Structural insight into the dimerization of human protein disulfide isomerase
Sara Bastos-Aristizabal, Guennadi Kozlov, and Kalle Gehring* sur la structure des prote ines, McGill University, 3649 Promenade Sir Department of Biochemistry, Groupe de recherche axe al, Que bec, H3G 0B1, Canada William Osler, Montre Received 31 December 2013; Accepted 10 February 2014 DOI: 10.1002/pro.2444 Published online 00 Month 2014 proteinscience.org
Abstract: Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). Here, it is shown that human PDI (PDIA1) dimerizes in vivo and proposed that the dimerization of PDI has physiological relevance by autoregulating its activity. The crystal structure of the dimeric form of noncatalytic bb0 domains of human PDIA1 determined to 2.3 A˚ resolution revealed that the formation of dimers occludes the substrate binding site and may function as a mechanism to regulate PDI activity in the ER. Keywords: crystal structure; thioredoxin-like domain; endoplasmic reticulum; protein disulfide isomerase; dimerization
Introduction Proper folding of nascent proteins that enter the secretory pathway often requires formation of disulfide bonds. Disulfide bonds can spontaneously form during folding, but this reaction is kinetically slow. In order to catalyze this process, the endoplasmic reticulum (ER) contains a large and fascinating family of oxidoreductases called protein disulfide isomerases (PDIs).1,2 These enzymes oxidize substrate proteins and then themselves have to be oxidized to complete the catalytic cycle.3 When the catalytic cysteines are reduced, PDIs are able to react with nonnative disulfides to form a mixed disulfide complex
Abbreviations: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; co-IP, co-immunoprecipitation; DTT, dithiothreitol; CHX, cycloheximide. Additional Supporting Information may be found in the online version of this article. Sara Bastos-Aristizabal and Guennadi Kozlov contributed equally to this work Grant sponsor: NSERC; Grant number: RGPIN-238873. *Correspondence to: Kalle Gehring; Department of Biochemis sur la structure des prote ines, try, Groupe de recherche axe al, McGill University, 3649 Promenade Sir William Osler, Montre bec H3G 0B1, Canada. E-mail: [email protected] Que
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and function as isomerases to rearrange protein disulfide bonds. PDI, also known as PDIA1, is the founding member and the most studied protein in the PDI family. PDI contains four thioredoxin-like domains abb0 a0 , where a denotes catalytic domains containing CxxC motifs and b stands for non-catalytic domains, with a 20-residue x-linker between b0 and a0 domain and a C-terminal acidic tail. The reduction potential of PDIA1 (2180 mV) is higher than this of other PDIs, meaning that it is a stronger oxidizer.4 The four thioredoxin-like domains arranged in a U shape with the b and b0 domains forming the base of the U and the catalytic sites of the a and a0 domains facing each other, as shown by crystal structures of human PDIA1 and its yeast homolog Pdi1p.5,6 PDIA1 is the most abundant protein present in the ER and is a major player in the oxidative pathway. The mechanism of PDIA1 catalysis has been widely studied and is thought to function as follows: PDIA1 catalyzes the oxidation and shuffling of disulfides in substrate proteins. In turn, PDIA1 is reoxidized by the ER sulfhydryl oxidase ERO1a, so it can interact with substrates again. Recent structural analysis indicates that ERO1a specifically interacts with the b0 domain of PDIA1 and preferentially oxidizes the a0 domain.7–9
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In vitro dimerization of PDIA1 has been reported repeatedly, making it possible that a similar phenomenon occurs in the ER of eukaryotes to regulate the levels of PDIA1 oxidation. Initial identification of a mixture of two different species of PDIA1 in solution was reported by gel-filtration analysis.10 Two years later, the same group reported the association/dissociation behavior of PDIA1 in different conditions, where both hydrophobic and ionic forces were responsible for dimer formation. They observed that the monomeric species were more active suggesting that dimerization decreased binding of substrates.11 More recently, studies employing analytical ultracentrifugation, small angle X-ray scattering (SAXS), and NMR12–15 provided additional evidence of in vitro PDIA1 dimerization. Freedman et al. reported that dimerization was primarily mediated by interactions between the hydrophobic pockets on the b0 domains.16 They proposed that the tendency of PDIA1 to dimerize could be relevant in vivo and that the high concentrations of PDIA1 in the ER might be balanced by the monomer/dimer ratio, which is influenced by the amount of PDI substrates present at any time. However, the structural mechanism of PDIA1 dimerization remained unanswered. Here, we show that dimers of full-length PDIA1 can be isolated from cultured cells and report the crystal structure of a dimeric form of the bb0 domains. The structure reveals that the formation of dimers blocks the substrate binding site and explains the slow kinetics of dimerization and the inhibition of dimerization by amphipathic molecules and substrate mimics observed in previous studies.
Results Human PDIA1 dimerizes in vivo The ability of PDIA1 to dimerize in vivo was studied by co-immunoprecipitation (co-IP) of myc- and FLAG-tagged PDIA1. First, HeLa cells were transfected with PDIA1-myc [Supporting Information Fig. S1(A,B), lane 2], PDIA1-FLAG [Supporting Information Fig. S1(A,B), lane 3], or both PDIA1-myc and PDIA1-FLAG [Supporting Information Fig. S1(A,B), lane 4]. After expression for 24 hours, the total cell lysates were subjected to analysis. SDS-PAGE Western blot analyses with anti-myc and anti-FLAG monoclonal antibody established that both tagged proteins were expressed in HeLa cells. Once expressions of transfectants were established, the experiments probing dimerization were performed. In particular, the lysates were subjected to co-IP using an antibody against myc [Fig. 1(A,B), lanes 1–3] or an antibody against FLAG [Fig. 1(A,B), lanes 4–6]. The products from the co-IP were split into two fractions and loaded on two different SDS-PAGE gels in the same order, and then detected by Western blot-
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Figure 1. In vivo dimerization of PDIA1. co-IPs with anti-myc (lanes 1–3) and with anti-FLAG (lane 4–6) were analyzed by SDS-PAGE followed by Western blot using anti-myc (A) and anti-FLAG (B). Asterisks indicate bands that belong to the heavy chains of the antibodies used in the immunoprecipitation.
ting. After blotting, one membrane was probed for myc [Fig. 1(A)], and the other probed for FLAG [Fig. 1(B)]. The dimer is expected to appear as a band present in the doubly transfected cells in both gels. Importantly, dimerization was evident when PDIA1-FLAG was co-immunoprecipitated by antimyc [Fig. 1(B), lane 3], and conversely when PDIA1myc was co-immunoprecipitated by anti-FLAG [Fig. 1(A), lane 6] in the doubly transfected samples. This result indicates that PDIA1-myc and PDIA1-FLAG interacted and pulled down each other. As a control, anti-myc and anti-FLAG antibodies were able to immunoprecipitate its respective antigen, PDIA1myc [Fig. 1(A), lane 1] and PDIA1-FLAG [Fig. 1(B), lane 5], respectively. Also, the anti-myc and antiFLAG antibodies were specific as they failed to directly pull down PDIA1-FLAG [Fig. 1(B), lane 2] and PDIA1-myc [Fig. 1(A), lane 4], respectively. To determine whether PDIA1 dimerization occurs inside of the cells or only after lysis, HeLa cells were singly transfected with either PDIA1-myc or PDIA1-FLAG, and the tagged PDIA1 proteins were expressed separately. After expression, the cells were combined and immunoprecipitation experiments were performed. For this, plates transfected with PDIA1-myc were harvested and combined with cells expressing PDIA1-FLAG and then lysed together. The lack of dimerization in total cell lysates of single transfectants that were mixed after separate expressions [Fig. 2(A), lane 3 and Fig. 2(B), lane 1], combined with the positive control showing dimerization when double transfectants were expressed [Fig. 2(A), lane 4 and Fig. 2(B), lane 2], indicates that PDIA1 dimerization occurs inside of the cells and is not a product of a post-lysis interaction.
PDIA1 dimerization is not mediated by disulfides To determine whether PDIA1 autoregulates its activity via dimerization, changes in dimerization levels were evaluated during ER-stress and while protein synthesis was inhibited. We hypothesized higher levels of PDIA1 dimer under conditions of low substrate availability compared with conditions of ER-stress in which unfolded proteins promote
Crystal Structure of the bb0 Dimer of Human PDIA1
Figure 2. PDIA1 dimerization occurs in the cells. co-IPs with anti-myc (lanes 1, 2) and with anti-FLAG (lane 3, 4) were analyzed by SDS-PAGE and Western blot using anti-myc (A) and anti-FLAG (B). Indicated with an asterisk are the bands that belong to the antibodies used in the immunoprecipitation.
dissociation of PDIA1 dimers. HeLa cells expressing both PDIA1-myc and PDIA1-FLAG were treated with 10 mM of dithiothreitol (DTT) and 50 mg/mL of cycloheximide (CHX). When supplied to cells, DTT acts as a strong reducing agent and is commonly used when studying ER-stress and the unfolded protein response (UPR).17 On the other hand, CHX is widely used for its ability to inhibit protein synthesis in eukaryotic cells.18 Unexpectedly, levels of substrate availability did not have an effect on the amount of PDIA1 dimers formed in the cells [Fig. 3(B), lanes 1–4]. However, the unchanged levels of PDIA1 dimerization in the presence of 10 mM DTT demonstrated that the dimerization mechanism does not involve intermolecular disulfides supporting previous data.11
Crystallization of the bb0 domains of human PDIA1 and structure determination In order to obtain structural insight into the mechanism of PDIA1 dimerization, we performed crystallographic studies of the bb0 domains of human PDIA1 and obtained a crystal that diffracted to
Figure 3. PDIA1 dimerization is disulfide independent. co-IPs with anti-myc (lanes 1–4) and with anti-FLAG (lane 5–8) were analyzed by SDS-PAGE followed by Western blot using antimyc (A) and anti-FLAG (B). Cross-reactive bands from antibody proteins are marked by an asterisk.
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˚ (Table I). The construct (residues 135–357) 2.3 A includes the bb0 domains and half of the x-linker and yields high-quality NMR spectra that were used to determine the NMR solution structure of the monomeric form.14 The initial attempts to phase the data by molecular replacement failed to produce solutions, even using structures with 100% sequence identity: the crystal structure of the b0 x fragment of human PDIA1 and the NMR structure of the bb0 domains. This suggested that the structure in our crystal deviated significantly from previous structures. The crystals appeared in a 9-month-old drop and we were not able to reproduce them with selenomethionine-labeled protein for experimental phasing of the diffraction data. An attempt to use the anomalous signal of sulfur was also unsuccessful. Eventually, a weak molecular replacement solution for two b0 domains was obtained using the b0 x crystal structure with loops and helices a1 and a3 removed. We then were able to locate the b domains of PDIA1 using b domain of ERp72 as a search model. Numerous rounds of manual model building and refinement were then used to improve the map and to arrive to the final structure (Supporting Information Fig. S2). No electron density was observed for P135, E348–S357 (the x-linker), the N-terminal GPLGS cloning linker and, additionally, residues E323–L334 for chain A and E322–A336 for chain B that correspond to the strand b5 and b5–a4 loop in the b0 x crystal structure.
Structure of the bb0 dimer of human PDIA1 The structure displays two bb0 molecules in the asymmetric unit (Fig. 4). In each molecule, the b
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Table I. Data Collection and Refinement Statistics bb0 fragment
Data collection Space group Cell dimensions ˚) a, b, c (A a, b, g ( ) ˚) Resolution (A Rpim CC (1/2) I/rI Completeness (%) Redundancy Refinement ˚) Resolution (A No. reflections Rwork/Rfree No. atoms Protein Water B-factors Protein Water RMS deviations ˚) Bond lengths (A Bond angles ( ) a
P1 40.52, 45.72, 55.15 81.85, 92.15, 80.02 50–2.30 (2.38–2.30)a 0.035 (0.194) 0.995 (0.856) 23.5 (5.4) 95.5 (92.7) 7.5 (7.0) 50–2.30 15,880 0.244/0.297 3103 3036 67 42.4 40.6 0.007 1.29
Highest resolution shell is shown in parentheses.
domain shows a characteristic thioredoxin-like fold with a five-stranded b-sheet in the b1–b3–b2–b4–b5 arrangement that is flanked by two a-helices on both sides. Unexpectedly, the b0 domain demonstrates surprising deviation from the canonical fold, as the strand b5 is missing in the model. Instead, the b4 strand forms an antiparallel b-sheet with the same strand of another bb0 molecule. The residues corresponding to the b5 strand and the following
Figure 4. Crystal structure of PDIA1 bb0 dimer. A ribbon diagram showing PDIA1 dimer in two different orientations. The dimer is colored by chain in which one is represented by yellow and the other one in magenta and the domains are labeled.
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b5–a4 loop are absent from the electron density map due to disorder. Structural overlay with the b0 x structure shows that the distance between the a1 and a3 helices is bigger in the dimeric form of the b0 domain (Fig. 5) resulting in a more open conformation with larger hydrophobic cavity. This difference is unlikely to be due to the I289A mutation in the crystallized b0 x domain, because the area of the mutation shows nearly identical local structure. As a consequence of the open conformation, the aromatic ring of F304 from second protomer inserts into the ligand-binding ˚ site of the first protomer by approximately 4 A deeper than W364 of the x-linker in the capped conformation of the monomeric b0 domain. Taken together, the two structures may provide an example of breathing-like motions in the ligand-binding site of the b0 domain that could be important for PDIA1’s ability to adjust to a wide variety of protein substrates with solvent-exposed hydrophobic stretches. We also compared the structure with the NMR structure of monomeric bb0 structure and crystal structures of human and yeast PDI, in order to evaluate whether dimerization results in different relative orientation of the b and b0 domains (Fig. 6). Structural overlay of the dimeric form to the bb0 fragments from monomeric PDI structures shows
Figure 5. Comparison of the b0 substrate-binding sites in dimeric and monomeric structures. (A) F304 and F305 of the b0 domain occupy the substrate-binding site of another b0 domain in the dimeric structure. (B) L343 and W347 of the X-linker interact with monomeric b0 domain (PDB code 2BJX).
Crystal Structure of the bb0 Dimer of Human PDIA1
side chains of F240, H256, L258, I289, I291, I301, F304, F305, and L320 of another protomer [Fig. 7(A)]. Next to F304, the side chain of F305 sits between the side chains of F304 and L320 while also reaching I318. A half-turn earlier, the side chain of I301 interacts with the aromatic ring of F304. In the middle of the helix a3, the side chain of R300 is involved in the network of intermolecular hydrogen bonds with carbonyl of D297 and side chains of E242 and N298, while maintaining a salt bridge with E303 of its own protomer. Despite being in the close proximity to G306, F249 does not participate in the dimer formation, as its aromatic ring is not present in the electron density map due to multiple
Figure 6. Dimerization does not affect relative orientation of the b and b0 domain. Orientation of the bb0 domains in the dimeric form (in yellow) is very similar to that in reduced monomeric PDIA1 (PDB code 4EKZ; in green) (A) and differs in oxidized PDIA1 (PDB code 4EL1; in blue) (B).
˚ to reduced human PDIA1 (PDB code RMSD of 1.5 A ˚ to oxidized human 4EKZ, 180 Ca atoms), 2.4 A ˚ to PDIA1 (PDB code 4EL1, 179 Ca atoms), 2.7 A NMR structure of identical construct of human ˚ PDIA1 (PDB code 2K18, 185 Ca atoms), and 3.3 A to yeast Pdi1p (PDB code 3BOA, 177 Ca atoms). The lowest RMSD value corresponds to the most similar inter-domain orientation, while the higher RMSDs indicate variability in relative orientation of the domains. High structural similarity of the dimeric form to the reduced monomeric form of PDIA1 demonstrates that the dimerization is not accompanied by changes in the interdomain orientation. It should be noted that the highest RMSD to yeast Pdi1p also reflects low sequence identity between the human and yeast proteins, leading to noticeable differences in domain structures.
Structural determinants of dimerization There are three main factors causing the b0 domain to dimerize. One of the main contributing factors is the mostly hydrophobic interactions between the substrate-binding a1–a3 surfaces. The second half of helix a3 is especially important for these interactions, as it contains several residues engaging the substrate-binding hydrophobic cavity. In particularly, the side chain of F304 of one protomer inserts into the middle of hydrophobic cavity formed by the
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Figure 7. Structural determinants of PDIA1 bb0 dimerization. Dimer forms via interactions of one molecule (yellow) with another (magenta) through their hydrophobic pockets. Dimerization is mediated by hydrophobic interactions (A), polar interactions (B) and swapping of the C-terminal a-helices (C).
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conformations and was omitted from the model. This hydrophobic nature of the dimeric interaction is supported by previous studies.11,16 Another significant contribution to dimerization comes from an intermolecular antiparallel b-sheet between strands b4 of each protomer. In the center of this b-sheet, carbonyl and amide of T319 of one protomer make hydrogen bonds with the same residue of the other protomer [Fig. 7(B)]. The b5 strand, which normally forms b-sheet with the b4 strand, is disordered in the dimeric structure. Besides backbone hydrogen bonds, the newly formed b-sheet is stabilized by isoleucine zipper on the buried side through side chains of I318 and by intermolecular hydrogen bonds between side chains of E321 and T319 on the solvent-exposed side. Finally, it appears that the swapping of the Cterminal helices a4 takes place between the protomers [Fig. 7(C)]. While the connection between the strand b4 and helix could not be traced in the model as this region is disordered in the crystal, the length of the missing region (12 residues in chain A and 15 residues in chain B) in comparison with the distance ˚ between carbonyl of between the visible ends (15 A residue 322 of chain A and the backbone nitrogen of ˚ between residue 335 of chain B and more than 33 A the same carbonyl and the backbone nitrogen of residue 337 of the same chain A) makes it mechanistically unlikely for the C-terminal helix to bind the same PDI molecule. The connecting regions would also have to run across each other if no swapping takes place making it even less probable. The swapping of C-terminal helices is indirectly supported by earlier NMR studies. NMR spectra of PDIA1 fragments containing the b0 domain show peaks corresponding to both monomeric and dimeric forms.14 Swapping would significantly slow down the monomer–dimer interconversion and is consistent with slow-exchange observed during NMR experiments. Interestingly, the thermal B-factors for these C-terminal helices are higher than for the rest of the domain indicating their relatively loose attachment to the rest of the structure. Furthermore, the residues involved in dimerization including F304, F305, L320, I318, I289, I301, R300, H256, L258, and E303 are highly conserved throughout many species (Supporting Information Fig. S3). Interestingly, some of these amino acids belong to the almost invariant RIXEFFG motif located in the dimer interface, where X represents a hydrophobic residue. We should note that it is difficult to correlate this conservation with an importance of PDIA1 dimerization because a majority of these residues make up the substrate-binding site. We also used the structure to evaluate the dimeric arrangement of the full-length protein. We made structural overlays of the dimeric bb0 fragment of PDIA1 with the ab fragments (PDB entry
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4EKZ) and a0 b0 fragments (PDB entry 3UEM) of human PDIA1. The resulting overlays show that the a domains of PDIA1 dimer would be positioned on the same side of the dimer with their catalytic sites roughly facing each other (Supporting Information Fig. S4). The a0 domains would be located on the other side of the dimer with their spatial position less defined due to longer linkers connecting b0 and a0 domains. We expect the catalytic and chaperone function of PDIA1 dimer to be severely impaired due to ligand-binding site being unavailable to bind substrates.
Discussion Previous studies have already shown in vivo dimerization for the PDI-family member PDIA7 and yeast Pdi1p using co-IP experiments.19,20 Here, we show that human PDIA1 also forms disulfide-independent dimers in vivo and propose that such dimerization may act as a regulatory control mechanism of protein folding in the ER or generate a reservoir of inactive protein that allows the ER to respond efficiently to a sudden increase in substrate availability. One of the Pdi1p crystal structures showed a different dimeric arrangement where the intermolecular interactions are mediated by extensive contacts as the two U-shaped proteins come together.20 Given the low sequence identity between human PDIA1 and yeast Pdi1p, it is unclear whether Pdi1p would be able to dimerize in a similar mode to the human PDIA1 b0 domain. Besides other members of the human PDI family, some PDI proteins from plants were previously shown to dimerize in vitro.19,21–23 In particular, studies on plant PDI have shown a tendency to dimerize at higher, but still physiologically relevant concentrations, which may reflect a concentration-depended mechanism similar to the one observed for PDIA1. Also, van Lith et al. showed that PDIA7 homodimers are not disulfide-linked.19 Interestingly, amino acids involved in PDIA1 dimerization are conserved between these two proteins, while the structure of PDIA7 b0 domain contains a PDIA1-like substrate-binding hydrophobic pocket (S. Bastos-Aristizabal, unpublished data), suggesting that their dimerization mechanism may be similar. Dimerization of PDIA1 inhibits substrate binding and therefore may function as a mechanism to regulate PDIA1 activity in the ER. Our structure shows that the dimerization involves substratebinding site explaining the molecular basis of this inhibition. Also, we showed that PDIA1 forms dimers in vivo using immunoprecipitation experiments, suggesting that the dimerization may have a physiological relevance by auto-regulating its activity. It is currently unclear whether the dimer observed in our crystal is responsible for in vivo dimerization of PDIA1; future studies are needed to confirm the mechanism in cells. If true, this
Crystal Structure of the bb0 Dimer of Human PDIA1
mechanism would allow the ER to maintain a balance between oxidation and reduction necessary for native disulfide bond formation. ER redox balance is important since the defective folding or localization of mutated proteins is responsible for a number of genetic diseases, known as protein trafficking diseases.
Materials and Methods Protein expression, preparation, and purification The bb0 fragment from human PDIA1 (residues 135–357) was cloned into pGEX-6P-1 vector (Amersham-Pharmacia) and expressed in Escherichia coli BL21 (DE3) in rich (LB) medium as N-terminal GST fusion. The GST fusion protein was purified by affinity chromatography on glutathione-Sepharose resin and tag was removed by cleavage with PreScission Protease, leaving Gly–Pro–Leu–Gly–Ser N-terminal extension. The cleaved protein was additionally purified using size-exclusion chromatography using HPLC buffer (10 mM HEPES, 50 mM NaCl, 3 mM DTT, pH 7.0).
Crystallization Crystallization conditions were identified utilizing hanging drop vapor diffusion with the PACT crystallization suite (QIAGEN). The best crystals were obtained by equilibrating a 0.6 mL drop of protein (10 mg/mL) in buffer (10 mM HEPES, 50 mM NaCl, 3 mM DTT, pH 7.0), mixed with 0.6 mL of reservoir solution containing 25% (w/v) polyethylene glycol 1500, and 25 mM sodium malonate, 37.5 mM imidazole, 37.5 mM boric acid, pH 4.0 and suspending over 0.6 mL of reservoir solution at 22 C. No additional cryoprotectant was necessary. For data collection, the crystals were picked up in a nylon loop and flash cooled in a N2 cold stream. The crystals contain two molecules in the asymmetric unit corresponding to ˚ 3 Da21 and a solvent content of 36.8%. Vm 5 1.95 A
model of the PDIA1 b domain. The initial model obtained from Phaser was extended with the program Coot26 and improved by multiple cycles of refinement using the program REFMAC 5.227 and model refitting. Local NCS restraints for residues 136–347 were used throughout the refinement. The refinement statistics are given in Table I. The final model has good stereochemistry (Table I) according to the program MolProbity28 with 95.4% of residues in favored regions and 100% of residues in allowed regions. The coordinates and structure factors have been deposited in the RCSB Protein Data Bank (accession number 4JU5).
Cell Lines Human cervical carcinoma HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 4 mM L-glutamine, 1 mM sodium pyruvate, and 4.5 g/L D-glucose. All media were supplemented with 10% fetal calf serum (Sigma), 100 units/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). HeLa cell lines were kept in a 5% CO2, 37 C incubator. For activity-regulation experiments, post-expression HeLa cells were incubated in the presence of 10 mM DTT or 50 mg/mL of CHX for 1 and 2 hours or for 2 and 4 hours, respectively.
Transfections Sub-Confluent HeLa cells in 10-cm dishes were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction. Briefly, cells were washed with 13 PBS and DMEM depleted of antibiotics was added. Transfections of the lipid–DNA mixture were performed using 24 mg of DNA for 6 hours in the presence of Opti-MEM I reduced serum-free medium (Invitrogen). After incubation for 6 hours at 37 C, the medium was replaced with fresh medium with antibiotics, and the cells were harvested for analysis by scraping them 24 hours post-transfection.
Structure solution and refinement Diffraction data from a single crystal of PDIA1 bb0 fragment were collected in the single-wavelength ˚ ) regime on an ADSC Quantum-210 CCD (1.7433 A detector (Area Detector Systems Corp.) at beamline F2 at the Cornell High-Energy Synchrotron Source (CHESS) (Table I). Data processing and scaling were performed with HKL2000.24 Initial attempts to phase the data using the NMR solution structure of bb0 domains from human PDIA1 (PDB entry 2K18) were unsuccessful. The structure was solved by molecular replacement with Phaser,25 using partial coordinates of domain b0 from human PDIA1 crystal structure (PDB entry 3BJ5) as a search model for the b0 domain. Coordinates of the b domain of rat ERp72 (PDB entry 3EC3) were used as a search
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co-IPs assays and antibodies Cells were lysed in 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5 mM EDTA, 10% glycerol, supplemented with 5 mM DTT and protease inhibitors cocktail (Complete, Mini, EDTA-free, Roche). Quantification of the total cell lysate was performed using Bradford (Bio-Rad). For the immunoprecipitations assay, 500 mg of lysate were incubated with 5 mg of anti-myc (Sigma) or anti-FLAG antibodies (Sigma) for 2 hours at 4 C with mixing. After that, Dynabeads protein G (Invitrogen) was added for two additional hours at 4 C. After extensive washing of the beads, immunoprecipitated proteins were eluted by adding SDS sample buffer.
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Western blot and detection Proteins present in total cell lysates were directly analyzed by SDS-PAGE for determination of the expression of tagged PDIA1 or subjected to co-IP to determine dimerization before analysis by SDSPAGE. To determine the presence of PDIA1-myc and PDIA1-FLAG tagged protein inside the cells, the total cell lysates were analyzed by SDS-PAGE. For this, equal amounts of lysates (50 mg) were initially run on SDS-PAGE gels. Proteins were then transferred to polyvinylidene difluoride membranes (Immun-Blot PVDF membrane) (BioRad) at 150 mA for 2 hours, and the membranes were blocked in 5% milk, PBS for 2 hours, followed by incubation with primary antibodies at the following concentrations: 1:5000 anti-myc and 1:10,000 anti-FLAG. After washing three times with 13 PBS, the membranes were incubated with secondary antibodies antimouse IgG, horseradish peroxidase conjugate (Sigma) for 1 hour, followed by extensive washing. Visualization was done by chemiluminescence using ECL plus Western Blotting Detection Reagents (Amersham) and then exposed to autoradiography film (HyBlot CL, Denville Scientific). Immunoprecipitated proteins were analyzed by 8% SDS-PAGE followed by the same protocol for Western blot described above.
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Acknowledgments The authors declare no conflict of interests. We thank Nozhat Safaee and Yogita Patel for assistance with the co-IP experiments. Data acquisition at the Macromolecular Diffraction (MacCHESS) facility of the Cornell High Energy Synchrotron Source (CHESS) was supported by the National Science Foundation award DMR 0225180 and the National Institutes of Health award RR-01646.
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regulated dynamic conformations of human protein disulfide isomerase. Antiox Redox Signal 19:36–45. Inaba K, Masui S, Iida H, Vavassori S, Sitia R, Suzuki M (2010) Crystal structures of human Ero1alpha reveal the mechanisms of regulated and targeted oxidation of PDI. EMBO J 29:3330–3343. Araki K, Nagata K (2011) Functional in vitro analysis of the ERO1 protein and protein-disulfide isomerase pathway. J Biol Chem 286:32705–32712. Frand AR, Kaiser CA (1999) Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum. Mol Cell 4:469– 477. Hu CH, Tsou CL (1992) C-terminal truncation of bovine protein disulfide isomerase increases its activity. Biochem Biophys Res Commun 183:714–718. Yu XC, Wang CC, Tsou CL (1994) Association and dissociation of protein disulfide isomerase. Biochim Biophys Acta 1207:109–113. Solovyov A, Gilbert HF (2004) Zinc-dependent dimerization of the folding catalyst, protein disulfide isomerase. Protein Sci 13:1902–1907. Li SJ, Hong XG, Shi YY, Li H, Wang CC (2006) Annular arrangement and collaborative actions of four domains of protein-disulfide isomerase: a small angle X-ray scattering study in solution. J Biol Chem 281: 6581–6588. Denisov AY, Maattanen P, Dabrowski C, Kozlov G, Thomas DY, Gehring K (2009) Solution structure of the bb’ domains of human protein disulfide isomerase. FEBS J 276:1440–1449. Nguyen VD, Wallis K, Howard MJ, Haapalainen AM, Salo KE, Saaranen MJ, Sidhu A, Wierenga RK, Freedman RB, Ruddock LW, et al. (2008) Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate binding b’ domain. J Mol Biol 383:1144–1155. Wallis AK, Sidhu A, Byrne LJ, Howard MJ, Ruddock LW, Williamson RA, Freedman RB (2009) The ligandbinding b’ domain of human protein disulphideisomerase mediates homodimerization. Protein Sci 18: 2569–2577. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2:326–332. Ennis HL, Lubin M (1964) Cycloheximide - aspects of inhibition of protein synthesis in mammalian cells. Science 146:1474–1476. van Lith M, Karala AR, Bown D, Gatehouse JA, Ruddock LW, Saunders PT, Benham AM (2007) A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells. Mol Biol Cell 18:2795–2804. Tian G, Kober FX, Lewandrowski U, Sickmann A, Lennarz WJ, Schindelin H (2008) The catalytic activity of protein-disulfide isomerase requires a conformationally flexible molecule. J Biol Chem 283:33630–33640. Rainey-Barger EK, Mkrtchian S, Tsai B (2007) Dimerization of ERp29, a PDI-like protein, is essential for its diverse functions. Mol Biol Cell 18:1253–1260. Gruber CW, Cemazar M, Mechler A, Martin LL, Craik DJ (2009) Biochemical and biophysical characterization of a novel plant protein disulfide isomerase. Biopolymers 92:35–43. Walker AK, Soo KY, Levina V, Talbo GH, Atkin JD (2013) N-linked glycosylation modulates dimerization of protein disulfide isomerase family A member 2 (PDIA2). FEBS J 280:233–243.
Crystal Structure of the bb0 Dimer of Human PDIA1
24. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326. 25. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Cryst 40:658–674. 26. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Cryst D60:2126–2132.
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27. Murshudov GN, Vagin AA, Lebedev A, Wilson KS, Dodson EJ (1999) Efficient anisotropic refinement of macromolecular structures using FFT. Acta Cryst D55: 247–255. 28. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50:437–450.
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Sara Bastos-Aristizabal, Guennadi Kozlov, and Kalle Gehring* sur la structure des prote ines, McGill University, 3649 Promenade Sir Department of Biochemistry, Groupe de recherche axe al, Que bec, H3G 0B1, Canada William Osler, Montre Received 31 December 2013; Accepted 10 February 2014 DOI: 10.1002/pro.2444 Published online 00 Month 2014 proteinscience.org
Abstract: Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). Here, it is shown that human PDI (PDIA1) dimerizes in vivo and proposed that the dimerization of PDI has physiological relevance by autoregulating its activity. The crystal structure of the dimeric form of noncatalytic bb0 domains of human PDIA1 determined to 2.3 A˚ resolution revealed that the formation of dimers occludes the substrate binding site and may function as a mechanism to regulate PDI activity in the ER. Keywords: crystal structure; thioredoxin-like domain; endoplasmic reticulum; protein disulfide isomerase; dimerization
Introduction Proper folding of nascent proteins that enter the secretory pathway often requires formation of disulfide bonds. Disulfide bonds can spontaneously form during folding, but this reaction is kinetically slow. In order to catalyze this process, the endoplasmic reticulum (ER) contains a large and fascinating family of oxidoreductases called protein disulfide isomerases (PDIs).1,2 These enzymes oxidize substrate proteins and then themselves have to be oxidized to complete the catalytic cycle.3 When the catalytic cysteines are reduced, PDIs are able to react with nonnative disulfides to form a mixed disulfide complex
Abbreviations: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; co-IP, co-immunoprecipitation; DTT, dithiothreitol; CHX, cycloheximide. Additional Supporting Information may be found in the online version of this article. Sara Bastos-Aristizabal and Guennadi Kozlov contributed equally to this work Grant sponsor: NSERC; Grant number: RGPIN-238873. *Correspondence to: Kalle Gehring; Department of Biochemis sur la structure des prote ines, try, Groupe de recherche axe al, McGill University, 3649 Promenade Sir William Osler, Montre bec H3G 0B1, Canada. E-mail: [email protected] Que
C 2014 The Protein Society Published by Wiley-Blackwell. V
and function as isomerases to rearrange protein disulfide bonds. PDI, also known as PDIA1, is the founding member and the most studied protein in the PDI family. PDI contains four thioredoxin-like domains abb0 a0 , where a denotes catalytic domains containing CxxC motifs and b stands for non-catalytic domains, with a 20-residue x-linker between b0 and a0 domain and a C-terminal acidic tail. The reduction potential of PDIA1 (2180 mV) is higher than this of other PDIs, meaning that it is a stronger oxidizer.4 The four thioredoxin-like domains arranged in a U shape with the b and b0 domains forming the base of the U and the catalytic sites of the a and a0 domains facing each other, as shown by crystal structures of human PDIA1 and its yeast homolog Pdi1p.5,6 PDIA1 is the most abundant protein present in the ER and is a major player in the oxidative pathway. The mechanism of PDIA1 catalysis has been widely studied and is thought to function as follows: PDIA1 catalyzes the oxidation and shuffling of disulfides in substrate proteins. In turn, PDIA1 is reoxidized by the ER sulfhydryl oxidase ERO1a, so it can interact with substrates again. Recent structural analysis indicates that ERO1a specifically interacts with the b0 domain of PDIA1 and preferentially oxidizes the a0 domain.7–9
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In vitro dimerization of PDIA1 has been reported repeatedly, making it possible that a similar phenomenon occurs in the ER of eukaryotes to regulate the levels of PDIA1 oxidation. Initial identification of a mixture of two different species of PDIA1 in solution was reported by gel-filtration analysis.10 Two years later, the same group reported the association/dissociation behavior of PDIA1 in different conditions, where both hydrophobic and ionic forces were responsible for dimer formation. They observed that the monomeric species were more active suggesting that dimerization decreased binding of substrates.11 More recently, studies employing analytical ultracentrifugation, small angle X-ray scattering (SAXS), and NMR12–15 provided additional evidence of in vitro PDIA1 dimerization. Freedman et al. reported that dimerization was primarily mediated by interactions between the hydrophobic pockets on the b0 domains.16 They proposed that the tendency of PDIA1 to dimerize could be relevant in vivo and that the high concentrations of PDIA1 in the ER might be balanced by the monomer/dimer ratio, which is influenced by the amount of PDI substrates present at any time. However, the structural mechanism of PDIA1 dimerization remained unanswered. Here, we show that dimers of full-length PDIA1 can be isolated from cultured cells and report the crystal structure of a dimeric form of the bb0 domains. The structure reveals that the formation of dimers blocks the substrate binding site and explains the slow kinetics of dimerization and the inhibition of dimerization by amphipathic molecules and substrate mimics observed in previous studies.
Results Human PDIA1 dimerizes in vivo The ability of PDIA1 to dimerize in vivo was studied by co-immunoprecipitation (co-IP) of myc- and FLAG-tagged PDIA1. First, HeLa cells were transfected with PDIA1-myc [Supporting Information Fig. S1(A,B), lane 2], PDIA1-FLAG [Supporting Information Fig. S1(A,B), lane 3], or both PDIA1-myc and PDIA1-FLAG [Supporting Information Fig. S1(A,B), lane 4]. After expression for 24 hours, the total cell lysates were subjected to analysis. SDS-PAGE Western blot analyses with anti-myc and anti-FLAG monoclonal antibody established that both tagged proteins were expressed in HeLa cells. Once expressions of transfectants were established, the experiments probing dimerization were performed. In particular, the lysates were subjected to co-IP using an antibody against myc [Fig. 1(A,B), lanes 1–3] or an antibody against FLAG [Fig. 1(A,B), lanes 4–6]. The products from the co-IP were split into two fractions and loaded on two different SDS-PAGE gels in the same order, and then detected by Western blot-
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Figure 1. In vivo dimerization of PDIA1. co-IPs with anti-myc (lanes 1–3) and with anti-FLAG (lane 4–6) were analyzed by SDS-PAGE followed by Western blot using anti-myc (A) and anti-FLAG (B). Asterisks indicate bands that belong to the heavy chains of the antibodies used in the immunoprecipitation.
ting. After blotting, one membrane was probed for myc [Fig. 1(A)], and the other probed for FLAG [Fig. 1(B)]. The dimer is expected to appear as a band present in the doubly transfected cells in both gels. Importantly, dimerization was evident when PDIA1-FLAG was co-immunoprecipitated by antimyc [Fig. 1(B), lane 3], and conversely when PDIA1myc was co-immunoprecipitated by anti-FLAG [Fig. 1(A), lane 6] in the doubly transfected samples. This result indicates that PDIA1-myc and PDIA1-FLAG interacted and pulled down each other. As a control, anti-myc and anti-FLAG antibodies were able to immunoprecipitate its respective antigen, PDIA1myc [Fig. 1(A), lane 1] and PDIA1-FLAG [Fig. 1(B), lane 5], respectively. Also, the anti-myc and antiFLAG antibodies were specific as they failed to directly pull down PDIA1-FLAG [Fig. 1(B), lane 2] and PDIA1-myc [Fig. 1(A), lane 4], respectively. To determine whether PDIA1 dimerization occurs inside of the cells or only after lysis, HeLa cells were singly transfected with either PDIA1-myc or PDIA1-FLAG, and the tagged PDIA1 proteins were expressed separately. After expression, the cells were combined and immunoprecipitation experiments were performed. For this, plates transfected with PDIA1-myc were harvested and combined with cells expressing PDIA1-FLAG and then lysed together. The lack of dimerization in total cell lysates of single transfectants that were mixed after separate expressions [Fig. 2(A), lane 3 and Fig. 2(B), lane 1], combined with the positive control showing dimerization when double transfectants were expressed [Fig. 2(A), lane 4 and Fig. 2(B), lane 2], indicates that PDIA1 dimerization occurs inside of the cells and is not a product of a post-lysis interaction.
PDIA1 dimerization is not mediated by disulfides To determine whether PDIA1 autoregulates its activity via dimerization, changes in dimerization levels were evaluated during ER-stress and while protein synthesis was inhibited. We hypothesized higher levels of PDIA1 dimer under conditions of low substrate availability compared with conditions of ER-stress in which unfolded proteins promote
Crystal Structure of the bb0 Dimer of Human PDIA1
Figure 2. PDIA1 dimerization occurs in the cells. co-IPs with anti-myc (lanes 1, 2) and with anti-FLAG (lane 3, 4) were analyzed by SDS-PAGE and Western blot using anti-myc (A) and anti-FLAG (B). Indicated with an asterisk are the bands that belong to the antibodies used in the immunoprecipitation.
dissociation of PDIA1 dimers. HeLa cells expressing both PDIA1-myc and PDIA1-FLAG were treated with 10 mM of dithiothreitol (DTT) and 50 mg/mL of cycloheximide (CHX). When supplied to cells, DTT acts as a strong reducing agent and is commonly used when studying ER-stress and the unfolded protein response (UPR).17 On the other hand, CHX is widely used for its ability to inhibit protein synthesis in eukaryotic cells.18 Unexpectedly, levels of substrate availability did not have an effect on the amount of PDIA1 dimers formed in the cells [Fig. 3(B), lanes 1–4]. However, the unchanged levels of PDIA1 dimerization in the presence of 10 mM DTT demonstrated that the dimerization mechanism does not involve intermolecular disulfides supporting previous data.11
Crystallization of the bb0 domains of human PDIA1 and structure determination In order to obtain structural insight into the mechanism of PDIA1 dimerization, we performed crystallographic studies of the bb0 domains of human PDIA1 and obtained a crystal that diffracted to
Figure 3. PDIA1 dimerization is disulfide independent. co-IPs with anti-myc (lanes 1–4) and with anti-FLAG (lane 5–8) were analyzed by SDS-PAGE followed by Western blot using antimyc (A) and anti-FLAG (B). Cross-reactive bands from antibody proteins are marked by an asterisk.
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˚ (Table I). The construct (residues 135–357) 2.3 A includes the bb0 domains and half of the x-linker and yields high-quality NMR spectra that were used to determine the NMR solution structure of the monomeric form.14 The initial attempts to phase the data by molecular replacement failed to produce solutions, even using structures with 100% sequence identity: the crystal structure of the b0 x fragment of human PDIA1 and the NMR structure of the bb0 domains. This suggested that the structure in our crystal deviated significantly from previous structures. The crystals appeared in a 9-month-old drop and we were not able to reproduce them with selenomethionine-labeled protein for experimental phasing of the diffraction data. An attempt to use the anomalous signal of sulfur was also unsuccessful. Eventually, a weak molecular replacement solution for two b0 domains was obtained using the b0 x crystal structure with loops and helices a1 and a3 removed. We then were able to locate the b domains of PDIA1 using b domain of ERp72 as a search model. Numerous rounds of manual model building and refinement were then used to improve the map and to arrive to the final structure (Supporting Information Fig. S2). No electron density was observed for P135, E348–S357 (the x-linker), the N-terminal GPLGS cloning linker and, additionally, residues E323–L334 for chain A and E322–A336 for chain B that correspond to the strand b5 and b5–a4 loop in the b0 x crystal structure.
Structure of the bb0 dimer of human PDIA1 The structure displays two bb0 molecules in the asymmetric unit (Fig. 4). In each molecule, the b
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Table I. Data Collection and Refinement Statistics bb0 fragment
Data collection Space group Cell dimensions ˚) a, b, c (A a, b, g ( ) ˚) Resolution (A Rpim CC (1/2) I/rI Completeness (%) Redundancy Refinement ˚) Resolution (A No. reflections Rwork/Rfree No. atoms Protein Water B-factors Protein Water RMS deviations ˚) Bond lengths (A Bond angles ( ) a
P1 40.52, 45.72, 55.15 81.85, 92.15, 80.02 50–2.30 (2.38–2.30)a 0.035 (0.194) 0.995 (0.856) 23.5 (5.4) 95.5 (92.7) 7.5 (7.0) 50–2.30 15,880 0.244/0.297 3103 3036 67 42.4 40.6 0.007 1.29
Highest resolution shell is shown in parentheses.
domain shows a characteristic thioredoxin-like fold with a five-stranded b-sheet in the b1–b3–b2–b4–b5 arrangement that is flanked by two a-helices on both sides. Unexpectedly, the b0 domain demonstrates surprising deviation from the canonical fold, as the strand b5 is missing in the model. Instead, the b4 strand forms an antiparallel b-sheet with the same strand of another bb0 molecule. The residues corresponding to the b5 strand and the following
Figure 4. Crystal structure of PDIA1 bb0 dimer. A ribbon diagram showing PDIA1 dimer in two different orientations. The dimer is colored by chain in which one is represented by yellow and the other one in magenta and the domains are labeled.
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b5–a4 loop are absent from the electron density map due to disorder. Structural overlay with the b0 x structure shows that the distance between the a1 and a3 helices is bigger in the dimeric form of the b0 domain (Fig. 5) resulting in a more open conformation with larger hydrophobic cavity. This difference is unlikely to be due to the I289A mutation in the crystallized b0 x domain, because the area of the mutation shows nearly identical local structure. As a consequence of the open conformation, the aromatic ring of F304 from second protomer inserts into the ligand-binding ˚ site of the first protomer by approximately 4 A deeper than W364 of the x-linker in the capped conformation of the monomeric b0 domain. Taken together, the two structures may provide an example of breathing-like motions in the ligand-binding site of the b0 domain that could be important for PDIA1’s ability to adjust to a wide variety of protein substrates with solvent-exposed hydrophobic stretches. We also compared the structure with the NMR structure of monomeric bb0 structure and crystal structures of human and yeast PDI, in order to evaluate whether dimerization results in different relative orientation of the b and b0 domains (Fig. 6). Structural overlay of the dimeric form to the bb0 fragments from monomeric PDI structures shows
Figure 5. Comparison of the b0 substrate-binding sites in dimeric and monomeric structures. (A) F304 and F305 of the b0 domain occupy the substrate-binding site of another b0 domain in the dimeric structure. (B) L343 and W347 of the X-linker interact with monomeric b0 domain (PDB code 2BJX).
Crystal Structure of the bb0 Dimer of Human PDIA1
side chains of F240, H256, L258, I289, I291, I301, F304, F305, and L320 of another protomer [Fig. 7(A)]. Next to F304, the side chain of F305 sits between the side chains of F304 and L320 while also reaching I318. A half-turn earlier, the side chain of I301 interacts with the aromatic ring of F304. In the middle of the helix a3, the side chain of R300 is involved in the network of intermolecular hydrogen bonds with carbonyl of D297 and side chains of E242 and N298, while maintaining a salt bridge with E303 of its own protomer. Despite being in the close proximity to G306, F249 does not participate in the dimer formation, as its aromatic ring is not present in the electron density map due to multiple
Figure 6. Dimerization does not affect relative orientation of the b and b0 domain. Orientation of the bb0 domains in the dimeric form (in yellow) is very similar to that in reduced monomeric PDIA1 (PDB code 4EKZ; in green) (A) and differs in oxidized PDIA1 (PDB code 4EL1; in blue) (B).
˚ to reduced human PDIA1 (PDB code RMSD of 1.5 A ˚ to oxidized human 4EKZ, 180 Ca atoms), 2.4 A ˚ to PDIA1 (PDB code 4EL1, 179 Ca atoms), 2.7 A NMR structure of identical construct of human ˚ PDIA1 (PDB code 2K18, 185 Ca atoms), and 3.3 A to yeast Pdi1p (PDB code 3BOA, 177 Ca atoms). The lowest RMSD value corresponds to the most similar inter-domain orientation, while the higher RMSDs indicate variability in relative orientation of the domains. High structural similarity of the dimeric form to the reduced monomeric form of PDIA1 demonstrates that the dimerization is not accompanied by changes in the interdomain orientation. It should be noted that the highest RMSD to yeast Pdi1p also reflects low sequence identity between the human and yeast proteins, leading to noticeable differences in domain structures.
Structural determinants of dimerization There are three main factors causing the b0 domain to dimerize. One of the main contributing factors is the mostly hydrophobic interactions between the substrate-binding a1–a3 surfaces. The second half of helix a3 is especially important for these interactions, as it contains several residues engaging the substrate-binding hydrophobic cavity. In particularly, the side chain of F304 of one protomer inserts into the middle of hydrophobic cavity formed by the
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Figure 7. Structural determinants of PDIA1 bb0 dimerization. Dimer forms via interactions of one molecule (yellow) with another (magenta) through their hydrophobic pockets. Dimerization is mediated by hydrophobic interactions (A), polar interactions (B) and swapping of the C-terminal a-helices (C).
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conformations and was omitted from the model. This hydrophobic nature of the dimeric interaction is supported by previous studies.11,16 Another significant contribution to dimerization comes from an intermolecular antiparallel b-sheet between strands b4 of each protomer. In the center of this b-sheet, carbonyl and amide of T319 of one protomer make hydrogen bonds with the same residue of the other protomer [Fig. 7(B)]. The b5 strand, which normally forms b-sheet with the b4 strand, is disordered in the dimeric structure. Besides backbone hydrogen bonds, the newly formed b-sheet is stabilized by isoleucine zipper on the buried side through side chains of I318 and by intermolecular hydrogen bonds between side chains of E321 and T319 on the solvent-exposed side. Finally, it appears that the swapping of the Cterminal helices a4 takes place between the protomers [Fig. 7(C)]. While the connection between the strand b4 and helix could not be traced in the model as this region is disordered in the crystal, the length of the missing region (12 residues in chain A and 15 residues in chain B) in comparison with the distance ˚ between carbonyl of between the visible ends (15 A residue 322 of chain A and the backbone nitrogen of ˚ between residue 335 of chain B and more than 33 A the same carbonyl and the backbone nitrogen of residue 337 of the same chain A) makes it mechanistically unlikely for the C-terminal helix to bind the same PDI molecule. The connecting regions would also have to run across each other if no swapping takes place making it even less probable. The swapping of C-terminal helices is indirectly supported by earlier NMR studies. NMR spectra of PDIA1 fragments containing the b0 domain show peaks corresponding to both monomeric and dimeric forms.14 Swapping would significantly slow down the monomer–dimer interconversion and is consistent with slow-exchange observed during NMR experiments. Interestingly, the thermal B-factors for these C-terminal helices are higher than for the rest of the domain indicating their relatively loose attachment to the rest of the structure. Furthermore, the residues involved in dimerization including F304, F305, L320, I318, I289, I301, R300, H256, L258, and E303 are highly conserved throughout many species (Supporting Information Fig. S3). Interestingly, some of these amino acids belong to the almost invariant RIXEFFG motif located in the dimer interface, where X represents a hydrophobic residue. We should note that it is difficult to correlate this conservation with an importance of PDIA1 dimerization because a majority of these residues make up the substrate-binding site. We also used the structure to evaluate the dimeric arrangement of the full-length protein. We made structural overlays of the dimeric bb0 fragment of PDIA1 with the ab fragments (PDB entry
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4EKZ) and a0 b0 fragments (PDB entry 3UEM) of human PDIA1. The resulting overlays show that the a domains of PDIA1 dimer would be positioned on the same side of the dimer with their catalytic sites roughly facing each other (Supporting Information Fig. S4). The a0 domains would be located on the other side of the dimer with their spatial position less defined due to longer linkers connecting b0 and a0 domains. We expect the catalytic and chaperone function of PDIA1 dimer to be severely impaired due to ligand-binding site being unavailable to bind substrates.
Discussion Previous studies have already shown in vivo dimerization for the PDI-family member PDIA7 and yeast Pdi1p using co-IP experiments.19,20 Here, we show that human PDIA1 also forms disulfide-independent dimers in vivo and propose that such dimerization may act as a regulatory control mechanism of protein folding in the ER or generate a reservoir of inactive protein that allows the ER to respond efficiently to a sudden increase in substrate availability. One of the Pdi1p crystal structures showed a different dimeric arrangement where the intermolecular interactions are mediated by extensive contacts as the two U-shaped proteins come together.20 Given the low sequence identity between human PDIA1 and yeast Pdi1p, it is unclear whether Pdi1p would be able to dimerize in a similar mode to the human PDIA1 b0 domain. Besides other members of the human PDI family, some PDI proteins from plants were previously shown to dimerize in vitro.19,21–23 In particular, studies on plant PDI have shown a tendency to dimerize at higher, but still physiologically relevant concentrations, which may reflect a concentration-depended mechanism similar to the one observed for PDIA1. Also, van Lith et al. showed that PDIA7 homodimers are not disulfide-linked.19 Interestingly, amino acids involved in PDIA1 dimerization are conserved between these two proteins, while the structure of PDIA7 b0 domain contains a PDIA1-like substrate-binding hydrophobic pocket (S. Bastos-Aristizabal, unpublished data), suggesting that their dimerization mechanism may be similar. Dimerization of PDIA1 inhibits substrate binding and therefore may function as a mechanism to regulate PDIA1 activity in the ER. Our structure shows that the dimerization involves substratebinding site explaining the molecular basis of this inhibition. Also, we showed that PDIA1 forms dimers in vivo using immunoprecipitation experiments, suggesting that the dimerization may have a physiological relevance by auto-regulating its activity. It is currently unclear whether the dimer observed in our crystal is responsible for in vivo dimerization of PDIA1; future studies are needed to confirm the mechanism in cells. If true, this
Crystal Structure of the bb0 Dimer of Human PDIA1
mechanism would allow the ER to maintain a balance between oxidation and reduction necessary for native disulfide bond formation. ER redox balance is important since the defective folding or localization of mutated proteins is responsible for a number of genetic diseases, known as protein trafficking diseases.
Materials and Methods Protein expression, preparation, and purification The bb0 fragment from human PDIA1 (residues 135–357) was cloned into pGEX-6P-1 vector (Amersham-Pharmacia) and expressed in Escherichia coli BL21 (DE3) in rich (LB) medium as N-terminal GST fusion. The GST fusion protein was purified by affinity chromatography on glutathione-Sepharose resin and tag was removed by cleavage with PreScission Protease, leaving Gly–Pro–Leu–Gly–Ser N-terminal extension. The cleaved protein was additionally purified using size-exclusion chromatography using HPLC buffer (10 mM HEPES, 50 mM NaCl, 3 mM DTT, pH 7.0).
Crystallization Crystallization conditions were identified utilizing hanging drop vapor diffusion with the PACT crystallization suite (QIAGEN). The best crystals were obtained by equilibrating a 0.6 mL drop of protein (10 mg/mL) in buffer (10 mM HEPES, 50 mM NaCl, 3 mM DTT, pH 7.0), mixed with 0.6 mL of reservoir solution containing 25% (w/v) polyethylene glycol 1500, and 25 mM sodium malonate, 37.5 mM imidazole, 37.5 mM boric acid, pH 4.0 and suspending over 0.6 mL of reservoir solution at 22 C. No additional cryoprotectant was necessary. For data collection, the crystals were picked up in a nylon loop and flash cooled in a N2 cold stream. The crystals contain two molecules in the asymmetric unit corresponding to ˚ 3 Da21 and a solvent content of 36.8%. Vm 5 1.95 A
model of the PDIA1 b domain. The initial model obtained from Phaser was extended with the program Coot26 and improved by multiple cycles of refinement using the program REFMAC 5.227 and model refitting. Local NCS restraints for residues 136–347 were used throughout the refinement. The refinement statistics are given in Table I. The final model has good stereochemistry (Table I) according to the program MolProbity28 with 95.4% of residues in favored regions and 100% of residues in allowed regions. The coordinates and structure factors have been deposited in the RCSB Protein Data Bank (accession number 4JU5).
Cell Lines Human cervical carcinoma HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 4 mM L-glutamine, 1 mM sodium pyruvate, and 4.5 g/L D-glucose. All media were supplemented with 10% fetal calf serum (Sigma), 100 units/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). HeLa cell lines were kept in a 5% CO2, 37 C incubator. For activity-regulation experiments, post-expression HeLa cells were incubated in the presence of 10 mM DTT or 50 mg/mL of CHX for 1 and 2 hours or for 2 and 4 hours, respectively.
Transfections Sub-Confluent HeLa cells in 10-cm dishes were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction. Briefly, cells were washed with 13 PBS and DMEM depleted of antibiotics was added. Transfections of the lipid–DNA mixture were performed using 24 mg of DNA for 6 hours in the presence of Opti-MEM I reduced serum-free medium (Invitrogen). After incubation for 6 hours at 37 C, the medium was replaced with fresh medium with antibiotics, and the cells were harvested for analysis by scraping them 24 hours post-transfection.
Structure solution and refinement Diffraction data from a single crystal of PDIA1 bb0 fragment were collected in the single-wavelength ˚ ) regime on an ADSC Quantum-210 CCD (1.7433 A detector (Area Detector Systems Corp.) at beamline F2 at the Cornell High-Energy Synchrotron Source (CHESS) (Table I). Data processing and scaling were performed with HKL2000.24 Initial attempts to phase the data using the NMR solution structure of bb0 domains from human PDIA1 (PDB entry 2K18) were unsuccessful. The structure was solved by molecular replacement with Phaser,25 using partial coordinates of domain b0 from human PDIA1 crystal structure (PDB entry 3BJ5) as a search model for the b0 domain. Coordinates of the b domain of rat ERp72 (PDB entry 3EC3) were used as a search
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co-IPs assays and antibodies Cells were lysed in 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5 mM EDTA, 10% glycerol, supplemented with 5 mM DTT and protease inhibitors cocktail (Complete, Mini, EDTA-free, Roche). Quantification of the total cell lysate was performed using Bradford (Bio-Rad). For the immunoprecipitations assay, 500 mg of lysate were incubated with 5 mg of anti-myc (Sigma) or anti-FLAG antibodies (Sigma) for 2 hours at 4 C with mixing. After that, Dynabeads protein G (Invitrogen) was added for two additional hours at 4 C. After extensive washing of the beads, immunoprecipitated proteins were eluted by adding SDS sample buffer.
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Western blot and detection Proteins present in total cell lysates were directly analyzed by SDS-PAGE for determination of the expression of tagged PDIA1 or subjected to co-IP to determine dimerization before analysis by SDSPAGE. To determine the presence of PDIA1-myc and PDIA1-FLAG tagged protein inside the cells, the total cell lysates were analyzed by SDS-PAGE. For this, equal amounts of lysates (50 mg) were initially run on SDS-PAGE gels. Proteins were then transferred to polyvinylidene difluoride membranes (Immun-Blot PVDF membrane) (BioRad) at 150 mA for 2 hours, and the membranes were blocked in 5% milk, PBS for 2 hours, followed by incubation with primary antibodies at the following concentrations: 1:5000 anti-myc and 1:10,000 anti-FLAG. After washing three times with 13 PBS, the membranes were incubated with secondary antibodies antimouse IgG, horseradish peroxidase conjugate (Sigma) for 1 hour, followed by extensive washing. Visualization was done by chemiluminescence using ECL plus Western Blotting Detection Reagents (Amersham) and then exposed to autoradiography film (HyBlot CL, Denville Scientific). Immunoprecipitated proteins were analyzed by 8% SDS-PAGE followed by the same protocol for Western blot described above.
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Acknowledgments The authors declare no conflict of interests. We thank Nozhat Safaee and Yogita Patel for assistance with the co-IP experiments. Data acquisition at the Macromolecular Diffraction (MacCHESS) facility of the Cornell High Energy Synchrotron Source (CHESS) was supported by the National Science Foundation award DMR 0225180 and the National Institutes of Health award RR-01646.
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Crystal Structure of the bb0 Dimer of Human PDIA1
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