The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)

Authors


S. Dhe-Paganon, Structural Genomics Consortium, Banting Institute, University of Toronto, 100 College Street, Room 511, Toronto, ON, Canada M5G 1L5
Fax: +1 416 946 0588
Tel: +1 416 946 3876
E-mail: sirano.dhepaganon@utoronto.ca

Abstract

Cyclophilins comprise one of the three classes of peptidylprolyl isomerases found in all eukaryotic and prokaryotic organisms, as well as viruses. Many of the 17 annotated human cyclophilins contain the catalytic domain in tandem with other domains, and many of the specific functions of a particular cyclophilin or its associated domains remain unknown. The structure of the isomerase domain from a spliceosome-associated cyclophilin, PPWD1 (peptidylprolyl isomerase containing WD40 repeat), has been solved to 1.65 Å. In the crystal, the N-terminus of one isomerase domain is bound in the active site of a neighboring isomerase molecule in a manner analogous to substrate. NMR solution studies show that this sequence binds to the active site of the cyclophilin, but cannot be turned over by the enzyme. A pseudo-substrate immediately N-terminal to the cyclophilin domain in PPWD1 could have wider implications for the function of this cyclophilin in the spliceosome, where it is located in human cells.

Abbreviations
Cyp

cyclophilin

IC50

50% inhibitory concentration

pNA

p-nitroaniline

PPIase

peptidylprolyl isomerase

PPWD1

peptidylprolyl isomerase containing WD40 repeat

suc

succinyl

Cyclophilins (Cyps) are one of three subfamilies of the peptidylprolyl isomerases (PPIases; E.C. 5.2.1.8), together with the structurally unrelated FK506-binding proteins and parvulins [1,2]. Biologists and clinicians initially focused on the specific and high affinity of PPIases for the immunosuppressants ciclosporin A, FK506 and rapamycin [3–5]. These immunosuppressive effects were eventually determined to be uncoupled from the enzymatic function of the PPIases, which involves the reversible cistrans isomerization of Xaa–Pro peptide bonds [2,6]. The evolutionary importance of this fold and/or function may be inferred from the broad distribution of PPIases throughout Eukaryota, Eubacteria, Archaea and, recently, a viral genome [2,7,8]. However, there is very little direct evidence to show that the enzymatic function encoded by the PPIases is the only, or even the primary, physiological role of these proteins in cells. There are a subset of proteins whose association with Cyps is necessary for correct folding and structural integrity: for instance, maturation of steroid receptor complexes in conjunction with Hsp90/Hsc70 [9–11] and the function of NinaA in Drosophila rhodopsin folding [12]. Cyp-catalyzed isomerization can also play a part in host response events, including the participation of host CypA in binding the capsid protein Gag of HIV-1 during infection in humans and packaging into HIV virions, and the association of a host CypB–viral polymerase complex leading to viral replication during infection by hepatitis C [13,14].

However, more recent studies of PPIases have focused on their roles as signal transducers rather than as chaperones or protein foldases. Specific examples include the participation of CypD in the mitochondrial permeability transition, leading to necrotic cell death [15], the isomerase-dependent regulation of ligand specificity for the SH2 domain of the non-receptor tyrosine kinase Itk [16,17], and the role of multiple Cyps in regulating transcriptional and spliceosomal events [18–20]. In these cases, the substrate proteins are already folded and performing a subset of their normal functions; an isomerase interacts with this substrate conformation, thereby allowing for a new set of molecular interactions to occur with the product conformer [1,17]. The molecular switch function of Cyps may well have different sequence determinants than their isomerization function, but very few studies have been performed to probe the sequence specificity of binding versus catalysis of Cyps. Early evidence indicated that CypA is able to catalyze the isomerization of a wide array of Xaa-Pro sequences with nearly identical catalytic efficiency [21]. In contrast, phage display and subsequent amino acid substitution analysis to identify CypA binding specificity identified a clear preference for sequences N- and C-terminal to the Xaa-Pro target culminating in a consensus sequence of FG*PXLp. This work indicates that preferential binding of target proteins may be dictated by a select subset of amino acids distinct from sequences that are substrates for catalysis [22]. The relevance of this finding is dependent on studies showing an in vivo context for peptides capable of binding to PPIases without being used as isomerization substrates; to date, no such peptide sequence has been described in the literature.

Further complicating the field of Cyp biology is the fact that many of the 17 Cyps annotated in the human genome have not been thoroughly studied in terms of either enzymatic or other functional significance. For some of these Cyps, their location inside the cell provides the only clue as to their function. The large subset of Cyps found to be stably associated with spliceosomes provides an example of the issues involved in studying the complexity of Cyp function. Spliceosomes are multi-megadalton complexes containing hundreds of proteins and five essential small nuclear RNAs, whose function is to excise out non-coding regions (introns) of translated pre-mRNAs [23,24]. Recent technological advances in the purification of spliceosomal complexes, coupled with advances in mass spectrophotometric methodology, have led to a massive increase in the identification of proteins found to be associated with spliceosomal complexes [25–28]. At least 11 of the 17 human PPIases have been found to be associated with intermediate spliceosomal subcomplexes [26,29]. One of the spliceosomal Cyps was identified using yeast two-hybrid screens for known spliceosomal components; another was recognized as a spliceosomal component based on sequence homology to an SR repeat containing splicing factors [20,30]. For two other spliceosomal Cyps, functional and structural studies eventually identified their cognate spliceosomal binding partners: PPIL1 binds to the SNW domain-containing protein 1 (SKIP), and CypH binds to the small ribonucleoprotein Prp4 [18,31–33]. Interestingly, in both of these cases, the interaction with spliceosomal components was found on surfaces not involving the isomerase active site and did not affect cistrans isomerase activity, indicating that the function of the isomerase in the spliceosomal complex may involve simultaneous active site and second-site interactions. The binding partners for the other seven spliceosomal Cyps are undetermined. Moreover, the physiological function of the spliceosomal Cyps remains unclear, which perhaps is not surprising, considering that so many proteins encoding the same enzymatic function are found simultaneously in spliceosomal complexes. It is theorized that this high level of complexity and seeming duplication of effort in the spliceosomal complexes are a function of the exquisite sophistication of dynamic networks needed to properly regulate and proofread the splicing process [23,34].

PPWD1 (peptidylprolyl isomerase containing WD40 repeat) was cloned in 1994 [35] and later purified as part of the catalytically competent form of the spliceosome C complex [26]. This polypeptide encodes an N-terminal WD40 repeat domain and a C-terminal domain homologous to Cyps. As part of an attempt to structurally characterize the spliceosomal Cyps, we have determined the high-resolution X-ray crystal structure of the isomerase domain of PPWD1 and monitored its activity via both UV kinetics and NMR solution experiments. In this structure, PPWD1 forms distinct intermolecular interactions within the asymmetric unit with an internal peptide containing a Gly–Pro sequence. Interestingly, the Pro residue is found in trans, an unusual circumstance for a substrate peptide. Further experiments have shown that PPWD1 is indeed a functional isomerase against a standard substrate sequence, but that, surprisingly, a peptide containing the internal sequence is able to bind, but is not catalyzed by the isomerase, suggesting that it is not a substrate. Both the intermolecular interaction and lack of enzymatic turnover were confirmed using NMR solution studies of the PPWD1 protein. This work represents the first structural and biochemical characterization of a WD40 repeat-containing spliceosomal Cyp, and the first instance of a Pro-containing sequence that is sufficient to bind specifically to the active site of a Cyp, but is not a substrate for cis–trans isomerization.

Results

The crystal structure of the isomerase domain of human PPWD1 (utilizing a construct encoding residues 473–646) was determined at 1.65 Å resolution. The final model of PPWD1 comprises three polypeptide chains; each chain is disordered from residues 473–482, such that the first interpretable density is for residue Gln483. All other residues encoded by the PPWD1 construct are present in the final model. In addition, the model contains a single glycerol molecule most probably contributed from the cryoprotectant and 388 water molecules. With an architecture consisting of eight antiparallel β-sheets forming a closed β-barrel with two α-helices packed against the core (Fig. 1A), the structure of PPWD1 is similar to the structure of the canonical CypA as well as to CypH and PPIL1, two other spliceosomal Cyps (Fig. 1B). The Cα atoms of CypA align to the isomerase domain of PPWD1 with an rmsd of 1.34 Å over 124 atoms, corresponding well to the 60% sequence similarity between the two isomerase domains. The main conformational differences lie in the α2–β8, β1–β2 and β4–β5 loops. The β1–β2 loop (corresponding to residues Thr498–Asp502) is shorter by five residues in PPWD1 compared with CypA. This deletion also occurs in six of the 17 currently annotated human Cyps, including PPIL1–PPIL4 (Fig. 2), but the significance of the resultant shorter β1–β2 loop is not understood.

Figure 1.

 Structure of PPWD1 and comparison with other Cyp structures. (A) The structure of the isomerase domain of PPWD1 is shown in ribbon format. The N-terminus and secondary structural elements are labeled. (B) Structural alignment of PPWD1 (magenta) with Cα stick representations of the canonical CypA (2RMA) in forest green and the spliceosomal CypH (1QOI) in light green. Unless otherwise mentioned, all figures were generated using PyMOL (http://pymol.sourceforge.net/). (C) The arrangement of molecules in the asymmetric unit in the PPWD1 crystal. N- and C-termini are labeled. The N-terminal peptide is bound in the active site of an adjacent protein. The homotypic interaction between the N-terminal peptide and the active site of a neighboring molecule could be a crystal contact; that is, it may be an artifact of the crystallization condition. Unfortunately, crystallographic methods cannot distinguish between crystal contacts and specific protein–protein interactions; however, the results presented here indicate that the homotypic interaction modeled in the crystal structure is supported by solution methods.

Figure 2.

 PPWD1 domain structure and sequence alignment of selected human isomerase domains. Top: a graphical overview of the PPWD1 gene. The internal peptide sequence QAEGP487KR is highlighted. Bottom: an alignment of selected human isomerase domains; output is from multalin [52]. Residues conserved at 90% or more are indicated by capital letters, and between 50% and 90% by lower case letters; (I/V) conserved positions are indicated by !; (N/D/E) conserved positions are indicated by #. All Cyps shown in this alignment (with the exception of CypA) have been experimentally described as being associated with spliceosomes, as referenced in the text. Green boxes highlight the conserved positions in the active site of CypA [2]; blue boxes outline the positions of the spliceosomal binding site on CypH [32]; pink boxes outline the main spliceosomal interaction regions on PPIL1 [31]. Asterisks (*) and crosses (+) above the alignment indicate the positions of the S1′ and S2′-S3′ subsites as described in [38].

PPWD1 crystallized with three protein molecules in the asymmetric unit. Unexpectedly, there was an intermolecular interaction, propagated throughout the crystal, in which the active sites of all three molecules in the asymmetric unit were bound to seven residues (QAEGP487KR) of an adjacent molecule (Fig. 1C). The three polypeptide chains in the asymmetric unit are conformationally identical, with rmsd values of 0.4 and 0.2 Å over all atoms in 176 residues. In addition, the N-terminal peptide is oriented in very similar fashion across all three molecules, also with rmsd values of 0.4 and 0.2 Å over all atoms for the first seven residues. Pro487 from one monomer is buried within the active site of another, and is less than 3 Å from the side-chain of the conserved Arg535 (Arg55 in CypA) (2.95 Å for NH1 and 2.85 Å for NH2). In addition, Pro487 of the peptide is bound in a hydrophobic pocket composed of Phe540, Phe593, Met541, Leu602 and His606, all of which are conserved between CypA and PPWD1 (Figs 2 and 3). The Nδ1 atom of the conserved Trp602 (Trp121 in CypA) is coordinating the backbone oxygen of Lys488 of the QAEGPKR peptide (Fig. 3A), and Phe540 is coordinating Lys488 through a stacking interaction. The other interactions between the side-chain of the QAEGPKR peptide in the active site are centered about residue Glu485; in addition to specific interactions between the backbone nitrogen of Glu485 with the oxygen of Gly551, the side-chain of Glu485 is nestled in a deep pocket of the enzyme (Fig. 3B). An oxygen of the γ-carboxyl group forms hydrogen bonds with the backbone amide nitrogen of Asn582 and a water molecule buried in the pocket; the other γ-carboxyl oxygen is involved in a network of water-mediated hydrogen bonds. Two residues in the PPWD1 active site, Gln543 and Gln591, contribute to the complementary polar nature of this pocket. The average B-factor for all three of the QAEGPKR regions is 48 Å2, and is comparable with an all-atom B-factor of 45 Å2 for all molecules in the asymmetric unit. These observations indicate a stable interaction, although PPWD1 runs as a monomer using size exclusion chromatography, which suggests that the interaction is either low affinity or has a high off rate.

Figure 3.

 Details of QAEGP487KR peptide interaction. (A) Cartoon model of PPWD1 and stick representation of the amino terminal peptide. Active site residues are shown in stick format and water molecules are shown as spheres. Note the water molecules in a hydrogen bonding network with Glu486 of the N-terminal peptide. (B) Electrostatic surface representation (+10 kT·e−1, red; −10 kT·e−1, blue) of PPWD1, generated by the apbs software package [53], showing the side-chains of Pro487 and Glu486 buried in deep pockets formed by the PPWD1 active site.

This mode of interaction and the trans conformation of the peptide in the active site mimic the enzyme–substrate interaction observed in the CypA–HIV-1 Gag complex [36]. Pro487 is found to be bound to PPWD1 in the trans configuration, which is also the conformer found in the complexes of the capsid protein Gag and in some peptides derived from GAG protein with CypA. For these complexes, the requirement for an X-Gly-Pro (X ≠ Pro) sequence was proposed to obtain stable binding of the trans isomer into the Cyp active site; the PPWD1 structure confirms this observation and, indeed, any other amino acid at that position would adopt φ,ψ angles such that Cβ would clash with the catalytic Arg535 and destabilize the trans conformation [36]. Although the backbone amide of the −2 position has been shown to be involved in a configuration-dependent hydrogen bond with the β4–β5 loop, the function of the side-chain at this position has not been exhaustively studied. As opposed to the mainly hydrophobic residues found in the context of HIV-1 capsid variants (containing Ala, Val, or Met at position −2), PPWD1 contains Glu at this position (Glu485), which is pointing into a region of charge complementarity in the PPWD1 active site that would not be well accommodated by hydrophobic side-chains. The relevance of finding a peptide bound in trans in the active site of isomerases is ambiguous; in the case of the HIV capsid, it has been proposed that an X-Gly-Pro sequence represents a poor substrate for catalysis, explaining the capture of the trans conformer in the crystal [36]. However, as the HIV capsid protein is indeed an efficient substrate for isomerization by CypA [37], it cannot yet be stated definitively that Gly-Pro sequences are the minimal determinant for binding versus catalysis for Cyps, or what role sequence variation amongst the Cyps may play. In summary, the interactions seen between the QAEGPKR peptide and PPWD1 active site in the current structure might be a product of an inability of the enzyme to catalyze the isomerization of this particular sequence; alternatively, the trans conformer of the substrate Gly–Pro sequence might simply have a higher affinity for the PPWD1 binding pocket. Further analysis is therefore necessary to attempt to distinguish between these two possibilities.

To determine whether PPWD1 is catalytically competent to isomerize Cyp substrates, we conducted biochemical and biophysical assays. Using a colorimetric coupled assay against succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (suc-AAPF-pNA), a well-characterized CypA substrate, we found that the isomerase domain of PPWD1 (using the crystallographic construct comprising residues 473–646) is indeed an active isomerase with a catalytic efficiency of 1.5 μm−1·s−1, similar to the values obtained in previous studies (Fig. 4A) [21,38]. This is not surprising as the active site residues of PPWD1 are nearly completely conserved when compared with CypA (Fig. 2). Furthermore, we found that ciclosporin A binds tightly to PPWD1 with a 50% inhibitory concentration (IC50) between 1 and 2 nm, a value similar to that for CypA (Fig. 4B). These enzymatic characteristics are not significantly different for a truncated PPWD1 construct without the N-terminal sequence (encoding residues 493–646), implying that this region does not interfere with the active site at the pico- to micromolar concentrations of protein used in the assay. As a result of the technical limitations of the enzyme coupled assay described above, we may not have detected enzymatic activity against low-affinity substrates in the millimolar KD range, nor could we detect binding without catalysis. To address this issue, we conducted direct NMR measurements, as described previously [39]. PPWD1 (residues 473–646) was active on the standard Suc-AAPF-pNA substrate, as indicated by a collapse of the peaks contributed by the cis and trans conformers (caused by enzyme-catalyzed isomerization that is rapid compared with the chemical shift differences between the cis and trans resonances) (Fig. 4C, red peaks). PPWD1 also bound to a synthetically derived QAEGPKR peptide, as shown by the small shifts for resonances on addition of the enzyme, especially for the +2 Arg resonances, which correlate well with the Arg interactions seen in the crystal structure. However, PPWD1 did not catalyze the isomerization of this peptide sequence, as it does for the model peptide substrate AAPF (Fig. 4C, black peaks; Fig. 4D). Finally, NMR-based experiments were undertaken to validate the intermolecular association of PPWD1 in the context of the protein construct used to obtain the crystal structure. 1H,15N-HSQC measurements were conducted on the PPWD1 construct which crystallized (residues 473–646), as well as an N-terminally truncated construct (residues 493–646). As expected, the overall spectra of these two constructs were similar. However, it is clear that the line widths of most resonances in the spectrum of the longer construct are broader than those in the shorter construct (Fig. 5). The simplest interpretation of this line broadening is that it is a result of a weak interaction between protein molecules, and it is quite possible that the interaction seen in solution using NMR is caused by the specific interactions captured in the crystal structure. Taken together, these data suggest that the intermolecular interaction trapped in the crystal structure can indeed be recapitulated using solution methods, and that this intermolecular interaction is a consequence of binding, but not efficient catalysis, of the QAEGPKR sequence by PPWD1.

Figure 4.

 PPWD1 is an active isomerase and binds to QAEGPK, but does not catalyze its isomerization. (A) Increasing amounts of PPWD1 accelerate the isomerization of suc-AAPF-pNA in a standard colorimetric assay. The bottom curve (squares) shows the unaccelerated cleavage of peptide relative to the catalyzed reaction (top curve, circles). (B) Ciclosporin A inhibits PPWD1 activity against suc-AAPF-pNA. (C) Binding and catalysis of isomerization for the standard substrate suc-AAPF-pNA. Black and red spectra of the Ala residues in the peptide are obtained from the uncatalyzed and catalyzed reaction, respectively. Acceleration of the cistrans isomerization of the peptide results in the collapse of these resonances into a single set of peaks. (D) Binding, but not isomerization, of the N-terminal peptide of PPWD1. A synthetic peptide corresponding to the seven residues seen in the crystal structure (QAEGPKR) was added to the PPWD1 protein (residues 473–646) to assess binding as in (C). Notice that the peak shift observed for Arg resonances is more pronounced than that for Lys (labeled with R and K, respectively), indicating a change in the chemical environment for this residue; this is confirmed by the model of the crystallographic data, which shows Arg489 pointing into the active site of the isomerase, whereas Lys488 points into the solvent. There are chemical shifts on addition of the enzyme, but no collapse of cis and trans peaks, suggesting that PPWD1 binds the N-terminal peptide but does not catalyze isomerization.

Figure 5.

 PPWD1 protein shows association in solution. Two overlain 1H,15N-HSQC spectra of PPWD1 proteins are shown. The 473–646 construct (black) contains the amino terminal sequence QAEGPKR, but 493–646 (red) does not. The 473–646 construct is the same protein that led to the crystal model. Experiments were conducted in parallel and under conditions of identical buffer and protein concentration.

Discussion

The QAEGPKR peptide is capable of binding the PPI domain of PPWD1 without being an efficient substrate for cis–trans isomerization. It is clear from earlier work that there is very little selectivity for substrates at the −1 or +1 position in the Cyp active site, with the caveat that Gly–Pro may promote the shift of equilibrium binding in the active site to the trans over the cis conformer (although with very little difference in catalytic efficiency) [21,22,38]. Therefore, it is reasonable to look N-terminal and C-terminal to these positions to find potential binding specificity in Cyps. Previous studies have delineated binding determinants for Cyps contained outside of the −1 and +1 positions using different methodologies. For instance, by comparing binding affinities with the ability to inhibit PPIase activity for peptide-based inhibitors, it was found that the active site of CypA could be separated into two distinct subsites (called S1′ and S2′–S3′), presumably indicating binding determinants on the protein distal to the −1 position [38]. In addition, a phage display experiment, which independently confirmed the preference for binding of Gly–Pro at the central position, described a binding preference for Phe at the −2 position and various amino acids at +3 and +4 for CypA [22]. These studies indicated that there might be separate sequence determinants for binding that are stricter than those for substrate turnover, and that these determinants are somehow dictated by residues outside the minimal active surface of the Cyps. The structure of PPWD1 indicates that the isomerase domain may bind tightly to a subset of protein targets with a polar side-chain at the −2 position, without losing the ability to be an efficient isomerase for substrates containing alternative residues at this position (A/G-P, for instance), as opposed to the hydrophobic preference of CypA at this position. Although residues that form the skeletal S2 pocket are identical between CypA and PPWD1, those adjacent to it, particularly in the β4–β5 loop (including residues Gly551–Gly553, Glu555 and Gly560–Glu561) are different between CypA and PPWD1, and may influence the side-chain specificity at the −2 position. Indeed, this region of the active site, including the β5–β6 loop (residues Ala583–Thr587), is the most divergent when comparing all Cyp active surfaces, and we therefore predict that these regions will dictate the greatest differences in specificity at the −2 position. In addition, using this structural rationale, there is some preference for a bulky side-chain at the +2 position because of potential stacking interactions with Trp601 and Phe540. Our current data cannot address positions C-terminal to +2, as our QAEGPKR peptide is constrained by its attachment to a neighboring Cyp molecule.

As mentioned earlier, many of the residues that make up the distal subsites on PPWD1 are conserved between the spliceosomal Cyps and CypA (Fig. 2). Two exceptions are Ala583, which is variously changed to Ser, Arg or Asn in spliceosomal Cyps, and Trp601 which is largely conserved amongst the non-spliceosomal Cyps, but is variably changed to His, glutamate, or Tyr in the spliceosomal subclass of Cyps (Fig. 2). Interestingly Ala583 is part of the S1′ subsite, and Trp601 and Phe540 are part of the S2′–S3′ subsite described previously [38]. It is reasonable to propose that the ability of PPWD1 to bind to sequence determinants that are not substrates for isomerization may be quite relevant to the larger biological function of Cyps if it is found to be a more general phenomenon.

The intermolecular interaction seen in the crystal structure of PPWD1 may imply a new function within the spliceosome, where it possibly plays a role in spliceosomal assembly or activity. Homotypic interactions are also observed in the crystal structure of another spliceosomal Cyp, CypH (SnuCyp-20), where isomerase domains interact with each other, again through an extended loop region N-terminal to the isomerase domain. PPWD1 and CypH have no sequence similarity in this region and crystallize under different conditions and with different spacegroups [32]. It is interesting in the context of this dissimilarity to observe that the two spliceosomal Cyps exhibit a similar type of intermolecular association in structural studies. Although the solution properties of the homotypic CypH interaction were not studied, the crystal structure of CypH bound to a peptide derived from the spliceosome shows that it binds a surface directly opposite to the isomerase active site (Fig. 2), suggesting that the homotypic interaction seen in the structure would not be impaired by spliceosomal association [32,33]. The overall sequence similarity between the isomerase domain of CypH and the isomerase domain of PPWD1 is reasonably high (55% over approximately 140 residues), but many of the residues that form the spliceosomal binding site of CypH are not conserved in PPWD1 (blue boxes, Fig. 2). In the case of CypJ (PPIL3), another spliceosomal Cyp whose interaction surface with the spliceosomal protein SKIP has been probed using NMR, the interaction with the spliceosomal component was again found to be distinct from the active site (pink boxes, Fig. 2) [31]. Again, the spliceosomal interacting region of CypJ is not well conserved amongst spliceosomal Cyps, including PPWD1. Although the spliceosomal target or interacting region of PPWD1 has not been isolated, it is reasonable to believe, based on the cases of CypH and CypJ, that this region may lie well outside the active site residues, and that these surfaces may be variable in terms of sequence amongst the spliceosomal Cyps in order to target isomerase binding to distinct spliceosomal substituents. It may be that the bifunctional isomerase domains of spliceosomal Cyps may be directed towards internal sequences in order to regulate the activity of these enzymes or to serve as a signal transduction element in addition to the isomerase function. Indeed, it may be that isomerization must be prevented in order for spliceosomal Cyps to perform these additional functions, and perhaps the homotypic interactions seen in the spliceosomal Cyps are indicative of peptide sequences that are binding determinants, but not efficient substrates, as in the case of PPWD1.

Experimental procedures

Cloning, expression and purification

Full-length cDNA encoding human PPWD1 was obtained from the Mammalian Gene Collection (BC015385). Constructs based around the predicted isomerase domain boundaries were cloned into pET28a-LIC and transformed into BL21 Gold (DE3) cells (Stratagene, La Jolla, CA, USA). Cultures were grown in Terrific Broth medium at 37 °C to D ∼ 6, and induced at 15 °C with the addition of 50 μm isopropyl thio-β-d-galactoside. Pellets were resuspended in 20 mL of lysis buffer (50 mm Tris, pH 8.0, 500 mm NaCl, 1 mm phenylmethanesulfonyl fluoride and 0.1 mL general protease inhibitor; Sigma P2714, St Louis, MO, USA) and lysed by sonication; lysates were then centrifuged for 20 min at 69 673 g. The supernatant was loaded onto nickel nitrilotriacetic acid resin (Qiagen, Valencia, CA, USA), washed with five column volumes of lysis buffer and five column volumes of low imidazole buffer (lysis buffer + 10 mm imidazole, pH 8), and eluted in 10 mL of elution buffer (lysis buffer + 250 mm imidazole, pH 8, and 10% glycerol). One unit of thrombin (Sigma) per milligram of protein was added to remove the tag overnight at 4 °C. For gel filtration, an XK 16 × 65 column (GE Healthcare, Piscataway, NJ, USA) packed with HiLoad Superdex 200 resin was pre-equilibrated with gel filtration buffer (lysis buffer + 5 mmβ-mercaptoethanol and 1 mm EDTA). Peak fractions were pooled and concentrated using Amicon concentrators (10 000 molecular mass cut-off; Millipore, Danvers, MA, USA). The protein was used at 15 mg·mL−1 for crystallization studies.

Crystallization, data collection and structure solution

A construct of the PPWD1 isomerase domain containing residues 473–646 crystallized in 1.7 m NH4SO4, 0.1 m sodium cacodylate, pH 5.7, and 0.2 m sodium acetate. Crystals were harvested into mother liquor with 15% glycerol and frozen in liquid nitrogen. Diffraction data were collected on an FR-E SuperBright Cu rotating anode/Raxis IV++ detector (Rigaku Americas, The Woodlands, TX, USA), and integrated and scaled using the hkl2000 program package [40,41]. For structure solution and refinement, the program phaser [42] was used as part of the ccp4 suite [43] to find the molecular replacement solution in the resolution range between 20 and 2.8 Å, using PDB ID 1XO7 as a search model. Following phaser, a round of maximum-likelihood refinement and phase extension to 1.65 Å was carried out with refmac5 [43,44], and the phases were then input into arp/warp [45] for automatic model building and iterative refinement. The models were completed using the graphics program o [46], and further rounds of refinement using refmac5 resulted in an R-factor of 19.9% (Rfree = 24.5%) for data from 44.32 to 1.65 Å. The model has excellent stereochemistry as judged by procheck [47], with no residues in disallowed or unfavorable regions of Ramachandran space. The final model of PPWD1 comprises three polypeptide chains corresponding to the three molecules in the asymmetric unit; each chain is disordered from residues 473–482, such that each model reflects residues Gln483–Lys646. Data collection and refinement statistics are provided in Table 1. Coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with ID 2A2N.

Table 1.   Data collection, phasing and refinement statistics [atomic coordinates were deposited in the Protein Data Bank (PDB) (http://www.rcsb.org): 2A2N].
Data setPPWD1
  1. a Highest resolution shell is shown in parentheses. b Rsym = 100 × sum(|I − <I>|)/sum(<I>), where I is the observed intensity and <I> is the average intensity from multiple observations of symmetry-related reflections. c Rfree value was calculated with 5% of the data.

Space groupC 1 2 1
Unit cell (Å)
Unit cell (deg)
139.658, 39.893, 115.638
90.00, 122.33, 90.00
BeamlineRigaku FR-E
Wavelength (Å)1.54178
Resolution (Å)23.20–1.65
Unique reflections59 800
Data redundancy (-fold)3.4 (2.6)
Completeness (%)a91.4 (63.1)
I/sigI34.86 (2.0)
Rsymb0.036
Refinement
 Resolution (Å)23.20–1.65
 Reflections used56 773
 All atoms {solvent}4259 {338}
 Rwork/Rfreec0.199/0.245
 rmsd bond length (Å)0.015
 rmsd bond angle (deg)1.368
 Figure of merit0.815
 Average B-factor (Å2)46.22
Ramachandran plot
 Favored (%)94.46
 Allowed (%)5.54
 Disallowed (%)0

PPIase colorimetric activity

PPIase activity was measured using a standard protease-coupled assay [3,48] adapted to a 96-well format. The reaction mixture contained 64 pm to 1 μm of PPIase and 200 nm chymotrypsin (Sigma) in reaction buffer (35 mm Hepes, pH 7.8, 368 mm trifluoroethanol, 50 mm NaCl2, 10 mm LiCl2, 5 mmβ-mercaptoethanol). The reaction was performed at 25 °C using 33–400 μm suc-AAPF-pNA (Bachem Americas, King of Prussia, PA, USA), and read at 390 nm on a BioTek Synergy 2 plate reader using flat-bottomed well plates (Costar 3695). Initial velocities were plotted against the substrate peptide concentrations to compare the uncatalyzed chymotrypsin rate with the isomerase-catalyzed reaction.

NMR experiments

Cells were inoculated into 20 mL of modified M9 medium containing (15NH4)2SO4, trace elements and Kao & Michayluk vitamin solution (Sigma). Growth and induction were performed as above, except that cells were induced at D > ∼ 3 with 100 μm isopropyl thio-β-d-galactoside. Protein was purified as above. 15N-labeled proteins at 1 mm in 20 mm Hepes, pH 7.5, 100 mm NaCl, 10 mm dithiothreitol and 10% D2O were pre-centrifuged at 35 000 g for 10 min, and then subjected to NMR using a cryoprobed Bruker AV500 spectrometer (Bruker, Milton, Canada). All spectra were recorded at 25 °C. For 1H,15N-HSQC experiments, a pulse sequence with ‘flip-back’ water suppression was used. Typically, sweep widths of 8000 and 2000 Hz were used for the F2 and F1 dimension, respectively. The data were processed with Topspin [49] or NMRpipe [50] software.

All samples aimed at assessing PPWD1 binding and catalysis of peptides were diluted to 300 μL with 5% D2O and placed into a Shigemi microcell (Allison Park, PA, USA) in 50 mm Hepes, pH 7, 500 mm NaCl and 1 mm dithiothreitol. Samples contained 3 mm peptide and either 0.5 mm suc-AAPF-pNA (Bachem) or TQAEGPKR (Sigma-Genosys), with and without 1 mm PPWD1-Cyp. Spectra were collected at 10 °C on a Varian 600 or 900 MHz spectrometer (Palo Alto, CA, USA). Spectra were acquired using standard Varian BioPack sequences, processed using NMRpipe software [50] and visualized using ccpn software [51].

Acknowledgements

Some of the NMR instrumentation used in the current study belongs to the Ontario Center for Structural Proteomics. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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