Crystal structures of the endoplasmic reticulum -1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Grazyna Kochana,1, Tobias Krojera,1, David Harveyb, Roman Fischerc, Liye Chend, Melanie Vollmara, Frank von Delfta, Kathryn L. Kavanagha, Matthew A. Brownb,e, Paul Bownessb,d, Paul Wordsworthb, Benedikt M. Kesslerc, and Udo Oppermanna,b,2

aStructural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington OX3 7DQ, United Kingdom; bBotnar Research Center, National Institute for Health Research Oxford Biomedical Research Unit, Oxford OX3 7LD, United Kingdom; cHenry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom; dWeatherall Institute for Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; and eUniversity of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane 4102, Australia

Edited* by Marc Feldmann, Imperial College London, United Kingdom, and approved March 15, 2011 (received for review January 26, 2011)

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunc- However, based on biochemical studies a model of ERAP1 activ- tional involved in trimming of peptides to an optimal ity was suggested that involves the binding of a 9–15-mer peptide length for presentation by major histocompatibility complex to the active site of ERAP1, where successively N-terminal resi- (MHC) class I molecules. Polymorphisms in ERAP1 have been asso- dues are cleaved off until the N-terminal part of the peptide is no ciated with chronic inflammatory diseases, including ankylosing longer reaching the active site. The rate at which this process spondylitis (AS) and psoriasis, and subsequent in vitro enzyme occurs appears to be peptide sequence specific (13, 14). studies suggest distinct catalytic properties of ERAP1 variants. To Recent genome-wide association studies (GWAS) have re- understand structure-activity relationships of this enzyme we vealed numerous ERAP1 polymorphisms to be associated with determined crystal structures in open and closed states of human (AS)—a chronic inflammatory disease ERAP1, which provide the first snapshots along a catalytic path. (15, 16) strongly associated with the HLA-B27 MHC I allele. ð Þ ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H- X 18-E Interestingly, recent GWAS and replication studies (17) reveal zinc binding and G-A-M-E-N motifs characteristic for members of a clear association of the rs30187 SNP in ERAP1, encoding a 528 the gluzincin family. The structures reveal extensive K R variant, with reduced AS risk, and, importantly, demon- domain movements, including an active site closure as well as three strate a haplotype of this SNP exclusively in the context of different open conformations, thus providing insights into the HLA B27. This is suggestive of an influence of ERAP1 in the catalytic cycle. A K528R mutant strongly associated with AS in development of AS by a mechanism involving peptide trimming. GWAS studies shows significantly altered peptide processing Further, it has recently been reported that ERAP1 associations in characteristics, which are possibly related to impaired interdomain psoriasis, an inflammatory skin disease commonly associated with interactions. AS, are also strongly correlated with MHC genotype (18). To understand the mechanism of ERAP1 catalytic activity and, antigen presentation ∣ ERAP1 mechanism ∣ MHC restriction possibly, to provide a structural explanation for the altered activ- ity observed in this variant, we determined crystal structures of ndoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi- human ERAP1 (wild-type allele) in several open forms as well Efunctional enzyme involved in regulation of immune and as an inhibitor bound closed conformation. inflammatory responses. Functions attributed to ERAP1 include Results and Discussion peptide trimming for antigen presentation on MHC class I molecules (1, 2), ectodomain shedding of receptors such Structure Determination of Human ERAP1. ERAP1 crystallized in as TNFR1(3), IL-6Ralpha, and IL-1RII (decoy IL-1 receptor) two different space groups, containing either one (P622) or three 2 2 2 (4, 5), as well as regulation of through inactivation molecules (P 1 1 1) in the asymmetric unit resulting in datasets of II and conversion of to in the with resolution limits of 2.7 and 3.0 Å, respectively (data collec- kidney (6). tion and refinement statistics are given in Table S1). Each of these Major histocompatibility complex (MHC) class I molecules are four molecules was present in a distinct conformation, which we expressed on the surface of most cells and present peptide frag- postulate to represent snapshots of the enzyme during the cata- BIOCHEMISTRY ments representing the cell repertoire to cytotoxic T lym- lytic cycle. The structure of the P622 crystal form was solved by phocytes. Display of abnormal (e.g., pathogen-derived) peptides single isomorphous replacement with anomalous scattering from a mercury derivative (SIRAS), whereas the structure of the sec- may lead to the recognition and killing of infected cells. The pep- 2 2 2 tides presented by MHC I molecules are 8–9 amino acid residues ond one (P 1 1 1) was determined by molecular replacement, long and are derived from proteolytic fragments generated by the proteasome and other cytosolic prior to transport to the Author contributions: G.K., T.K., B.K., and U.O. designed research; G.K., T.K., D.H., R.F., L.C., endoplasmatic reticulum through TAP1/2 (7). Here, the final M.V., and K.L.K. performed research; G.K., T.K., R.F., F.v.D., K.L.K., M.B., P.B., P.W., B.K., and N-terminal trimming to the correct size occurs in the endoplas- U.O. analyzed data; and G.K., T.K., P.B., P.W., B.K., and U.O. wrote the paper. matic reticulum by ERAP1 (1, 8–11). Epitope peptide precursor The authors declare no conflict of interest. molecules of 9–15 amino acids in length are rapidly trimmed by *This Direct Submission article had a prearranged editor. ERAP1, while its activity is greatly reduced toward shorter pep- Freely available online through the PNAS open access option. tides. The underlying mechanism for this remains unclear but has Data deposition: The structures have been deposited in the , been suggested to be a “molecular ruler” mechanism (2). Impor- www.pdb.org (PDB ID codes 2YD0 and 3QNF). tantly, ERAP1 and MHC I molecules appear to synergize in the 1G.K. and T.K. contributed equally to this work. generation of peptide repertoires that are appropriate for each of 2To whom correspondence should be addressed. E-mail: [email protected]. the polymorphic MHC I molecules (12). Lack of structural data This article contains supporting information online at www.pnas.org/lookup/suppl/ for ERAP1 has thus far greatly hampered mechanistic studies. doi:10.1073/pnas.1101262108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1101262108 PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7745–7750 Downloaded by guest on September 30, 2021 using individual domains of the previously solved monomer as parison of domain similarities cf. Table S2). Domain I (residues search models. Native crystals of space group P622 diffracted 46–254) of ERAP1 (Fig. 1A) is composed of a central, saddle- initially beyond 2.3 Å; however, the onset of radiation damage shaped eight-stranded β-sheet (β1, 2, 4, 5, 8 and β11, 13, 14). This limited the final resolution to 2.7 Å. Nevertheless, the electron central motif is on one end flanked by a three-stranded β-sheet density map after SIRAS phasing and solvent flattening was of (β3, 6, 7) and on the opposite end by a four-stranded β-sheet exceptional quality (an exemplary region of the electron density (β9 þ 10 and β12 þ 15) that packs against the catalytic domain map is given in Fig. S1). Although the general metalloprotease and interacts with domain IV via an elongated loop connecting inhibitor bestatin (N-(3R-amino-2S-hydroxy-1-oxo-4-phenylbu- strands 9 and 10. Domain II (residues 255–529), the thermolysin- tyl)-L-leucine) was not added to any crystallization trials, we like catalytic domain, consists of an N-terminal subdomain found it bound to the active site of the first, but not the second, composed of one alpha-helix (α4) and a five-stranded β-sheet crystal form. The apparent occupancy of the molecule is low as (β16–20). The exo-peptidase specific GAMEN motif (24) serves B judged from its increased thermal motion factors ( average pro- as the outermost strand of this sheet and creates one edge of the 14 9 2 B 58 6 2 α tease domain: . Å , average bestatin: . Å ) and the patchy substrate binding cleft. Helix 6 is the linker to the alpha-helical electron density. The most probable explanation for this observa- subdomain and contributes the two histidine residues, His353 and tion is that bestatin was present in the general protease inhibitor His357, which form part of the canonical zinc-binding motif mix added during purification and was thereby bound to the (H-E-X-X-H-X18-E). Moreover, this helix also forms the bottom protein and copurified. of a central channel that runs along the surface of the catalytic domain. In contrast to thermolysin, and similar to ePepN, F3, and Overall Structure of the Closed State. In space group P622 ERAP1 is LTA4, the N-terminal substrate binding region of this cleft is present in a closed conformation; i.e., the active site of the obstructed by domain I and therefore provides a steric block, molecule is occluded from the solvent. In contrast, space group which is crucial for the exo-peptidase activity. ERAP1, like its P212121 contains three molecules in the asymmetric unit, and structural homologues, lacks the calcium-binding loop of thermo- each molecule is present in slightly different open conformations lysin on the C-terminal side of the bound substrate so that the (Fig. 1). The overall four-domain architecture of ERAP1 is re- cleft expands toward this side of the domain. The catalytic markably similar to previously solved structures of Tricorn inter- domain and the large C-terminal domain are connected by a acting factor F3 (PDB ID code 1Z5H) (19), aminopeptidase N small domain III (residues 530–614), which is composed of from Escherichia coli (ePepN, PDB ID code 2ZXG) (20, 21), two β-sheets that form a beta-sandwich (β20–26). Domain IV and Leukotriene A4 /aminopeptidase (LTA4H, PDB (residues 615–940) consists solely of alpha-helices. It displays a ID codes 1HS6 and 3B7U) (22, 23), the latter being the closest bowl-like shape and in the closed state arches over the catalytic structurally characterized eukaryotic ortholog (39% overall domain and thereby forms a large central cavity that completely homology) that does not contain Domain III (Fig. S2; for com- seals off the active site. Like in F3 and ePepN, the helices adopt

Fig. 1. Overall structure and different conformational states of human ERAP1. (A) Closed form of ERAP1. ERAP1 consists of four domains (I: green, II: orange, III: yellow, IV: teal). The first crystal form showed one molecule where the active site of domain II is shielded by domain IV. N-glycosylation sites are colored in magenta. (B) Open form of ERAP1. A second crystal form contained three molecules of ERAP1 and revealed that it is indeed a highly dynamic molecule. Domain IV of molecule 2 moves away from the protease domain (II) and permits substrate access to the active site. Note that parts of domain IV, facing toward the catalytic domain, were disordered. (C) A distance plot shows the Cα-Cα distance of equivalent residues of the closed and the three open molecules. Conforma- tional changes are mostly confined to domain III and IV. Regions that were not defined by electron density in the open molecules are blank. The insets show superpositions of the closed state (gray) and each open molecule. The different rotary motion for the individual molecules is indicated by arrows.

7746 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1101262108 Kochan et al. Downloaded by guest on September 30, 2021 the form of a superhelix with an antiparallel topology. The inter- internal hydroxyl and carboxyl groups of bestatin in the closed  3 nal cavity has an approximate volume of 2;920 Å and is there- state (Fig. 2A and Fig. S3). The terminal amino group of the in-  3 fore considerably larger than the one found in ePepN (2;200 Å ) hibitor forms hydrogen bonds to Glu183 and Glu320, which form  3 and LTA4 (1;130 Å ). Two disulfides anchor alpha-helices to one the N-terminal anchor for any peptide substrate. Bestatin binding another, one within the thermolysin domain and the other within to ERAP1 is similar to the one observed for ePepN (20) where domain IV.Electron density interpreted as carbohydrate moieties the main-chain carbonyl group of the inhibitor, besides interact- were found attached to the following N-glycosylation sites: Asn70, ing with the zinc ion, also forms a hydrogen bond to the hydroxyl Asn154, and Asn414. group of Tyr438, which in turn is involved in polarizing the carbo- nyl group during the catalytic cycle (Fig. S4). Glu354 is the equiva- 298 266 The Open States of ERAP1. The second crystal form of ERAP1 con- lent of Glu in ePepN and Glu in F3 and is responsible for tains three molecules of ERAP1 in the asymmetric unit, which all activation of a water molecule, which in turn performs a nucleo- show the molecule in different open conformations. Except for philic attack on the carbonyl carbon of the scissile bond and specific parts of the structure (discussed below) we do not observe thereby triggers the catalytic mechanism (Fig. S4). The N-term- any significant conformational changes with respect to the closed inal phenyl ring of the bestatin moiety points into the S1 speci- state; the differences are confined to en bloc movements of do- ficity pocket (Fig. 2A), where it stacks against the side chain of mains III and IV (Fig. 1C). Domain III undergoes an approxi- Phe433. The pocket is further bordered by the side chain of mately 15 ° rotation away from the core of the protein during Met319, the aliphatic part of Glu183 and the carboxamide group opening, using the region around Gly529 at the beginning of of Gln181. A previous study showed that mutating Gln181 to glu- the domain as a hinge (Fig. 1B). As a result of this movement, the tamate increased the affinity of ERAP1 for basic N-terminal C-alpha atom of Asp614, which marks the end of domain III and residues (25). The end of the pocket is formed by the aliphatic the beginning of domain IV, has shifted by around 7 Å. The part of the side chain of Arg430 and its bottom is sealed via an relative movement of domain IV is different for the three mole- electrostatic interaction of the guanidinium group of Arg430 with cules in the open crystal form (Fig. 1C). In molecule 1 domain IV the carboxylate of Glu865 of domain IV, which stacks against the swings 28 ° away from the protease domain, followed by a 12 ° imidazole ring of His160 from domain I. Further interactions of lateral rotation, which leads finally to a 10-Å shift of the center ERAP1 with bestatin are shown in Fig. S3. In summary, ERAP1 of the domain away from the core. The different rotational move- forms less of a well-defined pocket, but rather a delimited area ments for molecules 2 and 3 are highlighted in Fig. 1C. It should that may be instrumental for the protein to degrade peptides with be noted that the tips of the helices that interact with domain I different N termini (20). and II in the closed state show different degrees of disorder as they are not stabilized by crystal contacts, whereas the part of ERAP1 Is Inactive in the Open Form. The region consisting of resi- domain IV that interacts with domain III is ordered. Hence it dues 425–434 of the catalytic domain forms part of the sidewall of is domain III that acts as a lever and pulls domain IV away from the S1 pocket and is wedged between domains I and IV in the the active site and thereby makes it accessible to substrates. The closed state. But as domain IV moves away in the open forms observed flexibility of the C-terminal domain is a common theme of ERAP1, these residues become dynamic and are no longer among similar like ePepN, LTA4H, and F3. To our defined by electron density. Additionally, Tyr438, which forms knowledge, ERAP1 is so far the only one where both extremes a hydrogen bond to the carbonyl group of bestatin in the closed —i.e., either open or completely closed states—have been shown. form via its hydroxyl group, is liberated and the side chain under- APN was reported in closed conformations, whereas F3 was goes a 6-Å movement and now points away from the active site reported in different open states that were postulated to be (Fig. 2B). Because the homologous residue in ePepN is involved inactive (see below). in stabilizing the tetrahedral transition state during proteolysis Interestingly, crystals of ERAP1 in the open state were (20), this argues for the inactivity of the open states. The observed obtained by cocrystallizing the protein in the presence of a sub- conformation of Tyr438 is similar to the one observed in F3, which strate peptide. However, there is no evidence for a peptide bound was also postulated to be inactive (19). Based on these observa- to the protein, and there is also no evidence for the presence of a tions we postulate that the binding affinity of bestatin or short bestatin molecule, which was found in the active site of the closed peptides must be low in the open state due to lack of a structured conformation. The functional implications for this observation binding pocket. The initial binding energy for longer peptides will be discussed below. could be conferred by a secondary binding site, presumably located within the internal cavity. However, we cannot exclude The Active Site of the Closed State. ERAP1 belongs to the gluzincin an induced-fit mechanism where an interaction of the phenyl ring family of metalloproteases and shares the common H-E-X-X-H- of the bestatin moiety with the side chain of Phe433 helps to X18-E zinc-binding motif (24). The catalytic zinc ion is coordi- stabilize the closed conformation. The presence of bestatin may nated by His353, His357, Glu376 (Fig. 2), and additionally by the therefore be due to altered affinities of the inhibitor in the two BIOCHEMISTRY

Fig. 2. Active site and internal cavity of ERAP1. (A) Bestatin binding to the active site of ERAP1. Bestatin is colored in orange and the surrounding electron density is an Fo-Fc map calculated in the absence of the molecule and drawn at 3.0σ.(B) Superposition of open (gray) and closed (colored) state of ERAP1. The movement of Tyr438 is highlighted and Phe433, which forms part of the S1 specificity pocket in the active protease is disordered in the open state. (C) Putative C-terminal substrate binding site within the internal cavity of ERAP1.

Kochan et al. PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7747 Downloaded by guest on September 30, 2021 different crystallization conditions, which differ significantly in encompass at least eight or nine residues to reach the active site, pH and composition. Recently, Thunnissen et al. proposed such depending on the primary structure of the peptide. Longer pep- a mechanism for the yeast ortholog of LTA4H (26), although the tides can easily be accommodated due to the large volume of the described structural rearrangements do not apply to ERAP1 and internal cavity. Though our modeling is tentative and the specific the resulting domain movements are very different for the two interactions may depend on the nature of a certain substrate, such proteins. Either way, the presence of an additional site of inter- a model would explain substrate binding in case of a disordered action would favor longer peptide substrates by an increase in the active site. Such a mechanism would also explain the marginal overall binding energy and/or by increasing the local concentra- cleavage of shorter peptides, although it has been shown that tion of free N termini, thus increasing their binding propensity ERAP1 is able to hydrolyse synthetic substrates (25), albeit with into the active site. Taken together, ERAP1 is devoid of protease greatly reduced affinity. activity in the open state due to a nonfunctional proteolytic site, It is instrumental for proteolysis to take place that domain IV and its closure is mandatory for proteolysis to take place. swings back over the catalytic domain and in doing so activates the protease. After the N-terminal residue is cleaved, the cavity Structural Evidence for a Molecular Ruler Mechanism. It has been must open in order to release the products, which can then bind shown that ERAP1 has a strong preference for peptide substrates for another round of proteolysis. This is supported by occurrence that are 9–16 residues long and that those substrates are degraded of peptide intermediates when monitoring the shortening of in a nonprocessive manner (2). Moreover, it was postulated that ERAP substrates (see below, Fig. 3, and ref. 2). The “molecular ERAP1 functions as a molecular ruler because cleavage efficacy ruler” model introduced earlier (2) is fully supported by our struc- is significantly reduced for peptides shorter than eight residues. tural data: The non-processive nature of peptide trimming is a The open conformations of ERAP1 revealed that the S1 pocket is result of N- and C-terminal anchoring, requiring active site clo- not properly formed in the closed state, resulting most likely in sure, and subsequent release of the N-terminally cleaved amino reduced substrate binding affinities. In the absence of a cocrystal acid residue. For a new round of cleavage to occur, the trimmed structure of ERAP1 with a peptide, it is speculated that addi- peptide needs also to be released to allow another round of pro- tional binding energy is conferred by a secondary binding site ductive binding due to the necessity to attach the free amino- within the internal cavity where the C-terminal part of the sub- terminus at the S1 binding site. As shown in Fig. 3, all trimming strate is bound and hence defines the minimal product length. We intermediate products can be observed with different maxima at identified a putative C-terminal binding pocket at the intersection successive time points. That observation can only be explained by of helices α21, 31, and 35 of domain IV. We modeled a pheny- a bind-cleave-release mechanism in which only one N-terminal lalanine residue into the pocket and a tripeptide into the active residue is removed at the time before the substrate peptide is site in order to illustrate a possible binding mode of the C termi- released, thus supporting previous data (2). Furthermore, our nus and the resulting length constraints on a bound peptide data suggests that the 8-mer is still able to bind to ERAP1 but (Fig. 2C). The distance of the C-terminal Cα-atom to the Cα is not further processed and acts as a competitor for the 9-mer. of the P2′ residue of the modeled AAR-peptide is approximately The simple model of protease activation by cavity closure pro- 15 Å. This distance corresponds to about four residues and vides a straightforward explanation why peptides above a certain together with the residues bound to the active site adds up to size limit cannot be degraded because they prevent domain IV a 7 mer. However, such an extended peptide conformation is from sealing the proteolytic chamber, hence preventing the unlikely, and we therefore assume that the peptide needs to necessary activation events to happen. For peptides below this

Fig. 3. Altered N-terminal peptide trimming by the AS associated ERAP1 K528R mutant. (A) Extracted ion chromatograms (EIC) of the peptide cleavage product TANRELIQQEL after trimming of QITANRELIQQEL by ERAP1 wild type and the K528R mutant. Peptide precursor and were incubated for different sampling times revealing differential kinetic activity between both variants. (B) Relative quantitation of the EICs for the substrate peptide QITANRELIQQEL shows higher activity of the ERAP1 wild type. The intermediate product TANRELIQQEL is further processed by ERAP1 wild type while the K528R variant stops processing this peptide at the 11-mer stage. (C) Relative quantitation of the EICs for all observed peptide intermediates. While the wild type reaches equilibrium at the 8- and 9-mer, the K528R variant stops processing the 11-mer.

7748 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1101262108 Kochan et al. Downloaded by guest on September 30, 2021 size limit, successful cleavage will depend on whether they can (Roche) per 2 L of solution. After 4 hr of protein adsorption on the reach the active site from the point in the internal cavity where Ni-NTA resin, the suspension was loaded on a gravity column and washed they are anchored. with 20 bed volumes of washing buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol and 10 mM imidazole). The protein was eluted with elution buf- fer containing 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol and 250 mM The AS Protective ERAP1 Variant K528R Suggests a Defect in Open- 528 imidazole. Fractions containing protein were combined and applied to a Close Transitions. An ERAP1 variant (K R) associated with low- Superdex 200 16∕60 (GE Healthcare) gel-filtration column equilibrated in er risk of AS was purified and tested in N-terminal trimming 10 mM HEPES (pH 7.5), 500 mM NaCl, 5% glycerol and 1 mM TCEP. Fractions experiments using a peptide precursor containing the HLA- containing ERAP1 were analyzed by SDS/PAGE. The purified protein was con- B27 restricted Chlamydia derived epitope NRELIQQEL (27). centrated to 17 mg∕ml and used for crystallization or activity assays. A precursor peptide with a four amino acid N-terminal extension 528 was subjected to digestion with wild-type ERAP1 and the K R Crystallization. All crystals of ERAP1 were grown by using the sitting drop va- variant in a time-dependent fashion, and the product peptide por diffusion method. Crystals of the closed form were obtained by mixing fragments were analyzed by quantitative mass spectrometry 100 nl of protein solution (17 mg∕ml) and 50 nl of a precipitant consisting of 1.4 M KCitrate; 0.1 M Cacodylate pH 5.7 and 0.17 mM n-Dodecyl-Beta-D-mal- (Fig. 3). Wild-type ERAP1 protein was able to degrade the 2∶1 13-mer peptide immediately in a manner that the starting peptide toside. The open form of ERAP1 was crystallized by mixing ratio of pro- tein and buffer consisting of 0.1 M Tris pH 8.0 and 20% PEG3350. Before substrate was barely detected within a time frame of 30 min. 528 crystallization the protein was mixed with a peptide WRVYEKCALK (molar However, the ERAP1 K R variant was less efficient in peptide ratio 1∶10). A solution containing mother liquor, supplemented with 20% processing suggesting a defect in the catalytic process. As with ethylene glycol was added to both crystal forms before they were flash fro- 528 other reported polymorphisms, the Lys residue is distant to the zen in liquid nitrogen. A mercury derivative of the closed crystal form was active site (Fig. 4A), located on the surface of domain III, where it prepared by adding 1 μl of reservoir solution supplemented with 10 mM forms polar interactions in the closed state with surrounding Thiomersal to a drop containing native crystals of space group P622. After residues Asn414, Ser415, and Ser416 (Fig. 4B) from the catalytic 10 min, a crystal was transferred to a cryo solution containing reservoir domain which are further stabilized by Arg906, Gln910 and Glu913 solution supplemented with 25% ethylene glycol and the crystal was from domain IV. In the open conformation, Lys528 interacts only immediately flash frozen in liquid nitrogen. with Asn414 and Ser416 (Fig. 4C). It is possible that substitution of Data Collection and Structure Solution. Data on native and derivative crystals Lys with Arg at this position results in additional formation of were collected on beamline I03 at the Diamond Light Source. All datasets hydrogen bonds or polar interactions with surrounding amino were integrated with XDS (29) and scaled with SCALA (30). One mercury site acids, or through affecting interactions due to increased side- was identified with the program HYSS (31) followed by phase refinement chain size. Both perturbations could result in defective transitions with SHARP (32) and solvent flattening with SOLOMON (33). Automated from the open to the closed form, thus affecting catalysis. model building was performed with ARP/wARP (34), resulting in a more than The differential activity patterns observed for the reported 85% complete model. After several rounds of manual rebuilding in COOT ERAP1 variants (28), ranging from no obvious change in activity (35) and subsequent cycles of refinement with REFMAC (36), the model of as determined in the Chlamydia peptide assay to severe proces- the closed form converged to a final Rfactor and Rfree of 15.5% and sing defects as observed with the K528R variant (Fig. 3), supports 21.5%, respectively. The structure of the open crystal form was determined by molecular replacement using the program PHASER (37). Individual the hypothesis that reduced trimming of peptides and consequent domains of the previously solved closed state were used as search models. altered antigen presentation on MHC class I molecules is a Alternating rounds of refinement with REFMAC and rebuilding with COOT mechanism involved in AS development. These structure-activity gave a final Rfactor and Rfree of 22.9% and 28.3%, respectively. The program relationships and association to disease are currently being VOIDOO (38) was used to calculate cavity volumes and the program LSQMAN further investigated in our laboratories. (39) was used to generate distance plots.

Materials and Methods Peptide Trimming Assays. 100 nmol of the Chlamydia (protein accession no. Cloning and Purification of Human ERAP1. Full-length recombinant ERAP11–941 C4PLH3) derived peptide 110–122 QITANRELIQQEL containing the HLA-B27- (MGC collection) was amplified and cloned into the FastBac vector containing restricted epitope NRELIQQEL were incubated with 3.5 μg of wild-type ERAP1 a tobacco etch virus (TEV) protease cleavable C-teminal 10x-histidine tag. or the AS protective variant K528R in 1ml of 50 mM Tris pH 7.8 containing Mutants were produced using Quick Change Mutagenesis kit from Strata- 0.5 μg protease free bovine serum albumin (Sigma) in a time course experi- . Generation of recombinant baculoviruses, insect cell culture, and ment at 30 °C. The reaction was stopped by adding formic acid (1% final con- infections were performed according to the manufacturer instructions centration) and the samples kept at −80 °C until analysis. Samples were (Invitrogen). The cultures were collected 120 h postinfection, and cells were desalted using C18 Zip-tip (Millipore) and analyzed by nano-LC-MS using a removed by centrifugation, the supernatant was used as a source of protein. Chipcube coupled to an Agilent 6520 quadrupole time-of-flight (Q-Tof) tan- Supernatants were supplemented with Tris buffer pH 8.0 to a final concen- dem mass spectrometer (Agilent). Peptide separation was performed using a

tration of 50 mM, NaCl to a final concentration of 300 mM, and NiSO4 to a 10 min gradient from 3% acetonitrile, 0.1% formic acid in H2O to 45% acet- final concentration of 1 mM. This solution was supplemented with PMSF to a onitrile, 0.1% formic acid in H2O at a flow rate of 600 nl∕ min. Data was ac- BIOCHEMISTRY final concentration of 1 mM and 1 tablet of EDTA-free protease inhibitors quired in MS only mode. Quantitative data was obtained by the generation

Fig. 4. Polymorphism associated with ankylosing spondylitis. (A) Surface representation of ERAP1 Mutations associated to AS are colored in red. Localization of Met349Val in the substrate pocket is only visible in the open conformation. (B and C) Interactions of Lys528 with surrounding amino acids in the closed state (B) and the open state (C). The side chain of Asn414 was disordered in the open state, as were interactions with domain IV. Parts of the surface carbohydrate moiety (NAG1414) attached to Asn414 is depicted in the closed form (B).

Kochan et al. PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7749 Downloaded by guest on September 30, 2021 and integration of Extracted Ion Chromatograms (EIC) for the doubly Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut charged ions of each peptide intermediate using the Masshunter Qualitative and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Analysis Software (Agilent). As a normalization, the areas of all EICs observed Ministry for Research and Innovation, Merck and Co., Inc., the Novartis in each time point were added together, and the signal of each peptide Research Foundation, the Swedish Agency for Innovation Systems, the intermediate was calculated as a percentage of the sum of all EICs. Swedish Foundation for Strategic Research, and the Wellcome Trust. M.A.B. is funded by a National Health and Medical Research Council (Australia) Principal Research Fellowship. This study was also funded, in part, Note. During typesetting of this manuscript a bestatin-bound open structure by the Arthritis Research UK (grants 19536, 18599, and 18797), by the Well- of ERAP1 was disclosed (40), supporting the conclusions described in the cur- come Trust (grant 076113), and by the National Institute for Health rent article. Research Oxford Comprehensive Biomedical Research Centre ankylosing spondylitis chronic disease cohort (theme code A91202). D.H. was in part ACKNOWLEDGMENTS. We thank the staff at Diamond Light Source for expert funded by the National Ankylosing Spondylitis Society (UK), R.F., P.B., and help at the beamline. The Structural Genomics Consortium is a registered B.M.K. are supported by an Action Medical Research Grant (charity no. charity (no. 1097737) funded by the Canadian Institutes for Health Research, 208701 and SC039284). The study was supported by the Oxford National the Canadian Foundation for Innovation, Genome Canada through the Institute for Health Research Biomedical Research Unit.

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7750 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1101262108 Kochan et al. Downloaded by guest on September 30, 2021