Peptide Binding by Catalytic Domains of the Protein Disulfide Isomerase-Related Protein ERp46

Peptide Binding by Catalytic Domains of the Protein Disulfide Isomerase-Related Protein ERp46

Andreas Funkner1†, Christoph Parthier2†, Mike Schutkowski2, Johnny Zerweck3, Hauke Lilie2, Natalya Gyrych1, Gunter Fischer1, Milton T. Stubbs2 and David M. Ferrari1 1 - Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany 2 - Institute of Biochemistry and Biotechnology, Martin Luther University of Halle Wittenberg, Kurt-Mothes-Straße 3, 06120 Halle (Saale), Germany 3 - JPT Peptide Technologies GmbH, Volmerstraße 5, 12489 Berlin, Germany

Correspondence to David M. Ferrari: [email protected] http://dx.doi.org/10.1016/j.jmb.2013.01.029 Edited by I. Stagljar

Abstract The protein disulfide isomerase (PDI) family member ERp46/endoPDI/thioredoxin domain-containing protein 5 is preferentially expressed in a limited number of tissues, where it may function as a survival factor for nitrosative stress in vivo. It is involved in insulin production as well as in adiponectin signaling and interacts specifically with the redox-regulatory endoplasmic reticulum proteins endoplasmic oxidoreductin 1α (Ero1α) and peroxiredoxin-4. Here, we show that ERp46, although lacking a PDI-like redox-inactive b′-thioredoxin domain with its hydrophobic substrate binding site, is able to bind to a large pool of peptides containing aromatic and basic residues via all three of its catalytic domains (a 0, a and a′), though the a 0 domain may contain the primary binding site. ERp46, which shows relatively higher activity as a disulfide-reductase than as an oxidase/isomerase in vitro compared to PDI and ERp57, possesses chaperone activity in vivo, a property also shared by the C-terminal a′ domain. A crystal structure of the a′ domain is also presented, offering a view of possible substrate binding sites within catalytic domains of PDI proteins. © 2013 Elsevier Ltd. All rights reserved.

Introduction Members of the protein disulfide isomerase (PDI) family are predominately located within the endoplasmic reticulum (ER) where they are involved in the oxidative folding of newly synthesized membrane proteins and secretory proteins. 1,2 Nonnatively folded proteins are recognized by the ER quality control system and may be translocated out of the ER where they are targeted for proteasomal degradation. 3,4 Although there are 20 different human PDI proteins known to date, only few of them have been characterized in more detail. ERp46 [also known as endoPDI or thioredoxin (Trx) domain-containing protein 5] was first reported in two parallel studies in 2003. 5,6 It carries a C-terminal KDEL retrieval signal and is accordingly enriched in ER fractions, but it has also been detected at the plasma membrane. 6,7 ERp46, with an apparent molecular mass of 48 kDa, is expressed in various tissue types 5,6,8 but is

enhanced in liver and plasma endothelial cells 5,6 and differentially expressed in pancreatic β-cells under varying glucose concentration. 9 To date, however, the physiological role of ERp46 remains unclear. A protective effect of the protein against nitrosative stress and against hypoxia in endothelial cells has been reported 5,10 and it can substitute for the essential PDI thiol-disulfide oxidoreductase activity in yeast. 6 Indeed, an involvement of ERp46 in the folding of disulfide-containing proteins is supported by its increased expression in immunoglobulin-secreting cells as well as by its regulatory effect on insulin secretion. 8,9 Only little is known about interaction partners of ERp46 in vivo and in vitro. By using cysteinetrapping mutants, it could recently be demonstrated that ERp46 specifically interacts with several substrate proteins including ER oxidoreductin 1α (Ero1α). 11 Moreover, ERp46 shows specificity toward the Trx-peroxidase peroxiredoxin-4, 12 oxidative

0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved.

J. Mol. Biol. (2013) 425, 1340–1362

1341

Peptide Binding by PDI-Related Protein ERp46

Fig. 1. Purification and structural integrity of ERp46. (a) Purity of ERp46 variants used in this work was determined by SDS-PAGE and Coomassie Brilliant Blue staining to N 95%. (b) Analytical size-exclusion chromatography shows anomalous migration profiles for full-length ERp46 (continuous line) and a1 (broken line), but not a2 (dot/dash line) and a3 (dotted line) variants. The a2 and a3 variants migrate as expected for globular, monomeric species. (c) Far-UV CD spectra of full-length ERp46 (continuous line) and a3 (broken line) compared to that of PDI (dotted line). All proteins show spectra typical for mixed content of α-helices and β-strands. ERp46 and a3 display a minimum at 219 nm, whereas PDI has its minimum at 208 nm.

recycling of which depends on the catalytic activity of several PDI family members. 12,13 Indeed, ERp46 is a preferred reductant of oxidized peroxiredoxin-4 in vitro and appears to enhance recycling of peroxidase disulfides in vivo. 12 It was also reported that ERp46 might modulate adiponectin signaling via interactions with the adiponectin receptor AdipoR1, though it remains unclear how this should happen as the proposed binding region involves residues of the cytoplasmic domain. 7 The different members of the PDI family are composed of one or more Trx-like domains that either contain the catalytic CXXC motif and therefore are presumed to exhibit redox activity 1,14–17 or lack such a motif and hence are redox inactive. Whereas some PDI-related proteins are composed of only single Trx-like domains (ERp18, AGR2-3, TMX1-2 and TMX4-5), others contain multiple copies, ranging from two (ERp27) to five (ERp72). 15 The prototype of the PDI family, PDI itself, has four Trxlike domains aligned in the order a-b-bx′-a′ (where a denotes catalytic and b denotes non-catalytic domains). 1,14,15,17 There is still uncertainty about the requirement for the large number of PDI proteins and their variable domain compositions, but different spatial domain arrangement seems to play a role in selective substrate binding. 15 ERp46 is the only member of the PDI family that is exclusively composed of multiple, presumably catalytic Trx-like domains, with an a 0-a-a′ architecture followed by a C-terminal acidic tail. 5,6,8 Much attention has been dedicated to the substrate binding mechanism of the b′ domain of PDI that was shown to contain the major substrate binding site in the human protein. 18,19 This non-catalytic domain was reported to be essential and sufficient for binding of small peptides, whereas the catalytic a and a′ domains also contribute to the binding of larger peptides and nonnative proteins. 19 However,

there is only sparse knowledge about the exact mechanism of substrate interaction of the redoxactive domains of PDI proteins. In this study, we show that ERp46 possesses significant chaperone and peptide binding activity despite lacking a b-type Trx domain, and we investigate the individual contribution to peptide binding by each a-type domain. In addition, we present a crystal structure of the C-terminal a′ domain of ERp46, which sheds light on structural restraints for substrate binding by the catalytic domains of PDI proteins.

Results Biophysical characterization In order to analyze the secondary and tertiary structural features of full-length ERp46 and the a3 variant [a1, a2 and a3 in this work indicate recombinant variants of the first (a 0), second (a) and third (a′) ERp46 domains, respectively], we performed far-UV CD and fluorescence measurements. ERp46 displays a far-UV CD spectrum typical for a mixed α-helical and β-sheet content (Fig. 1c), closely resembling that of Trx. 20 Interestingly, the pronounced minimum at 219 nm, compared to the 208 nm minimum for PDI, suggests a higher relative content of β-sheet and lower α-helical content within ERp46. Determination of the oligomerization state PDI is known to exist as a mixture of both monomer and dimer in solution. Comparable to PDI, ERp46 shows anomalous elution behavior in size-exclusion purification. This prompted us to

1342

Peptide Binding by PDI-Related Protein ERp46

Fig. 2. Determination of the reducing and oxidizing activity of ERp46 variants. (a) The reducing activity of ERp46 (gray continuous line) and the a1, a2 and a3 variants (gray broken line, gray dotted line and gray dot/dash line, respectively) compared to PDI (black continuous line) and ERp57 (black broken line) determined by following the precipitation of the insulin B-chain at an absorbance of 650 nm. All proteins show reducing activity at 0.28 μM, higher than in a reaction mixture without PDI protein (control, black dotted line). (c) Lag-times and relative activity for the catalyzed insulin reduction assay. Highest relative reactivity per active site is displayed by PDI and lowest by ERp57, with intermediary values for ERp46 variants. The non-catalyzed reaction was too slow to allow lagtime computation. (b) Oxidizing and isomerase activity of ERp46 (gray continuous line) and of the a1, a2 and a3 variants (gray broken line, gray dotted line and gray dot/dash line, respectively) compared to PDI (black continuous line) and ERp57 (black broken line) in a reduced, denatured RNase refolding assay, determined by measuring the RNase-Acatalyzed conversion of cCMP to CMP at 296 nm. Protein concentrations were 2 μM (PDI) and 5 μM (ERp46 variants, ERp57). Full-length ERp46 and its variants show oxidizing/isomerase activity significantly above control (black dotted line, reaction mixture without PDI protein). (d) Lag-times and relative activity for the oxidative RNase refolding assay. PDI displays the highest relative reactivity, averaged per active site, while ERp46 displays the lowest. All three single domain variants of ERp46 show a higher relative reactivity than the full-length protein. Error values indicate the standard deviation from two independent experiments.

determine the monomer/dimer status of full-length ERp46. In reducing SDS-PAGE (Fig. 1a) and analytical size-exclusion chromatography (Fig. 1b), the indicated apparent molecular masses displayed by ERp46 are approximately 45 kDa and 73 kDa, respectively. The latter suggests an oligomerization factor of 1.7, which is similar to that obtained for the a1 variant. Both a2 and a3 elution profiles suggested an oligomerization factor of 1.1, indicating that both variants are exclusively monomeric in solution. The oligomerization factor of 1.7 for wild-type (WT) ERp46 and a1 is below the expected factor of 2.0 for a pure dimer and might be caused by a nonglobular shape or for a1 perhaps due to protein truncation. We then applied analytical ultracentrifugation to better determine the monomer/dimer status of ERp46 (Supplementary Fig. 1). A molecular mass of 45.8 kDa was obtained, consistent with a monomeric species. The standard sedimentation coefficient (s) was experimentally determined as 3.0S, which in comparison with the expected s-value of about 3.5S for a similarly sized globular protein indicates a more extended arrangement of ERp46 domains, suggesting an ellipsoid with axial lengths of approximately 5–10:1. Although these values are approximations since the precise hydration value of

ERp46 is not known, it is likely that the non-globular shape of ERp46 rather than protein dimerization is responsible for the anomalous elution profile by sizeexclusion chromatography. Characterization of the catalytic activity In order to characterize the catalytic activity of ERp46 and its individual domains, we used in vitro assays employing the precipitation of the B-chain of native proinsulin to determine reducing activity and the refolding of reduced and denatured RNase A to measure the oxidizing activity. Both assays measure the catalytic activity indirectly and with complex kinetics. 16 Here, for a comparison of the relative activity of the PDI proteins, we measured the reaction lag-times for both reactions. 16 It was shown previously that ERp46 is able to catalyze reduction of disulfide bonds. 8 When we compared reductase activities of the ERp46 variants with PDI and ERp57, we found the lag-time for WT ERp46 to be longer than that of PDI but shorter than that of ERp57, whereas a1, a2 and a3 variants all showed longer lag-times (Fig. 2a and c). In terms of relative reactivity (per active site), this suggests each catalytic site of ERp46 and of the single domain variants a1 and a3 to be roughly equally active to

1343

Peptide Binding by PDI-Related Protein ERp46

Fig. 3. Chaperone activity of ERp46. (a) Inhibition of aggregation of thermally denatured citrate synthase (CS) was followed by measuring light scattering at 500 nm. Both WT ERp46 (dark gray, bottom curve) and ERp46 a3 (light gray) inhibit aggregation of CS. CS without any PDI protein was used as a control (black, dotted line, top). (b) ERp46 (gray) inhibits aggregation of CS more effectively than ERp57 (black, broken line, top) and the negative control (black, dotted line) but less than PDI (black, continuous line, bottom). Different batches of CS were used for (a) and (b). (c) ERp46 decreases the susceptibility of E. coli cells toward heat shock. The number of colonies surviving on LB agar with antibiotics (equal-sized strips from LB plates laden with an equal OD of cells are shown) after treatment for 20 min or 60 min at 50 °C followed by overnight growth at 37 °C is higher for cells transformed with an ERp46 expression construct than for cells transformed with an ERp57 expression vector or vector alone (control). PDIp shows a more pronounced effect.

those of ERp57 but substantially less active than the average activity of catalytic sites in PDI. The a2 variant displayed a somewhat higher reactivity (Fig. 2c) compared to a1 and a3. To verify whether ERp46 also exhibits oxidase activity, we employed an RNase oxidative refolding assay. Here, the reaction lag-time for ERp46 is roughly similar to ERp57 but longer than PDI, whereas the lag-times for the three ERp46 single domain variants are longer. Indeed, the relative reactivity of the active sites of ERp46 is much lower than that of PDI but also significantly lower than that of ERp57 (Fig. 2b and d). The ERp46 single domain variants displayed relative (per active site) reactivity about twice that of the full-length protein, indicating that interaction of the full-length protein with larger substrates such as RNase A cannot occur unimpeded at all three active sites simultaneously. Taken together, the data indicate that ERp46 has oxidative activity but that the full-length protein might preferentially catalyze reduction of disulfides, relative to other PDI family members.

thermally induced aggregation of citrate synthase (CS) (Fig. 3). A comparison of the slope of the aggregation curves suggests that the chaperone activity of ERp46 is substantially higher than that of ERp57 but lower than that of PDI (Fig. 3). Recently, it was shown that the PDI family member PDIp can act as a chaperone independently of its catalytic activity. 22 The authors found a protective effect of the recombinant protein against heat-shockinduced cellular damage and reduced growth rate in Escherichia coli cultures. We applied this method to investigate a general chaperone activity of ERp46 in vivo, compared to PDIp and ERp57. Expression of ERp46 led to a significantly reduced susceptibility of E. coli cells to heat-shock-induced damage. This protective effect was significantly higher than observed for ERp57, which displayed nearly no effect on E. coli cells, but was less pronounced than in the case of PDIp (Fig. 3c). All recombinant proteins were expressed in roughly equal amounts, as determined by SDS-PAGE (Supplementary Fig. 2). We therefore conclude that ERp46 may possess chaperone activity in vivo and in vitro.

Characterization of the chaperone activity Analysis of the peptide binding selectivity Beside their redox activities, PDI proteins can also act as molecular chaperones. 1,21 To determine whether this also applies to ERp46, we employed the in vitro aggregation assay for citrate synthase, a protein lacking disulfide bonds. We find that both WT ERp46 and the a3 variant are able to prevent

In analogy to other chaperones, peptide binding by PDI is very weak and the Kd generally lies in the micromolar range. 21 Due to this complicating issue, identification of PDI peptide complexes and peptide binding selectivity have preferentially been

1344

Peptide Binding by PDI-Related Protein ERp46

Fig. 4. Peptide binding to dyelabeled ERp46. ERp46 was labeled with Cy5 or DyLight549 NHS-esters. As control, GST labeled with DyLight549 was used. Fluorescent signal intensity was recorded on a microchip reader and visually confirmed spots with identical peptides averaged. (a) Plot of binding signal intensities of Cy5-labeled versus DyLight549-labeled ERp46. The dotted line indicates an R 2 coefficient value of 1.0. The experimental R 2 value is 0.82. (b) Plot of binding signal intensities of DyLight549-labeled ERp46 versus GST. In comparison to ERp46, labeled GST produces significantly fewer signals.

investigated by cross-linking with radioactively labeled model peptides, for example, Δ-somatostatin, mastoparan, the glucocorticoid receptor fragment and the bovine trypsin inhibitor fragment in former studies. 19,23,24 Additionally, cross-linking to various pentapeptides were used to identify the preferred peptide binding motif of PDIp, 25 whereas surface plasmon resonance was used to investigate the peptide binding property of P5. 26 More recently, NMR titration experiments were used to analyze the interaction of PDIb′, PDIbb′ and ERp18 with model peptides. 27–29 Finally, we have previously shown that the peptide binding selectivity of the PDI family member ERp29 and its Drosophila melanogaster orthologue Wind could be deduced using immobilized peptides and a far-Western approach. 30,31 A limiting aspect of these studies is the lack of statistical significance and robustness due to the limited number of peptides that can feasibly be studied. We therefore decided to exploit recent advances in peptide microarray technology to determine the peptide binding selectivity of ERp46 using a library of several thousand peptides. Our implementation of the microarray approach uses fluorescently labeled proteins for rapid detection of interacting peptides. To verify the appropriateness of the fluorescent-labeled protein approach to detect binding to peptides, we compared binding to 9200 15-mer peptides on a microarray chip using two different dyes. We selected DyLight549 for its excellent solubility and signal stability and Cy5 for its neutrality, small size and effective application in sensitive quantification protocols such as twodimensional difference gel electrophoresis. WT ERp46 or glutathione S-transferase (GST) minimally labeled (dye label-to-protein molar ratio, 1:5) with Cy5 or DyLight549 NHS-esters was applied to peptide microarrays at 50 nM, the microarrays were washed and scanned as previously described and the resulting peptide binding pattern was compared.

Although fewer spots were detected using DyLight549 rather than Cy5 as label, DyLight549 provided 1.65-fold stronger signals (Fig. 4) and over 93% of the detected spots were confirmed with Cy5. In the inverse case, confirmation was significantly below 90%. Furthermore, only 3 peptides bound significantly to ERp46-DyLight549 and not at all to ERp46-Cy5 versus 102 for the converse case. Hence, the probability of a spot detected with a single dye label being a true ERp46 binder is higher if the dye label used is DyLight549 despite an overall lower yield in the number of binding peptides. Therefore, DyLight549 was selected for the remainder of the microarray analyses. Here, we analyzed the binding of labeled, fulllength ERp46 as well as the single domain variants a1, a2 and a3 to 3283 different peptides. Although reproducible, the weak binding signals for the single domain variants prevented any meaningful gradation of detected positive signals. However, the consistency with WT ERp46 binding patterns, low background noise and statistical significance of data enabled satisfactory classification as binder or nonbinder, allowing us to map peptide binding among the different domains. Domain involvement in peptide binding WT ERp46 bound significantly to 556 peptides, corresponding to ca 17% of the total effective pool (Table 1). Overall, 62% of the binding peptides also bound one or more of the single domain variants. Only 2 of the 63 peptides with binding rated strong or above (signal N 4000 counts, marked ****+) were not recognized by a single domain variant. Roughly 60% (334 peptides) of all WT binding peptides were also bound by the N-terminal a1 domain variant alone, whereas a2 and a3 variants bound considerably fewer at 22% and 7%, respectively (Table 2). Both a2 and a3 variants showed little to no binding of peptides not recognized by WT ERp46, and of the 51

1345

Peptide Binding by PDI-Related Protein ERp46

Table 1. Detection of binding peptides by single domain variants correlates with WT binding avidity % of peptides that also bind Avidity class *****+ **** *** ** * All classes

WT binders 4 59 166 222 105 556

WT + any variant WT + a1 WT + a2 WT + a3 100 97 83 50 34 62

100 95 82 47 31 60

100 53 27 16 9 22

100 29 4 4 2 7

The number of peptides binding to WT ERp46 within the indicated avidity class is shown, followed by the percentage thereof that is also recognized by the given single domain variants.

peptides additionally recognized by the a1 variant, 19 in fact did bind WT ERp46 but fell below the significance threshold (data not shown). Hence, the proportion of false positives for a1 would be in the range of 8–13%. Compositional analysis Overall, peptides containing Arg and to a lesser extent His (but not Lys) are enriched in the bound pools, whereas acidic residues and Met are somewhat disfavored (Fig. 5 and Tables 2 and 3). Phe, Tyr and Trp are favored, with Trp content displaying a strong variation. Indeed, although between 89% and 95% of all binding peptides carried aromatic residues, the average number per peptide varied considerably (Table 2). Tyrosines were the most enriched, from 33% in the effective full pool to up to 76% in the a3 binding pool. The roughly doubled occurrence of Trp in the WT pool

can be attributed to its binding to the a1 variant. In contrast, no enrichment of the amino acid was found in the a2 binding pool and it was largely excluded from the a3 binding pool (Fig. 5 and Table 2). However, as indicated in Table 2, the distribution of the data shows that many of these findings mainly reflect trends rather than absolute properties. It would nevertheless appear that aromatic residues are especially important for peptide binding by ERp46 and that the enrichment of Trp-containing peptides in the WT pool is mainly due to a 0 domain binding. In contrast, peptide binding to the a′ domain might be reduced in the presence of Trp-containing residues. To verify whether ERp46 recognizes a particular motif in binding peptides, we first analyzed the binding patterns for the individual domains, making use of peptides that bound to WT ERp46 and only one of the three single domain variants. Of the 221 peptides that bound to a1 alone and the 10 peptides binding to a2 alone (Tables 4 and 5 and Supplementary Tables 2 and 3), no highly conserved motif could be detected, indicating that conformation of the peptide rather than sequential juxtaposition might play a role. However, a1 binders tended to have Tyr, Phe and Trp aromatic residues spread further apart and most frequently contain an Arg residue, while a2 binders tended to have a residue with a side-chain carbonyl group (carboxyl or amide in Asp, Glu, Asn or Gln) following a hydrophobic patch of 3–4 residues containing Tyr or Phe but not Trp. No peptides that bound to a3 alone were found, but 37 peptides that also bind a1 and a2 in addition to a3 were found (Table 6 and Supplementary Table 4). Alignment of these 37 sequences shows a larger and more coherent hydrophobic stretch, almost

Table 2. Binding of ERp46 variants to microarray peptides WT (a0-a-a′)

a1 (a0)

a2 (a)

a3 (a′)

385 124 60 10 221

140 37 22 4 10

37

(3281, 100)b

556 334 100 17

(1958, 60)c (1088, 33)c (487, 15)c (1150, 35)c

91 60 29 61

89 57 31 59

90 57 14 67

95 76 3 68

(906, 27)c (665, 20)c (284, 9)c (103, 3)c

22 31 25 12

20 30 25 13

20 32 24 14

14 38 19 24

Bound, unique peptides Total bound, above cutoff Thereof WT binders Proportion of WT pool (%) Proportion of effective pool (3298 × 3 spots) (%)a Thereof with binding to a single domain only Relative composition (%) F/W/Y residues Tyr Trp Phe Average F/W/Y content (%) Single 2 3 4 or more

7 1 —

Values in parentheses refer to total unique microarray peptides. The effective pool is the total microarray pool minus damaged or unreadable spots and is determined visually. Relative composition indicates the proportion of each binding pool (or total chip content) containing at least one of the indicated residues. Average content reflects the proportions with the indicated number of occurring residues. a Total microarray pool. b Effective pool. c Total chip content.

1346

Peptide Binding by PDI-Related Protein ERp46

(a)

(b)

Binding per protein ERp46 GST DL549 Cy5 DL549 827 1283 433

Spot confirmation1 ERp46 ERp46/ GST DL549/Cy5 DL549 771 93,2% 90 10,9%

Protein Dye very weak or better

*+

weak or better

** +

679

1012

61

653

96,2%

89 13,1%

moderate or better

*** +

376

504

9

371

98,7%

28 7,4%

strong or better

**** +

157

210

1

155

98,7%

2 1,3%

very strong or better

***** +

46

57

1

45

97,8%

1 2,2%

9

8

0

9

100%

0

extremely strong or better ****** +

Dye Specific2 DL549 3

Cy5 102

0

5

0%

Fig. 5. Occurrence of aromatic and hydrophobic residues in ERp46 binding pools. (a) In the WT ERp46 binding pool, aromatic residues Phe, Tyr and Trp and the basic residue Arg are favored whereas acidic residues and Met are disfavored (red boxes), compared to the occurrence in non-binding peptides (dotted line, normalized values). (b) Peptides binding to WT ERp46 (black) or a1, a2 and a3 variants (white, consecutively a1, a2 and a3) generally contained between 1.8-fold and 2.5-fold more Phe and Tyr residues compared to the occurrence on chip (dotted line, normalized values). Trp was similarly enriched in WT and a1 pools, remained largely unchanged in the a2 pool and appeared strongly reduced in the a3 pool. In contrast, overall content of Val, Ile and Leu residues do not increase, although Val content increases in a2 and a3 binding pools. Lower panel: ranking of binding peptides in order of binding avidity. The total number of peptides detected per labeled protein within the six binding avidity classes is shown in the three numerical columns to the left. Spot confirmation 1 shows the number and proportion of the given DyLight-labeled ERp46 binding peptides that are also recognized by Cy5labeled ERp46 or DyLight-labeled GST, respectively. Dye specific 2 indicates the number of cases where peptides appeared to be recognized only by one dye-labeled protein, with no detectable signal for the second dye-labeled variant. It does not indicate whether bias favors the binding or non-binding event.

always lacking Trp residues. Interestingly, sequence analysis indicated what might be a recurring motif (hereafter referred to as the a3 binding motif): a stretch of about six residues containing one or more Phe or Tyr and other large hydrophobic residues but usually interrupted by a Lys, Arg or His residue is present in almost all binding peptides (Table 6 lists strong binders). A small, polar or hydrophobic residue (Ser, Gly, Pro or Ala) is most frequently found at the N-terminal end of the sequence. To verify whether the aromatic residues are important for WT ERp46 binding, we compared signal intensities for WT ERp46 binding to peptides with or without one or more aromatic residues. As

shown in Fig. 6, basically all peptides show an aromatic residue-dependent binding that was lost in peptides with alanine substitution. Peptide origins Peptide binding to PDI has generally been exemplified by binding to a C-terminal fragment of somatostatin (Δ-somatostatin) and mastoparan and binding of these peptides to PDI can inhibit its redoxdependent refolding activity. 28 Hence, the corresponding peptides were included on the peptide microarray. Indeed, we find significant binding of PDI to both Δ-somatostatin and mastoparan peptides but

1347

Peptide Binding by PDI-Related Protein ERp46

Table 3. Average residue frequency in ERp46 variant binding peptides WT

A D E F G H I K L M N P Q R S T V W Y D,E K,R S,T F,Y F,W,Y I,L,V I,L,V,M,A,P K,R-D,E

a1

a2

a3

Non-binders

Av

±

Av

±

Av

±

Av

±

1.01 0.51 0.62 0.89 1.06 0.53 0.65 0.95 1.08 0.14 0.56 0.65 0.70 1.13 1.64 0.84 0.88 0.38 0.86 1.13 2.07 2.48 1.75 2.13 2.61 4.41 0.94

1.09 0.75 0.83 0.87 1.05 0.70 0.78 0.93 0.96 0.38 0.78 0.73 0.82 0.87 1.26 0.84 0.92 0.66 0.86 1.16 1.18 1.40 1.15 1.23 1.43 1.59 1.78

1.08 0.43 0.55 0.89 1.08 0.55 0.68 1.08 1.00 0.14 0.50 0.64 0.67 1.30 1.56 0.76 0.90 0.42 0.85 0.98 2.38 2.32 1.74 2.16 2.58 4.45 1.40

1.13 0.73 0.80 0.89 1.10 0.70 0.80 0.99 0.92 0.38 0.78 0.73 0.81 0.88 1.24 0.78 0.95 0.69 0.90 1.16 1.16 1.29 1.22 1.31 1.42 1.63 1.78

0.97 0.45 0.64 1.09 1.15 0.56 0.87 1.13 1.06 0.11 0.39 0.60 0.57 1.02 1.31 0.73 1.36 0.14 0.94 1.09 2.15 2.05 2.02 2.16 3.29 4.97 1.06

1.07 0.67 0.77 0.94 1.31 0.69 0.80 1.00 0.91 0.39 0.65 0.65 0.69 0.97 1.14 0.84 1.01 0.35 1.00 1.13 1.20 1.24 1.26 1.25 1.33 1.59 1.91

0.70 0.49 0.43 1.16 1.51 0.43 0.73 1.08 1.05 0.05 0.27 0.73 0.73 0.78 1.27 0.59 1.68 0.03 1.35 0.92 1.86 1.86 2.51 2.54 3.46 4.95 0.95

0.91 0.65 0.65 0.96 1.71 0.65 0.77 1.06 0.85 0.23 0.65 0.69 0.77 0.85 1.22 0.76 0.85 0.16 1.09 1.01 1.00 1.38 1.33 1.35 1.24 1.41 1.60

Av 1.52 0.93 1.11 0.37 1.10 0.36 0.61 0.92 1.19 0.28 0.73 0.78 0.71 0.53 1.65 0.97 0.84 0.13 0.33 2.04 1.45 2.61 0.70 0.83 2.65 5.22 −0.60

± 1.29 0.98 1.14 0.64 1.09 0.61 0.75 0.95 1.04 0.53 0.83 0.88 0.84 0.70 1.33 1.00 0.88 0.38 0.60 1.50 1.16 1.69 0.88 0.96 1.32 1.75 1.90

Average values (Av) and standard error (±) are shown. WT, WT ERp46. The most significant changes compared to the unbound pool are in boldface.

no binding to either by GST (Fig. 6a). In contrast, ERp46 bound to Δ-somatostatin with similar avidity as PDI but did not bind the mastoparan fragment. Interestingly, Ala substitution of the most C-terminal Phe residue in Δ-somatostatin leads to loss of binding by ERp46, but not by PDI. In contrast, substitution of an alanine in mastoparan with a Phe residue did not lead to ERp46 binding and did not change PDI binding significantly. The protein origins of peptides that bound ERp46 with higher avidity than Δ-somatostatin are listed in Table 7. Of the secreted proteins, the signaling protein Notch1 is most represented whereas ERp29 is the most represented of the ER residents. In Notch1, the strongest binding peptides cover sequences containing at least two Cys in the original protein, and it is interesting to note the similarity in some of these peptides [Table 7, UIDs (Unique Identifiers) 6325, 6342 and 6346]. The UID 5945 peptide corresponds to K106–G120 of the growthfactor-like domain of APP, in which Cys117 forms a disulfide bond to Cys73. 32 The second APP-derived peptide (UID 6024) contains residues close to the extended dimer interface of the E2 domain. 33 In the case of ERp29, the binding peptides contain sequences from two regions of interest: the first is the area at the dimer interface and the reported b domain peptide binding site (Table 7, peptide UIDs

4256, 4259 and 4261) whereas the second contains residues of the region corresponding to the peptide binding site of the PDI b′ domain (peptide UIDs 4286 and 4288). Several of the binding peptides contain an a3 binding motif-like sequence (e.g., see ERp29 peptides UIDs 4256, 4259 and 4261 with sequence –SKFVLV-), and many are overlapping (Table 7 and Supplementary Table 5), further supporting binding specificity. It remains to be shown whether the listed proteins are bona fide ERp46 interaction partners, whether interactions occur with extended polypeptide regions or also with folded domains and what role the binding events might have. Crystal structure of the ERp46 a′ fragment In order to better understand the mechanism of substrate binding by ERp46, we attempted to crystallize the full-length protein (residues 33–432). From a single protein crystal obtained after approximately 4 weeks of incubation, the 2.65-Å-resolution structure of residues 323–428 of ERp46 constituting most of the C-terminal a′ domain was solved after molecular replacement (Fig. 7a). No electron density was observed for the a 0a fragment or for the Cterminal KDEL retrieval sequence. Apparently, the a′ fragment arose over time from proteolytic cleavage of human ERp46 by low levels of an unknown

1348

Peptide Binding by PDI-Related Protein ERp46

Table 4. ERp46 WT binders that are unique for the a1 variant UID 6647

R

F

I

S

L

A

K

Y

S

H

Y

Q

Y

F

S

6648 6649

R R

A A

I I

S S

L L

A A

K K

Y Y

S S

H H

Y Y

Q Q

Y Y

F A

S S

N

R

N

L

S

Y

A

N

T

I

W

K

N

S

T

Q

S

L

Q

S R

W W

K Y

Y G

F P

K

Y

Q Q

R R

H H

H H

P P

M M

S S

Y Y

W W

W W

S S

M M

G G

D

E

A

Y

A

Y

R

Y

T

N

Q A

A M

Q V

R L

K D

R V

D E

W F

Q L

W Y

Y H

G G

F A

Y Y

P P

W W

K K

P P

T T

K

G S

Y F

W Y

A R

S Y

H V K

3515 6354 3730

P

6656 6657 6661 6670 3924

T

D R

T G

W Y

Y R

4893 4894 3359 3295 4822 5015

G

V

5571 6247

S R V

K

L

I

T

H

S

I

H

V V

F A T

P

K

W

F

I I

A A

S S

P P

Y A

R R

Y Y

L

A

G

R

K

Y

H

I

S

G

Y

W

A

S

F

L

S

D

M

P E

M

D

G

Y

R

F

W

R

A R

G A

K K

R L

T R

R A

P N

P T

A T

Y W

I N

P

V

Y

Q

G A

A S

A N

R T

L A

T R

A G

L F

R I

I S

K R

K

T

L

A

N V

S

N

T

A

R

G

F

I

S

R

S

P

A

G

F

S F

E E

N S

P S

F Y

Y I

R S

S R

L S

S P

P P

A G

K F

F

L

T P

A

K

P S

P R

A K

V S

F Y

T V

R K

I V

S F

H S

Y P

R K

K

K

M

E

S F

H M

Y Y

R Y

P R

W T

I Y

N H

K I

I T

L N

R I

E G

N

K

I W

S

Q

A

S

Y

H

L

W

S

6346 6342 6639 6518

N

6642 6678 S

K

K

6325

6647

W

S

3357

A

Q

T

W

R

G

L

N

N

E

S

Aromatic residues that when exchanged to Ala lead to loss of binding to both WT and a1 are indicated in black; those not analyzed, in gray. Frequently occurring Arg residues are boxed. Peptides listed are those that bind strongly or better.

contaminating protease, an unexpected process we could verify by SDS-PAGE analysis of similarly treated protein samples (Supplementary Fig. 3) and

by limited proteolysis of the recombinant protein with trypsin (data not shown). Here, full-length ERp46 was completely cleaved to produce two major

Table 5. ERp46 WT binders that are unique for the a2 variant UID 4662 3774

A

4472

A

4775

T

R

A

F

I

F

I

D

E

S

H

L

V

G

G

I

F

F

S

D

S

E

E

I

A

A

H

E

F

A

S

L

S

K

S

Y

I

Y

L

D

T

E

P

A

I

V

K

V

Y

D

Y

Y

E

T

D

E

E

D

F

L

Y

V

D

I

K

G

P

T

V

S

P

S

K

R

E

F

R

F

L

L

D

V

S

6025

M

T

H

L

R

V

I

Y

E

R

M

N

Q

S

L

3407

T

I

Q

E

V

A

G

Y

V

L

I

A

L

N

T

V

M

H

V

6087

5341

T

Q

4360

6415

I

N

L

N

H

L

K

A

G

L

Q

A

F

F

Q

K

A

G

T

S

L

A

G

G

L

R

F

T

V

Aromatic residues that when exchanged to Ala lead to loss of binding to both WT and a2 are indicated in black; those not analyzed, in gray. A residue with a carbonyl oxygen side chain of the C-terminus (boxed) frequently follows the hydrophobic stretch containing the aromatic residue.

1349

Peptide Binding by PDI-Related Protein ERp46

Table 6. ERp46 WT binders that also bind the a3 variant UID 3361 3601

K

Y

H

I

S

A

L

Y

V

V I

D

L

K

K

F

G

D

V

K

L

T

Q

S

M

A

I

I

R

Y

N

E

T

G

L

Y

F

V

Y

S

K

V

Y

F

3986

L

Y

F

V

Y

S

K

V

Y

F

R

G

Q

S

S

3987

L

A

A

V

A

S

K

V

Y

F

R

G

Q

S

S

K

V

I

P

K

S

K

F

V

L

K

V

I

P

K

S

K

F

V

L

V

K

F

D

T

S

K

F

V

L

V

K

F

D

T

Q

Y

V

K

F

R

V

V D

T

E

3983

4256

I

T

V

T

F

Y

4259 4261 4372

K

P

V

Y

A

D

S

4471 4437

K

P

G

Q

S

A

V

H

F

Y

S

L

S

K

S

Y

I

Y

L

R

H

G

I

P

F

F

V

K

V

5198

G

R

P

Q

E

V

G

R

V

Y

I

Y

L

Q

R

5222

G

G

E

A

Q

Q

G

V

V

F

I

F

P

G

G

L

D

K

E

S

Y

P

V

F

Y

L

F

R

E

S

Y

P

V

F

Y

L

F

R

D

G

D

L

E

4286

Y

K

4288 5836

Y

Y

R

G

G

5839 5945

6638

S

H

I

G

V

Y

Y

R

G

G

H

I

G

V

Y

Y

R

G

G

A

L

L

T

P

Y

R

S

L

V

G G

E

R

T

Y

Y

V

K

T

H

T

H

I

V

I

Q

F

E

D

F

T

V

Y

L

G

L

I

G

P

L

I

V

S

R

K

S

Y

V

F

Y

V

H

R

N

A

K

P

P

A

V

F

T

6488 6517

G G

P

Y

G

S

R

Aromatic residues that when substituted with Ala lead to loss of binding to both WT and a3 are indicated in black; those not, analyzed in gray. Trp residues are excluded. A recurring sequence containing hydrophobic residues, one or more Phe or Tyr and usually interrupted by a Lys or Arg is underlined. The sequence generally begins after a small, polar residue (Ser, Gly) or a small, hydrophobic one (Pro, Ala). Peptides listed are those that bind strongly or better.

fragments of approximately 17 kDa and 33 kDa, which we believe to comprise the C-terminal a′ fragment and the N-terminal a 0a fragment, respectively. Despite further attempts, crystals of full-length ERp46 could not be obtained. The resistance of ERp46 to form crystals possibly lies in its intrinsic flexibility. The crystal of the ERp46 a′ domain belongs to the P3221 space group and contains one molecule in the asymmetric unit (Table 8). The structure could be refined to an R-factor of 0.23 and an Rfree of 0.26. Modification of the crystallization conditions did not result in crystals with improved diffraction properties. Unsurprisingly, the crystal structure of the ERp46 a′ domain shows strong similarity to catalytic domains of other PDI family members 15 and reveals a typical β-α-β-α-β-α-β-β-α Trx-like fold with five β-strands, the fourth of which runs antiparallel, surrounded by four α-helices (Fig. 7b). The loop region between α2 and β2 is disordered in the crystal as the residues 366–372 show no electron density. Strong similarity in structure was observed with an unpublished NMR structure for human ERp46 a′ [residues 322–432, Protein Data Bank (PDB) ID: 2DIZ, serving as the search model for molecular replacement in the ERp46 a′ crystal structure solution]. Interestingly,

the ERp46 a′ variant studied by NMR closely resembles the fragment generated by in situ proteolysis during ERp46 a′ crystallization. Superposition of both structures shows nearly identical positions of the secondary structure elements (Fig. 7b), with an RMSD over all atoms of approximately 1.1 Å, with most of the differences in loop regions. Typically, the Cys residues of the active site (C350 and C353) are located at the N-terminus of the long α2-helix (Fig. 7a). Both residues are clearly in the reduced state, which is confirmed in the corresponding NMR structure (PDB ID: 2DIZ). Although the protein was purified in the presence of 0.1% (v/v) 2-mercaptoethanol, no reducing agent was present during the extensive crystallization period, suggesting that the reduced state of C350 and C353 reflects the thermodynamically preferred physiological state of the molecule. Interestingly, ERp46 contains two additional, non-catalytic, surface-accessible Cys in each single domain, and in the a′ domain, these are clearly in the oxidized state (Fig. 8). Non-catalytic Cys residues, which are also found in the a domain of ERp57 and in the a′ domain of PDIr, are not believed to have a structural role since redox-active domains that lack such

1350

Peptide Binding by PDI-Related Protein ERp46

Fig. 6. Ala substitution of aromatic residues abrogates ERp46 peptide binding. (a) ERp46 binds to the classical PDI binding peptide Δ-somatostatin but not to the peptide mastoparan, which lacks aromatic residues. PDI binds both peptides as expected. For ERp46, binding is lost when the C-terminal Phe in Δ-somatostatin is replaced by Ala but is not gained by substituting Phe and Tyr into the mastoparan peptide. PDI binding is not affected by the changes. (b) Peptides that bind strongly to ERp46, and their derivatives. Top panel: representative microarray spots, with identical settings for parent and derived peptide signal detection. White spots indicate fluorescence signals and positive binding events. Black indicates no binding. Middle panel: statistical analysis of microarray data for full-length ERp46. Bottom panel: corresponding peptide sequences and their UIDs. The a3 binding motif-like sequences can be seen in several of these peptides (UIDs 3986, 3987, 4256, 4259, 4286, 4372 and 5198).

1351

Peptide Binding by PDI-Related Protein ERp46

Table 7. Protein origins of peptides binding to ERp46 and its individual domains Peptide KTHTHIVIPYRS LVG IRSQVMTHLRVIYER LHFIFRHKNPKTGVY FYVHRNAKPPAVFTR NAKPPAVFTRISHYR AVFTRISHYRPWINK QFEDFTVYLGERTYYV RIYTFHAHGVTYTKE SGLIGPLIVS RKSYV VSVASRRSPHVRVNFFG VYVWSRRSPHVRVNAAG NRNLS YANTINWKKL GHIGVYYRGGALLTS YYRGGGHIGVYYRGG TVTFYKVIPKSKFVL KVIPKSKFVLVKFDT SKFVLVKFDTQYPYG YKLDKESYPVFYLFR ESYPVFYLFRDGDLE INETGLYFVYSKVYF LYFVYSKVYFRGQSS LAAVASKVYFRGQSS GDVKLTQSMAIIRYI EGAVLDIRYGVSRIA DIRYGVSRIAYSKDF GSLFGFSVQAARPGR GRPQEVGRVYIYLQR GGEAQQGVVFIFPGG TRGYRAMVLDVEFLYH GYRGVKLADWVS LAQ KPVYKPGQSVKFRVV ADSHFRHGIPFFVKV RHASAKHVAYAVYSL AVYSLSKSYIYLDTE AVASLSKSYIYLDTE VS AVASNTARGFIS R LPFESSYIS RS PPGF SNTARGFIS RS PAGF TSENPFYRS LS PAKF FYRS LS PAKFNGLLS PWKNS TQSLQS WKYF WQSS EALRVMAKIMR GYWASHLAGRKYHIS KYHISALYVVDLKKF GFYPWKPTIASPYRY GAYPWKPTIASPARY SPYRYYFS VDRDLSV RAKLRANTTWNPVYQ RWYGPKYITHSIHNE RKAGS KNFFWKTFTS

WT **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** ***** **** **** ***** **** **** **** **** **** ******* **** **** **** ***** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** ***

GST

** *

**

*

** *

* *

a1

a2

a3

UID

Origin

Region

Type

Note

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ? ++ ++ ++ ++ ? ++ ++ ++ ++ ? ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ? ++ ++ ++ ++ ++ ++

++

5945 6024 4084 6638 6639 6640 6488 6375 6517 6746 6747 3515 5839 5836 4256 4259 4261 4286 4288 3983 3986 3987 3601 3608 3610 5083 5198 5222 3924 3882 4372 4437 4467 4471 4472 6247 6342 6325 6346 6347 6354 5565 3359 3361 4893 4894 4899 5015 3730 3694

APP APP Calnexin Chymase I Chymase I Chymase I Cpl Cpl Cpl Derlin3 Derlin3 EGFR Erlin2 Erlin2 ERp29 ERp29 ERp29 ERp29 ERp29 FasL FasL FasL GST GST GST Integrina Integrina Integrina insp3r Lysozyme mug1 mug1 mug1 mug1 mug1 Notch1 Notch1 Notch1 Notch1 Notch1 Notch1 TGFb UGGT UGGT vp1 vp1 vp1 vp1 VSVG Δ-som

K106–G120 I526–R540 L201–Y215 F104–R118 N109–R123 A114–K128 Q716–V731 R115–E129 S886–V900

P P P P P P P P P D 6744 D 6744 P P D P P P P P P P D P P P D 5082 P P P P P P P P D 4471 P P P P P P P P P P D P P P P

1 1 1 1 1 1

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ? ++ ++ ++ ++ ++

++ ++ ++

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ? ++

++ ++ ++

++

N466–L480 G29–S43 Y34–G38+ T45–L59 K50–T64 S55–G69 Y110–R124 E115–E129 I181–F195 L186–C200 G61–I75 E96–A110 D101–F115 G360–R374 G420–G434 T2379–H2394 G37–Q51 K116–V130 A326–V340 R411–L425 A421–E435 V1306–R1320 L141–F155 S1311–F1325 T1391–F1405 F1396–C1410 P1471–F1485 W436–R450 G1377–S1391 K1387–F1401 G341–Y355 S351–V365 R676–Q690 R87–E101 R101–S115

1 1 1 1 1 1 1, 5 1, 3 1, 3 2, 4 2 2 1 1 1 4 1, 3, 4 1, 3, 4 1 1 1 1 1 1 1

1 1 1 1 1

With the exception of Δ-somatostatin (Δ-som) that bound with moderate avidity, all peptides bound strongly or very strongly. Where binding to GST was observed, the avidity class is given. Significant binding to the single domain variants is indicated (++). The a3 binding motif-resembling sequence in the Δ-somatostatin peptide (UID 3694, underlined) does not enable binding to a3. The type of peptide is either parent (P) or derived (D). In the latter case, the parent UID is provided if available. S indicates Cys in original sequence. For all peptides, n ≥ 2. Cpl, ceruloplasmin; na, not available. Notes are as follows: (1) an overlapping peptide also binds (see Supplementary Table 5), (2) multimerization interface residue(s), (3) dimerization interface residue(s), (4) substrate binding site residue(s) and (5) peptide that contains sequence duplication (underlined).

disulfides still adopt the equivalent tertiary structure. Nevertheless, it has been speculated that this disulfide bond destabilizes the oxidized state of the active-site Cys. 34 The primary sequence of the ERp46 a′ domain closely resembles that of the redox-active domains

of other PDI family members (Supplementary Fig. 4). An important difference occurs in the sequentially opposite occurrence of two buried, charged residues (K344 and E378 in the a′ domain versus E47 and K81 in the PDIa domain) believed to modulate the catalytic activity of Trx. 1,35 The distance between

1352

Peptide Binding by PDI-Related Protein ERp46

Fig. 7. Domain arrangement of human ERp46 and crystal structure of the C-terminal fragment. (a) Fulllength ERp46 (UniProt ID: Q8NBS9, residues 1–432) contains a sequence of three catalytic domains (a 0, a and a′) each with a CGHC active-site motif within a Trx fold (black box). The primary sequence and secondary structure elements of residues observed in the crystal (residues 323–428) are displayed. The CGHC active-site motif at the N-terminus of α-helix 2 is underlined. (b) In the tertiary structure of the ERp46 a′ domain, five central β-strands (β1–β5) are flanked by four α-helices (α1–α4), in a βαβαβαββα fold typical for Trx-like domains. Superposition of the ERp46 a′ crystal structure (green) and the unpublished NMR structure (cyan; PDB ID: 2DIZ) shows few conformational differences, mainly restricted to the loop regions. N- and C-termini are indicated.

K344 and C353 side chains in the crystal structure is 6.7 Å, too wide for direct electrostatic interactions unless both residues were brought closer together for instance via conformational change. Conformational change in the active site of the ERp46 a′ domain Other residues that are structurally conserved between ERp46 a′ and the catalytic domains of other PDI proteins map to the vicinity of the active site. This includes residues directly adjacent to the catalytic Cys (W349, G351 and H352) as well as residues of the loop beneath the catalytic Cys (G395, Y396 and P397) in which P397 shows the typical cis-conformation (cis-proline loop). The side chain of W349 has a torsion angle of − 67° (gauche −) compared to + 53° (gauche +) in the NMR structure, resulting in a movement of approximately 8.5 Å at the indole moiety nitrogen atom (Fig. 8a). As a result, W349 is no longer in the proximity of Y396 and C350 becomes more solvent exposed (Fig. 8). Hence, W349 might act as a lid capable of adopting two extreme conformations, an “open” conformation and a “closed” conformation, as has been shown for Trx and the PDI bb′xa fragment. 36,37 This would allow control of access to the active site, especially to the N-terminal reactive Cys.

Table 8. Statistics of crystallographic data processing and structure refinement Data processing Radiation source Wavelength (Å) Resolution range (Å) Space group Cell dimensions a, b, c (Å) α, β, γ (°) Rmerge (%) Completeness (%) Multiplicity 〈I/σ(I)〉 Wilson B factor (Ų) Structure refinement No.of reflections (working set/test set) Rwork (%) Rfree (%) No. of atoms Protein Water Average B factors (Ų) Protein Water RMSD Bond lengths (Å) Bond angles (°) Ramachandran plot (%) Favored Allowed Outlier

BESSY BL14.1 0.91840 55–2.65 (2.79–2.65) P3221 63.6, 63.6, 68.7 90, 90, 120 7.7 (41.4) 98.9 (99.0) 3.7 (3.7) 12.0 (3.5) 61.2 4602/239 22.9 25.9 772 23 55.3 49.1 0.018 1.79 95.8 4.2 0

Values for the highest-resolution shell (2.79–2.65 Å) are given in parentheses. The structure has been assigned the PDB ID: 3UJI.

Peptide Binding by PDI-Related Protein ERp46

1353

Fig. 8. Conformational differences in the vicinity of the active site. (a) Superposition of the crystal structure (green) and the NMR structure (cyan) of the ERp46 a′ domain. Residues in the vicinity of the active site are shown as stick representations. Differences in side-chain conformations of His352 and Trp349 are shown. In the crystal structure alone, the indole moiety of Trp349 is flipped out and is distant from Tyr396, resulting in enhanced solvent exposure of Cys350. Arg415, the corresponding residue of which is implicated in regulating catalytic activity in the PDI a domain, forms a stable hydrogen bond with the carbonyl oxygen of the polypeptide backbone of Pro397 (broken line). A salt bridge exists between the buried residues K344 and E378. The catalytic Cys are in the reduced state, whereas the non-catalytic Cys (Cys381 and Cys388) are in the oxidized state in both the crystal and the NMR structure. (b) Trp349 may act as a flexible lid that controls access to Cys350, adopting an “open” or a “closed” conformation as in the crystal (green) or NMR structure (blue), respectively.

A putative peptide binding site, based on similarity with b and a domains We show here that ERp46 has major peptide binding activity, and the question then arises where the corresponding a-type domain binding sites might be. Analysis of peptide binding to the a′ domain of ERp46 suggests that the binding site might be close to or contain one or more acidic and polar residues in addition to hydrophobic character. However, in comparison to the a domain, the reduced number of binding peptides also suggests that the binding site may be less pronounced in the a′ domain.

Hence, in the absence of further experimental data, the localization of a candidate site can only be considered a qualified guess. The two most plausible possibilities would appear to be that (1) the a domains use a b′-domain-like binding surface or (2) peptide binding may reflect substrate binding for redox reactions at the active site itself. In regard to the first option, we first note that, in PDI, the b′ domain peptide binding site involves the residues Phe223, Ala228, Phe232, Ile284, Phe287, Phe288, Leu303 and Met307 side chains. 27 Interestingly, the corresponding surface of the ERp46 a′ domain displays a cleft with a hydrophobic base as

Fig. 9. A putative ERp46 a′ domain peptide binding site, based on that of the PDI b′ domain. Residues corresponding to the PDI b′ domain peptide binding site (a) (yellow) form a cavity in the ERp46 a′ domain (b) that is bridged by Asp333 and Lys390 via a bound water molecule (light blue). The split cavity has a hydrophobic base and is flanked by polar and charged residues (Asp, Glu in red, Lys, Arg in blue). Corresponding residues at the PDI b′ domain and ERp46 a′ domain putative peptide binding sites are listed (c).

1354

Fig. 10. A putative interaction site for peptides at the catalytic site of the a′ domain. The association of a symmetry equivalent (light blue) in the crystal structure of ERp46 a′ (green) suggests a possible interaction of protein or peptide substrates with Trp349, which inserts into a hydrophobic region of the putative substrate analogue (a and b). The interaction surface in the a′ domain (c) is primarily hydrophobic (white), surrounded by several basic residues (blue). Coordination of the symmetry equivalent D334, marked with a yellow asterisk in (c), and the activesite C350 appears to closely resemble the active-site juxtaposition of a Cys residue in, for example, ERp57 bound substrates; hence, the binding of Cys-containing peptides to ERp46 might employ similar interactions to those seen here.

well, but here, the cleft is bridged by Asp333 and Lys390 via an intervening bound water molecule (Fig. 9). The site is flanked by two further acidic residues, explaining perhaps the preference for basic peptides. The Asp333–Lys390 bridge thus obstructs a portion of the cleft and may explain the inability to

Peptide Binding by PDI-Related Protein ERp46

bind peptides with Trp. Indeed, such a bridge is unlikely in a 0 or a domains as the corresponding residues (Thr72 and Ala130 and Glu200 and Gly256, respectively) are not charged pairs. The situation appears similar for yeast PDI 38 but may involve additional residues for protein interaction as is the case in the b domain of the PDI-related protein ERp29. 30 In support of the second option, we observed crystal contacts between the two symmetry-related molecules in the crystal that may identify candidate residues. Including W349 and residues located in or near the active site (Fig. 10a and b) and the cisproline loop, the residues form a small hydrophobic patch in the neighborhood of C350, comparable to the case in catalytic domains of other PDI family members. 15 Although it is conceivable that the observed protein interactions result from crystal packing, the involvement of W349 in the interactions suggests that we might fortuitously be witness to a state resembling a substrate bound state at the active site. Indeed, a similar interaction has been suggested between heterologous Trx domain proteins and clients. 36,37 Here, W349 is inserted into a hydrophobic groove formed by F332 from α1-helix and I387 and Y391 from α3-helix of the symmetry-related molecule (Fig. 10c), and the interaction appears stabilized by additional hydrogen bonds and electrostatic interactions. Hence, W349 may function as a lid and/or a “grappling hook” as it inserts into hydrophobic areas of a substrate, perhaps bringing a substrate into register for redox reactions or as part of a chaperone:substrate binding-release cycle. A more detailed view of these interactions is presented in Supplementary Fig. 5. Whether this might reflect the principle mode of interaction with small peptides as well is unclear, as it does not account for the general decrease in acidic content in binding peptides (the requirement of the Arg residue is more likely to stabilize aromatic side chains to allow an optimal fit in the binding pocket, and Lys residues are not enriched) as the site is flanked by positively charged residues (Fig. 10c) and would essentially mean that a and b domains have diverged to create independent peptide binding sites. Both binding sites may of course exist simultaneously. It is of interest to note here that several of the a-type domain binding peptides described in this work span sequences containing Cys residues in the protein of origin, which may perhaps form transient disulfides with the active-site cysteines (Table 7).

Discussion It is currently unclear why the domain composition and the domain arrangement of the individual PDI

Peptide Binding by PDI-Related Protein ERp46

family members are so diverse. Whereas some PDI proteins contain catalytic and non-catalytic domains, others are exclusively composed of either catalytic or non-catalytic domains. 15 Much attention has been dedicated to the substrate binding mechanism of the b′ domain suggested to contain the major substrate binding site in human PDI and other PDI proteins. 18,19,22,27,39 A major breakthrough came with the elucidation of the crystal structure of full-length yeast PDI where a continuous hydrophobic patch formed mainly by the b and b′ domains was observed. 38 In addition, recent structural investigations on the b′x and bb′ fragments of human PDI identified a large hydrophobic patch in a cleft between helices α1 and α3 as the major substrate binding site. 27,28,40 However, catalytic domains may also be involved in some redoxindependent substrate binding, as suggested for the ERp57/tapasin complex and for the ERp72 a 0-a domains, 41–44 and a small hydrophobic patch at the active site including the CGHC-preceding Trp residue has been implicated in direct interactions with a binding protein. 41 We wished to determine whether redox-independent peptide binding is a conserved property of PDI Trx domains. We used ERp46 as a model protein since it is the only member of the PDI family that is exclusively composed of catalytic domains, arranged in an a 0-a-a′ architecture. 5,6 The interaction of PDI proteins with their ligands is very weak, 28 and the various methods previously employed to study such interactions are disadvantaged by relatively limited peptide pool sizes. Recently, our group succeeded in the development of a peptide microarray approach suitable for the detection and analysis of peptide binding by full-length PDI proteins and mutant variants (A.F., unpublished results). We first verified that the expressed full-length protein and single domain variants were functionally intact. Indeed, all three isolated catalytic domains of ERp46 display significant reducing and oxidizing activity, though relative to other PDI proteins, the reducing activity of ERp46 appeared higher. This is fully consistent with recent findings on the high reducing activity of full-length ERp46 on peroxiredoxin-4 in vitro. 8,12 Furthermore, we found that fulllength ERp46 displays chaperone-like activity in vivo in E. coli, rescuing heat-shocked E. coli cells in a manner shown to be redox independent for PDIp. 22 This indicated that one or more of the ERp46 domains should indeed display peptide binding activity. In agreement, the full-length protein and the expression variant for the third catalytic domain (a3) also exhibit chaperone activity in vitro, albeit less pronounced than PDI, in the suppression of aggregation of citrate synthase. Hence, we concluded that the a′ domain should harbor a peptide binding site.

1355 We then employed a peptide microarray screening method to investigate binding to the individual domains. Here, we found that ERp46 binds to a large pool of peptide substrates despite lacking a PDI b′-like domain. Indeed, a well-studied PDI b′ domain substrate, Δ-somatostatin, bound both PDI and ERp46 with roughly equal avidity, and more than 40% of binding peptides showed similar or stronger binding avidity. Notably, for about 60% of the recognized peptides, binding could be attributed to the N-terminal a 0 domain, whereas a smaller proportion appear to also interact, mainly nonexclusively, with the a or a′ domains in isolation. The main peptide binding motif is likely to be significantly influenced by peptide conformation and overall composition, with the primary requirement being the occurrence of one or more (preferentially two or more) aromatic side chains. For the a′ domain, the closest approximation to a peptide binding motif involved a group of 2–4 hydrophobic and aromatic residues following upon a Ser, Gly, Ala or Pro and usually with a Lys, Arg or His residue within or immediately juxtaposed to the hydrophobic core. However, the motif does not seem to be the major binding determinant for a 0 or a domains. In almost all cases, binding is lost upon alanine substitution of one or more of the aromatic residues. Such substitution also eliminates binding by ERp46 to a Δ-somatostatin peptide but does not affect binding by PDI, indicating possible differences in motif recognition between these proteins. This is in agreement with an earlier report that Tyr-containing pentapeptides do not compete with Δ-somatostatin for PDI binding, although they do for binding to PDIp, indicating different modes of interaction between the various proteins. Arg residues appear especially important for a 0 domain binders, whereas a domain binders may require a side-chain carbonyl oxygen shortly after the Phe- or Tyr-containing sequence. Interestingly, peptides with Trp residues appear to be enriched in the pool of a 0 binding candidates but are largely excluded from a′ binding peptides. Whether this constitutes a tryptophan-specific exclusion criterion for peptide binding to a PDI a-type domain remains to be confirmed by independent means. It is also interesting to note that both a1 and a2 variants used here include the subsequent linker regions that are slightly reminiscent of the x-region following the b′ domain in PDI. Such a region, absent at the C-terminus of a3, may cap the binding sites as in PDI and hence modulate peptide binding (L. W. Ruddock, personal communication). We can exclude overall loss of structural integrity in the ERp46 variants as a likely cause of the reduced binding to the second and third catalytic domains, as (1) all three domains display roughly equal redox properties in reduction and oxidation of substrates, (2) the a′ equivalent used in this study retains overall secondary structure typical for Trx

1356 domain containing proteins and very similar to fulllength ERp46 and (3) only the soluble, monomeric species were observed for a and a′ equivalents by size-exclusion chromatography. A presumably mild denaturing effect of the detergent Triton X-100 at 0.4% (v/v) as used for blocking of unspecific interactions to the peptide microarray cannot be excluded. Indeed, it has been reported that Triton X-100 interferes with both catalytic and chaperone activities of PDI 19,24,45 and that binding to Δsomatostatin and mastoparan is inhibited with increasing concentrations of the detergent, 19 with almost all peptide binding abolished at concentrations of 1% (v/v). 24 However, binding to Δ-somatostatin, which is known to be based on hydrophobic interactions, 16 was still observed at 0.3% (v/v) Triton X-100 as it is in this work with the comparable concentration of 0.4% (v/v). Hence, the high detergent concentration does not prevent the hydrophobic interactions between Δ-somatostatin and other peptides such as mastoparan and PDI, and therefore, a protein denaturing effect is likely to be minimal. Indeed, in separate work, we show that, despite a reduction in signal amplitude upon increase of Triton X-100 from 0.2% to 0.4% (v/v), over 90% of binding peptides for a PDI-related protein were retained (A.F., unpublished results). Therefore, Triton X-100 at 0.4% (v/v) and above its critical micelle concentration does not substantially affect the overall peptide binding selectivity of ERp46 and is unlikely to account for the preferential binding of peptides containing Trp to the a 0 domain but not to a or a′ domains. In contrast, it may account in part for the reduced binding signal intensity and, perhaps, the reduced overall binding pool sizes for the individual domains compared to the full-length protein. The broad substrate binding specificity of ERp46 is reminiscent of previously published data on the binding preference of PDIp, which includes peptides containing Tyr and Trp residues with no adjacent negative charge 25 and suggests a conserved property of Trx domains in these and other PDI-related proteins. 19,25,30,46,47 However, the different requirements for recognition of the PDI substrate Δ-somatostatin and the differences in the putative peptide binding site of ERp46 a′ and PDI b′ also suggest that peptide binding is likely to vary among PDI-related proteins and between their individual domains. This would also offer an explanation for the occurrence of multiple Trx domains in most PDI proteins. Our data also lend support to the idea that redox-independent peptide binding by both catalytic and non-catalytic Trx domains lies at the heart of substrate selection for oxidative protein folding in the ER. As many of the detected peptides could bind to more than one domain, it is possible that the higher avidity of peptide binding to the full-length ERp46 is a product of multiple possible interactions of a common peptide. However, from our work, it cannot be

Peptide Binding by PDI-Related Protein ERp46

excluded that the high detergent concentration used might result in reduced avidity relative to the fulllength protein. Hence, whether the full-length protein can better stabilize interactions by shielding peptides and peptide binding site from the environment, by offering a higher concentration of binding sites or by regulating access to the binding site as in the case of the PDI x-linker region for the b′ domain will require further study to verify. 40 Unfortunately, full-length ERp46 proved to be quite resilient toward crystallization. Nevertheless, we did obtain a single protein crystal that structure solution showed to be composed mainly of the C-terminal catalytic a′ domain. An NMR structure of the third domain of human Trx domain-containing protein 5 (PDB ID: 2DIZ) had already been deposited in the Research Collaboratory for Structural Bioinformatics PDB, but the data remain unpublished to date. A further crystal structure (PDB ID: 3UVT) was reported by Gulerez et al. shortly before submission of this manuscript. 48 All three structures show a characteristic Trx fold, with a typical βαβαβαββα sequence. Significant conformational differences in the vicinity of the active site, mainly for the side-chain conformations of W349 and H352, can be seen in the NMR structure, but both crystal structures are highly similar. Although the effect of crystal packing might lead to misinterpretation, the striking association of a symmetry-related molecule in both crystal structures with the active-site surface and the positioning of W349 into a hydrophobic groove within the symmetry-related molecule 48 is reminiscent of enzyme/ substrate complexes with several members of the Trx family including ERp57/tapasin, Trx/BASI, DsbA/ DsbB, Trx/Trx-reductase and Trx/Trx-reductase/ferredoxin, respectively. 36,41,49–51 In all these complexes, the interaction is mediated to a significant extent through residues within or immediately adjacent to the CXXC motif including the conserved Trp and residues from the cis-proline loop. 37,52,53 The conserved Trp predominantly adopts a “closed” conformation in most structures of Trx-related proteins, but the “open” conformation can be observed in the crystal structure of the Trx/Trx-reductase/ferredoxin complex (PDB ID: 2PUK). 36 Here, the corresponding Trp inserts into a hydrophobic groove in ferredoxin/Trx-reductase. 41 It is thus possible that the ERp46 a′ domain W349 residue might act as a flexible lid in that it can adopt “open” and “closed” conformations, controlling substrate binding and/or access to the N-terminal reactive Cys. It is unclear to what extent the active site might contribute to peptide binding in the a3 domain. Plausibly, peptides might bind in a manner somewhat analogous to that of the Trx domain: interacting protein complexes described above and indeed, it would seem logical that some interacting protein substrates for redox catalysis would make contact with residues other than cysteine near the active site.

1357

Peptide Binding by PDI-Related Protein ERp46

However, the ERp46 a′ domain does contain a pocket around Ile387 similar to PDI b′ domain peptide binding site, where the composition of flanking residues appears to better complement content of residues in the putative peptide binding motif. Indeed, the reduced binding surface area of the pocket in the ERp46 a′ caused by equatorial bridging by Asp333 and Lys390 might explain an apparent inability of the a′ domain to bind Trp residues. It would be interesting to learn, in future work, whether the Asp333–Lys390 bridge may be reversibly disrupted by certain clients of the fulllength ERp46 protein and whether this might affect the peptide binding spectrum of the a′ domain. In the ERp46 a′ domain, both catalytic Cys are in the reduced state and analysis by Ellman's assay suggests that this is also the case for the other catalytic domains of ERp46 (data not shown). In crystal structures of other PDI-related proteins, the active-site Cys residues may be oxidized (as in the yeast PDI a domain and ERp57 a′ domain, PDB IDs: 2B5E and 3F8U), 41 may be reduced (as in the yeast PDI a′ domain 38) or may display both redox states (as in ERp18, PDB ID: 1SEN). 29 It seems plausible that the reduced state of the catalytic Cys observed in the crystal structures of the a′ domain of ERp46 as observed in this work and by others 48 represents the major physiological state of the protein, which would agree with the observed preferred reducing activity. Interestingly, in both crystal structures, the active-site proximal Cys381–Cys388 pair forms a disulfide bond, which may play a structural role. 48 In silico simulations suggest that R120 in the a domain of PDI is able to modulate the pKa of the Cterminal catalytic Cys by moving in the vicinity of the active site. 16,54,55 Support for such a mechanism was recently provided with the crystal structure of the a domain of ERp72 where the equivalent R270 forms a salt bridge with the buried E200. 42 Although this Glu is highly conserved among most of the PDI family members, it is substituted by Lys344 in the ERp46 a′ domain. Additionally, the R270 equivalent R415 in the ERp46 a′ domain is not inserted into the hydrophobic core but is hydrogen bonded to the carbonyl oxygen of P397, similar to the case in the ERp72 a 0 domain, and entry of R415 into the active-site region would likely be impeded by unfavorable interactions with K344. The pKa value of the catalytic C353 in the ERp46 a′ domain is therefore unlikely to be modulated by the conserved Arg. It is however possible that this role is performed by the buried ε-amino group of K344 (A. K. Lappi, personal communications).

Closing remarks In summary, we were able to show for the first time experimentally that a PDI protein that is exclusively composed of multiple catalytic domains is able to bind

to a large pool of peptide substrates. Using a novel peptide microarray technique for PDI-related proteins, we determined the peptide binding selectivity of ERp46 and its individual a-type domains. Importantly, this indicates that loss of the CGHC motif during evolution as in the b′ domain is per se not necessary for peptide binding and suggests that redox-independent peptide binding may be an inherent feature of the Trx fold in PDI proteins. However, further work will be required to more precisely define the peptide binding sites and peptide binding selectivity of ERp46 and other PDI-related proteins.

Materials and Methods Bacterial expression vector constructs PDI protein expression vectors were kind gifts from L. W. Ruddock, University of Oulu. Bacterial pET23 expression vectors contained the coding sequences of mature human ERp46 (R33–L432, expressed protein WT ERp46) or that of the corresponding a 0-(R33–Q189), a-(G190–G322) and a′-(R296–A428) (expressed proteins a1, a2 and a3, respectively) domains with an N-terminal (His) 6-tag (MHHHHHHM). The expression vectors of hPDIp, hERp57 and hPDI contained the (His)6-tagged proteins but lacked a signal sequence as previously described. 54,56–58 The GST control construct was derived from the pGEX-4T-2 vector without any insert (GE Healthcare). All constructs were verified by sequencing analysis. Recombinant protein expression and purification The expression and purification of PDIp, ERp57, PDI and the a domain is described elsewhere. 54,56–58 ERp46 and its single domain variants were expressed in E. coli Rosetta (DE3) [pLysS] or BL21 (DE3) [pLysS] cells, respectively, in the presence of the required antibiotics (50 μg/ml ampicillin and 30 μg/ml chloramphenicol). GST was expressed in BL21 cells using ampicillin only. After incubation at 37 °C, protein expression was induced at OD600 nm ~ 0.4 with 0.5 mM IPTG, and expression was continued overnight at 30 °C. Recombinant proteins were harvested by sonication of lysozyme and DNase (Sigma) treated cells in 20 mM Tris–HCl (pH 8), 300 mM NaCl and 8 mM imidazole buffer with benzenesulfonyl fluoride hydrochloride protease inhibitor (AppliChem) on ice. The (His)6-tagged proteins were then purified by incubation with Ni-NTA Sepharose beads (GE Healthcare) for 2 h at 4 °C. After washing in the presence of 0.1% (v/v) Triton X100, we eluted the protein from the beads with 300 mM imidazole. After dialysis against 20 mM Tris–HCl/2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (pH 6.7–7.2) (dependent on pI value), 0.1% (v/v) βmercaptoethanol and 0.01% (v/v) Triton X-100, the proteins were further purified over a MonoQ Sepharose column (GE Healthcare) with increasing concentration of NaCl. Pooled fractions were further purified by sizeexclusion chromatography. Protein purity was consistently ≥ 95% as determined by SDS-PAGE analysis and

1358 Coomassie Brilliant Blue staining. Bovine RNase A was purchased from Sigma. Analytical size-exclusion chromatography The molecular weight of ERp46 and its individual domains was estimated by analytical size-exclusion chromatography. Proteins were applied to a Superdex 200 prep-grade HiLoad 26/60 size-exclusion column (GE Healthcare) with a geometric column volume of 318 ml and a void volume of 110 ml, at a flow rate of 1 ml/min. Column calibration was achieved with low- and high-molecular-weight calibration kits (GE Healthcare). Determination of the specific elution volume allowed calculation of the Kav, which was then plotted against the logarithm of the molecular weight to provide a calibration curve. Experimental molecular weight values were determined using the calibration curve. The oligomerization factor was calculated as the ratio of the experimental and theoretical molecular weights. Analytical ultracentrifugation Analytical ultracentrifugation was carried out with a Beckman Optima XL-A ultracentrifuge and an An-60 Ti rotor at 20 °C. The ERp46 concentration was adjusted to 0.3 mg/ml in 20 mM Tris–Cl (pH 7.4) and 150 mM NaCl. Sedimentation runs were performed at 40,000 rpm, and equilibrium measurements were performed at 12,000 rpm for 45 h. Data were collected and analyzed with the Beckman XL-A data acquisition and analysis program provided by the manufacturer. Peptide synthesis and printing of peptide microarrays Peptide microarrays were produced by JPT Peptide Technologies GmbH (Berlin, Germany). Briefly, approximately 3283 unique 13-mer to 15-mer peptides (Supplementary Table 1) derived from ER resident and secretory protein sequences were synthesized on cellulose membranes in a parallel manner using SPOT synthesis technology. For this process, Cys residues were exchanged for Ser. The peptides constitute roughly one-half of a more expansive set from 57 different proteins, details of which are as presented elsewhere (A.F., unpublished results). Following side-chain deprotection, we transferred the solid-phase bound peptides into 96-well microtiter filtration plates (Millipore, Bedford, MA, USA) and treated them with 200 μl of aqueous triethylamine [2.5% (v/v)] to cleave the peptides from the cellulose membrane. Peptidecontaining triethylamine solution was filtered off and used for quality control by liquid chromatography–mass spectrometry. Subsequently, solvent was removed by evaporation under reduced pressure. Resulting peptide derivatives (50 nmol) were re-dissolved in 25 μl of printing solution [70% dimethyl sulfoxide, 25% of 0.2 M sodium acetate (pH 4.5) and 5% glycerol; by volume] and transferred into 384-well microtiter plates. Peptide microarrays were generated by contact printing on epoxy-modified slides (Corning; Germany). All peptides and controls were deposited in triplicates for quality control of the results. Printed peptide microarrays were quenched, washed with water followed by ethanol and dried using microarray centrifuge. Resulting peptide microarrays were stored at 4 °C.

Peptide Binding by PDI-Related Protein ERp46

Peptide interaction by microarray chip analysis To analyze the interaction of ERp46 and its individual domains with different peptides displayed on peptide microarray, we labeled ERp46 variants and GST with 1:5 molar ratio of Cy5-NHS-ester (GE Healthcare) or DyLight549-NHS-ester (Thermo Scientific) for 30 min at room temperature. After quenching with 0.1 M glycine and separation from residual dye over NAP5 desalting columns (GE Healthcare) using 20 mM Hepes (pH 7.0), 150 mM NaCl, 0.4% (v/v) Triton X-100 and 1 mM DTT, we applied labeled proteins to the peptide microarrays at a final concentration of 0.1–1.0 μM and incubated them at room temperature and in the dark for 2 h. Unbound protein was removed by several washes first with incubation buffer and then with water. Peptide microarrays were dried by centrifugation and dye-labeled protein was detected on an ArrayWorkx scanner (Applied Precision, LLC) equipped with the appropriate filters. Qualitative and quantitative evaluation of peptide interactions Signal intensities were analyzed by GenePix Pro Software (Molecular Devices) using a local background calculation algorithm and statistically evaluated with Microsoft Excel VBA-based analysis programs. Spot fluorescence intensity data for each peptide were averaged across all sub-arrays. For multiple microarrays, the slope of all signals was normalized to unity. All spots were manually verified. A fluorescent signal was considered positive and significant if (1) it was reproduced on at least one additional sub-array, (2) it could be visually confirmed and (3) the averaged signal was measured above 3× the standard deviation value for noise levels after automated local background subtraction. For confirmation of peptide binding, either protein variants were labeled with alternative dyes as previously described or single domain variants similarly labeled were used for mapping. Labeling, assay, detection and quantification parameters were kept constant for all measured proteins. Importantly, the detectable signal range allows reproducible detection of multiple peptides including somatostatin and mastoparan. 27,30,31 As a doubling of signal intensity could be visually readily confirmed, signals were graded into avidity classes as follows: nonbinding or not significant (below cutoff), very weak (equal to or above cutoff but less than 1000 counts, designated *), weak (1000–2000 counts, **), moderate (2000–4000 counts, ***), strong (4000–8000 counts, ****), very strong (8000–16,000 counts, *****) and extremely strong (above 16,000 counts, ******). The cutoff value for signal significance for WT ERp46 was fairly high at 637 counts due to elevated noise but fell nevertheless into the “very weak” signal range. The same cutoff value was applied for signals obtained with labeled GST. CD and fluorescence spectroscopy To analyze the secondary and tertiary structure of ERp46 and its individual domains, we dialyzed the proteins into 50 mM Na-phosphate buffer (pH 7.4) and centrifuged them to remove slight precipitate. Far-UV CD spectra were collected between 190 and 260 nm at 20 °C with a CD

1359

Peptide Binding by PDI-Related Protein ERp46

spectropolarimeter J-710 (Jasco) using 0.20 mg/ml protein solution in 1-mm cuvettes (Hellma). Spectra were scanned 15 × at 50 nm/min and a bandwidth of 1 nm. Fluorescence spectra were collected using a Fluoromax-2 fluorescence spectrometer (Jobin Yvon, Horiba) equipped with filters for excitation (280 nm and 295 nm) and emission (305– 400 nm) and a bandwidth of 5 nm, using a protein concentration of 2–5 μM in 1-cm micro-cuvettes (Hellma). Spectra were accumulated 5 × each. Sample intensities were subtracted from buffer intensities and normalized to the protein concentration. Oxidative refolding of denatured RNase A Reduced and denatured bovine RNase A (Sigma) was used as a substrate to determine the oxidase and chaperone activities of a protein, as described elsewhere. 59 Briefly, RNase A was denatured and reduced in 100 mM Tris–Cl, 2 mM ethylenediaminetetraacetic acid (EDTA), 6 M GdnCl and 140 mM DTT at room temperature, overnight. The protein was rebuffered into 0.1% (v/v) acetic acid and then added to a final concentration of 5 μM to a solution of PDI proteins at 2– 5 μM in 100 mM Tris–Cl (pH 8.0), 2 mM EDTA, 1 mM GSH and 0.2 mM GSSG. The reaction was started by addition of the RNase A substrate cCMP to a final concentration of 4.5 mM. RNase-A-catalyzed hydrolysis of cCMP to CMP was followed spectrophotometrically at 296 nm at 25 °C. Spontaneous refolding of denatured RNase A and hydrolysis of cCMP was used as negative control. For semiquantitative evaluation, the lag-time was measured, that is, the time before an apparent increase in absorbance of 0.1 was recorded. Relative activities are obtained upon normalization of reciprocal lag-times against the molar concentration of protein active sites. 16 Reduction of natively folded insulin Reductase activity was monitored using the insulin reduction assay. 60 Briefly, insulin from a 10-mg/ml stock was diluted to 1 mg/ml in 0.1 M potassium phosphate and 2 mM EDTA (pH 6.5). For measurements, 500 μl of the insulin solution was added to 0.28 μM respective PDI protein, with samples without PDI protein serving as negative control. The reaction was started by adding 4 mM GSH in a final volume of 600 μl and followed spectrophotometrically at 650 nm. Determination of the lag-time allowed approximate quantification of the reaction. 16 Inhibition of aggregation of thermally denatured citrate synthase Inhibition of the aggregation of thermally denatured citrate synthase can be used to determine the in vitro redoxindependent chaperone activity of a PDI protein. 61 To start the reaction, we diluted citrate synthase (Sigma) at 1 mg/ml 1:100 (v/v) in pre-warmed 40 mM Hepes–KOH (pH 7.5) together with 0.15–0.25 μM PDI protein or without PDI protein as control and incubated it at 43 °C with constant stirring in 1-cm cuvettes (Hellma). Precipitation was detected by light scattering with a Fluoromax-2 fluorescence

spectrometer (Jobin Yvon, Horiba) at an emission and excitation wavelength of 500 nm and a bandwidth of 1.5 nm. Protective effect of ERp46 in E. coli cells To analyze the in vivo chaperone activity of ERp46 and other PDI proteins, we used the susceptibility of E. coli cells to heat shock, as previously described. 22 E. coli Rosetta (DE3) [pLysS] cells transfected with the corresponding vector were grown overnight on LB plates with antibiotics at 37 °C. Single colonies were transferred into 100 ml LB media with antibiotics and incubated at 37 °C until the OD600 nm reached 0.4 AU. Induction was performed with 0.5 mM IPTG and the proteins were expressed overnight at 30 °C. The cell suspension was centrifuged 20 min at 4000g and 4 °C and the cells were resuspended into ice-cold LB media containing antibiotics. The OD600 nm was adjusted to 0.2 AU. Aliquots of the cell suspension were incubated for 0 min, 20 min and 60 min at 50 °C, respectively. We plated 100 μl of several dilutions onto LB plates with antibiotics and incubated them overnight at 37 °C. E. coli cells transfected with the empty pET23 vector were used as a negative control.

Crystallization and data collection Full-length ERp46 purified by size-exclusion chromatography was dialyzed in 10 mM Hepes (pH 7.1) and 10 mM NaCl. The protein was concentrated to approximately 20 mg/ml using centrifugation filter devices (Millipore). Sitting-drop crystallization was performed in 96-well plates with different crystallization conditions. We placed 200 nl of a 1:1 (v/v) mixture of protein and crystallization buffer automatically over 110 μl reservoir solution by a pipetting robot (Cartesian). The plates were incubated at 288 K for several months. Crystal growth was detected using a crystallization plate-imaging system (Desktop Minstrel UV; Rigaku Europe). A single crystal that grew after approximately 4 weeks in 0.1 M Hepes (pH 7.5) and 20% (w/v) polyethylene glycol 10.000 (Hampton Research) was harvested, placed in cryobuffer [25% (v/v) ethylene glycol] and stored in liquid nitrogen. An initial data set of the trigonal crystal (space group P3221) was collected using an in-house X-ray source (rotating anode generator MM007; Rigaku Europe) with CCD detector (Saturn 944; Rigaku Europe), which gave a resolution of 3.1 Å. Final data collection (data set statistics given in Table 8) was performed under cryogenic conditions (100 K) at BESSY synchrotron beamline 14.1 (Helmholtz Zentrum Berlin) equipped with a fast scanning 225-mm CCD-mosaic detector (Marresearch). The recorded data set of oscillation images was processed to a resolution of 2.65 Å using the program XDS. 62

Structure solution and refinement Phases were calculated by molecular replacement using the program Phaser 63 with the solution structure of the third Trx domain of ERp46 (PDB ID: 2DIZ) as a search model. The structure was manually completed

1360

Peptide Binding by PDI-Related Protein ERp46

using the program COOT and refined using TLS and twin refinement in REFMAC5 (from the CCP4 suite, 64 as the analysis of the diffraction data with the program PHENIX.XTRIAGE 65 indicated a small fraction of merohedral twinning in the crystal). Refinement statistics are given in Table 8. Structural figures were created using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC).

Accession numbers The coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics PDB (PDB ID: 3UJ1).

Acknowledgements We thank L. W. Ruddock for the PDI protein expression vectors used in this work and for critical proofreading of this manuscript. We also thank the members of the BESSY synchrotron facility (Berlin) for measurement time and Stefan Zimmermann for valuable assistance in protein crystallization. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Ministry of Culture and Education of the State of Sachsen-Anhalt.

Supplementary Data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2013.01.029

Received 6 July 2012; Received in revised form 9 January 2013; Accepted 23 January 2013 Available online 30 January 2013

Keywords: chaperone; substrate binding; peptide microarray; X-ray crystal structure; thioredoxin fold † A.F. and C.P. contributed equally to this work. Abbreviations used: EDTA, ethylenediaminetetraacetic acid; ER, endoplasmic reticulum; GST, glutathione S-transferase; PDB, Protein Data Bank; PDI, protein disulfide isomerase; Trx, thioredoxin; WT, wild type.

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