Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution

The TM1394 gene of Thermotoga maritima encodes a heat shock protein (Hsp33) with a molecular weight of 32,407 Da (residues 1–290) and a calculated isoelectric point of 5.33. Hsp33 is a molecular chaperone, distinguished from all other known chaperones by its mode of redox-regulated activity.1 Hsp33 is a cytoplasmic protein with four highly reactive cysteines contained within a zinc finger that respond quickly to changes in the redox environment. Oxidation of these cysteines to form two disulfides releases the Zn2+, which drives conformational changes in the protein.2 According to the current model, Hsp33 proteins cycle between a reduced monomeric and an oxidized dimeric state with chaperone activity and the ability to bind protein substrates.3 Here, we report the crystal structure of TM1394, determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 Hsp33 from Thermotoga maritima (TIGR: TM1394, Swiss-Prot: Q9X1B4) was amplified by polymerase chain reaction (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 modified Terrific Broth using the Escherichia coli strain GeneHogs®. 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, 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 nickel-chelating resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. 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 target protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer-exchanged into Buffer Q [20 mM Tris pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl and applied to a RESOURCE Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The target protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate RESOURCE Q fractions were pooled, buffer-exchanged into Crystallization Buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 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 (Amersham Biosciences) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 7.9 and 150 mM NaCl 0.02% (w/v) sodium azide. The protein was crystallized using the nanodroplet vapor diffusion method5 with standard JCSG crystallization protocols.4 The crystallization reagent contained 15% glycerol, 8.5% isopropanol, 17% polyethylene glycol (PEG)-4000, 0.1 M HEPES, pH 7.5. Ten percent (v/v) ethylene glycol (final concentration) was included as a cryoprotectant. The crystals were indexed in the orthorhombic space group P212121 (Table I). Native diffraction data were collected on beamline 9-1 at the Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) using the BLU-ICE6 data collection environment. The dataset was collected at 100K using a Quantum 315 CCD detector. Data were integrated and reduced using Mosflm7 and then scaled with the program SCALA from the CCP4 suite.8 Data statistics are summarized in Table I. The structure was determined by molecular replacement (MR) using the program MOLREP from the CCP4 suite.8 The N-terminal domain (residues 1–177) of Hsp33 from E. coli [Protein Data Bank (PDB) code 1i7f] was used as a search model. Multiple MR searches with different values of similarity and completeness parameters were performed. The best solution gave an Rfree = 48.8 and Rcryst = 46.4 after initial rigid-body and restrained refinement. Model building and refinement were performed using the program O9 and TLS refinement in REFMAC5.8 Refinement statistics are summarized in Table I. The final model includes two protein monomers (residues 1–287, chains A and B), two Zn2+ ions, two Cl− ions, three 1,2-ethanediol molecules, two unknown ligands (UNL), and 273 water molecules in the asymmetric unit. Electron density was also observed for three of the six histidine residues from the expression and purification tag, but not for residues 288–290. Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,10 SFcheck 4.0,8 and WHAT IF 5.0.11 Protein quaternary structure analysis used the Protein Quarternary Structure (PQS) file server (http://pqs.ebi.ac.uk/). Figure 1(a) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/), and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of TM1394 have been deposited in the PDB and are accessible under the code 1vq0. Crystal structure of Hsp33 from Thermotoga maritima. (A) Diagram showing the secondary structure elements in TM1394 (chain A) superimposed on its primary sequence. The α-helices, 310-helix, β-bulges, and γ-turns are indicated. The β-sheet strands are indicated by red letters (A, B, and C). β-Hairpins are depicted as red loops, and cysteine residues coordinating the zinc are marked with blue dots. No electron density was found for residues 288–290 as indicated by purple dots. (B) Stereo ribbon diagram of TM1394 color-coded from N-terminus (blue) to C-terminus (red), showing the domain organization. Helices (H1–H10) and β-strands (β1–β13) are indicated. The structure of TM1394 [Fig. 1(b)] was determined to 2.20 Å resolution by the MR method using the N-terminal domain of Hsp33 from E. coli (PDB code 1i7f) with a sequence identity of 22% as the search model. Data collection, model, and refinement statistics are summarized in Table I. The Matthews' coefficient (Vm)12 is 3.22 Å3/Da and the estimated solvent content is 61.5%. The Ramachandran plot, produced by MolProbity,10 shows that 97.05% and 2.95% of the residues are in favored and allowed regions, respectively. The TM1394 monomer contains 13 β-strands (β1–β13), 10 α-helices (H1–H10), and one 310-helix (H2′) [Fig. 1(a and b)]. The total β-strand, α-helical, and 310-helical content is 29.3%, 34.8%, and 0.7%, respectively. TM1394 folds into two domains. The N-terminal domain (residues 1–229) folds into a four-layer αβαβ domain with two β-sheets (A and B) and seven α-helices (H1–H7). The antiparallel β-sheet A comprises five β-strands (β1, β2, β8–β10) arranged in 51243 topology. β-Sheet A is sandwiched between helices H5–H7 on one side and helices H1–H4 on the other. β-Sheet B contains six β-strands (β3– β7 and β11) arranged in 632145 topology. Strands β3– β7 are antiparallel, whereas β5 and β11 are parallel. The long central helix, H2, is deeply buried between sheets A and B and helices H1 and H4. The C-terminal domain (residues 230–287) comprises three α-helices (H8–H10) that pack against a two-stranded (β12 and β13), antiparallel β-sheet C. The neck of this domain contains a ribbon-type zinc finger with a Zn2+ ion coordinated tetrahedrally by the four sulfhydryl side chains of Cys231, Cys233, Cys263, and Cys266 [Fig. 2(a)], indicating that the protein is present in its reduced state. The N- and C-terminal domains are connected via a short linker (residues 229–231) and interact through a small interface with a buried surface area of 464 Å2. Conserved residues in this interface that are involved in interdomain interactions are Arg29, Arg84, Tyr86, Lys228, Tyr229, and Trp265. (A) TM1394 C-terminal zinc-finger domain in ribbon representation with zinc (pink sphere) and coordinating cysteine residues (Cys231, Cys233, Cys263, and Cys266) depicted in sticks. (B) Superposition of the TM1394 crystallographic dimer (gray) with the biological dimer of oxidized Hsp33 from E. coli (chain A, blue; chain B, orange) that contains the domain swap for residues 178–233. (C) Superposition of zinc sites in TM1394 and Hsp33 from Bacillus subtilis (gray with labels in brackets). Zn2+ ions are shown as spheres, and the cysteines, as well as Tyr270(275) and Arg84(86) residues, are depicted in sticks. A structural similarity search, performed with the coordinates of TM1394 using the DALI13 server, shows highest homology to the recently determined structure of the reduced Hsp33 from Bacillus subtilis (PDB code 1vzy),14 with a root mean square deviation (RMSD) of 2.2 Å over 280 residues and a sequence identity of 37%. Oxidized Hsp33 from E. coli (PDB code 1i7f)15 aligns with an RMSD of 2.1 Å over 168 residues and a sequence identity of 22%. Interestingly, only the region between residues 1 and 168 could be aligned due to a structural rearrangement in E. coli Hsp33 [Fig. 2(b)]. The oxidized Hsp33 from E. coli forms dimers that contain a domain swap in the region that includes residues 178–233, which correspond to helices H5–H7 and strand β10 (residues Gln182–Glu226) in Thermotoga maritima. TM1394 forms a very similar dimer but without any domain swap. Ser147 and Gln149, located in the dimer interface of TM1394 and in the vicinity of the potential cross-over region, are conserved in all members of the family. Our results from analytical size exclusion chromatography and static light scattering indicate a monomer as the main species in solution, which supports the previous suggestion14 that formation of a stable dimer requires the domain swap. The active sites of Thermotoga maritima and Bacillus subtilis Hsp33, both in the reduced state, superimpose with an RMSD of 1.3 Å for the side-chain atoms of the four cysteine residues, Arg84, Tyr270, Trp265, and the zinc [Fig. 2(c)]. The four zinc-binding cysteines are strictly conserved, and Arg84 is conserved or substituted with lysine in all members of the Hsp33 family, suggesting that Hsp33 from Thermotoga maritima uses the same redox regulation mechanism as other Hsp33 members. It is noteworthy that, although TM1394 is a two-domain protein, no examples are found where either domain appears as a separate protein or as part of other multi-domain proteins. TM1394 has over 100 homologs in eubacteria and archaea, but it appears to be absent in eukaryotes. Models for TM1394 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1394. The TM1394 structure represents an Hsp33 protein whose structure has been determined by X-ray crystallography. The structural information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional role of this redox-regulated chaperone. This work was supported by NIH Protein Structure Initiative grant from the National Institute of General Medical Sciences (www.nigms.nih.gov). 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).