the imino nitrogen atom of the Xaa-Pro peptide bond, thus facilitating the cis-trans isomerization by deconjugating and, hence, weakening the peptidyl-prolyl amide bond.9 An Arg55 to Ala mutation in the human CyPA reduces its enzymatic activity 100-fold. TM1367, which exhibits a remarkable structural similarity to CyPA, does not have even a single residue of the PPIase active site conserved and, thus, it represents a novel member of the cyclophilin (peptidylprolyl isomerase) fold. Also, significant structural differences are observed in the loop regions that surround the active site. TM1367 contains a PEG-200 molecule bound in the location corresponding to the active site of human CyPA [Fig. 3(A)]. This PEG-200 molecule is stacked between the indole side-chains of Trp39 and Trp69. Other residues that contribute to this PEG binding site include Glu42, Tyr44, Pro91, Ala92, and Val95 [Fig. 3(A)]. The carboxamide side-chain of Asn37, which corresponds to the catalytic Arg55 of the human CyPA structure, points away from the PEG site and, hence, the biological function of this site is unclear. A: Structure of TM1367 with a PEG-200 molecule in the putative active site. Contact residues of chain B in a shell 4 Å from the PEG molecule are shown in ball-and-stick. B: Structural superposition of TM1367 (blue) and the C-terminal domain of a protein of unknown function from B. cereus (PDB: 1×7f, green), which is a seven- rather than eight-stranded, closed β-barrel. TM1367 also exhibits a strong structural similarity to the C-terminal domain of a protein of unknown function from B. cereus. However, this protein forms a seven-stranded β-barrel [Fig. 3(B)] rather than an eight-stranded β-barrel and lacks the corresponding N-terminal β-strand in the TM1367 structure. Despite lacking one β-strand of the β-barrel, the MCSG hypothetical protein superimposes remarkably well with CyPA and TM1367. Further, Arg272 of this protein aligns structurally with the catalytic Arg55 of human CyPA, although the other residues of the CyPA active site are not conserved. This Arg272 residue, which adopts a side-chain conformation that differs from CyPA Arg55, is conserved in all known homologues of the B. cereus protein. Analysis of the crystallographic packing of TM1367 using the PQS server17 indicates that a monomer is the biologically relevant form [Fig. 1(A)]. This finding is also consistent with results from analytical size exclusion chromatography in combination with static light scattering. The TM1367 protein presently contains about 25 close sequence homologues mainly of bacterial and archaeal origin. Structural models for these sequences are accessible at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1367. The TM1367 crystal structure reveals unexpected similarities to members of the cyclophilin (peptidylprolyl isomerase) fold of the SCOP database and to the C-terminal domain of a hypothetical protein of unknown function from B. cereus. In the absence of statistically significant sequence similarity, pronounced structural similarity such as a high DALI Z-score (Z > 9)18 and the conservation of subtle structural features between protein structures19 have been used to argue for a divergent relationship. We hypothesize such a divergent evolutionary relationship between TM1367, CyPA, and the B. cereus hypothetical protein on the basis of their extensive structural similarity. However, residues of the PPIase active site and the calcineurin binding site are not conserved in TM1367, and the possibility of a convergent relationship cannot be ruled out. At this stage, the available sequence and structure information cannot define the function of TM1367. It will be of interest to investigate whether TM1367 has rotamase activity, binds CsA, or acts as a regulator of calcineurin. Further biochemical and biophysical studies, in combination with additional sequence and structural information for proteins of this fold, should yield valuable insights into the precise functional role and evolutionary origin of TM1367. TM1367 from Thermotoga maritima (TIGR: TM1367, Swiss-Prot: Q9X187) was amplified by PCR from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli strain GeneHogs® (Invitrogen). Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze/thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 32,500g for 30 min. The soluble fraction was applied to nickel-chelating resin (GE Healthcare) pre-equilibrated with Lysis Buffer. The resin was washed with Wash Buffer [50 mM Tris pH 7.9, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP], and the target protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP]. The eluate was diluted 10-fold with Buffer Q [20 mM Tris, pH 7.9, 50 mM NaCl, 5% (v/v) glycerol, 1 mM TCEP] and applied to a RESOURCE Q column (GE Healthcare) pre-equilibrated with the same buffer. The flow-through fraction, which contained the target protein, was buffer exchanged with Crystallization Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 1 mM TCEP) and concentrated for crystallization assays to 15 mg/mL by centrifugal ultrafiltration (Millipore). Molecular weight and oligomeric state of the target protein were determined using a 1.0 × 30 cm Superdex 200 column (GE Healthcare) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl, 0.02% (w/v) sodium azide. The protein was crystallized using the nanodroplet vapor diffusion method20 with standard JCSG crystallization protocols.1 The crystallization reagent contained 50% polyethylene glycol 200 (PEG-200) 200, 0.2 M NaCl, 0.1 M phosphate/citrate pH 4.2. The crystals were indexed in orthorhombic space group P21212 (Table I). Multi-wavelength anomalous diffraction data were collected at SSRL (Stanford, CA) on beamline 9-2 at wavelengths corresponding to the high energy remote (λ1) and the inflection point (λ2) of a selenium MAD using the BLU-ICE21 data collection environment (Table I). All data sets were collected at 100K using a Mar 325 CCD detector. Data were integrated and reduced using Mosflm22 and then scaled with the program SCALA from the CCP4 suite.23 Data statistics are summarized in Table I. The initial structure was determined with the 1.90 Å selenium MAD data (λ1,2) using the CCP4 suite23 and SOLVE/RESOLVE.24 Model building and refinement were performed using O25 and REFMAC5.23 Refinement statistics are summarized in Table I. The final model includes three monomers (residues 1–124 for chains A and B and 1–123 for chain C), three residues of the His-tag for chains A and B, five residues of the His-tag for chain C, three PEG-200 ligands, one Ni2+ ion, and 243 water molecules in the asymmetric unit. Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool,26 MolProbity,6 SFcheck 4.0,27 and WHATIF 5.0.28 Protein quaternary structure analysis was performed using the PQS server.17 Figures were prepared with PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for TM1367 at 1.90 Å resolution have been deposited in the PDB and are accessible under the code 1zx8. This work was supported by NIH Protein Structure Initiative grants P50-GM62411 and U54-GM074898 from the National Institute of General Medical Sciences (http://www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS). The SSRL is 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). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.