Human type 1 tumor necrosis factor receptor shedding aminopeptidase regulator isoform a

Target ID:

ERAP1A

Aliases:

A-LAPALAPAPPILSARTS-1ARTS1ERAAPERAAP1ERAP1KIAA0525PILS-APPILSAP

Deposition Date:

Tuesday 8th February 2011

Families:

Miscellaneous

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

3QNF

Authors:

M.VOLLMAR,G.KOCHAN,T.KROJER,D.HARVEY,A.CHAIKUAD,C.ALLERSTON,J.R.C.MUNIZ,J.RAYNOR,E.UGOCHUKWU,G.BERRIDGE,B.P.WORDSWORTH,F.VON DELFT,C.BOUNTRA,C.H.ARROWSMITH,J.WEIGELT,A.EDWARDS,K.KAVANAGH,U.OPPERMANN

Site:

Oxford

3QNF

tabs

Structure Details

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1; ERAAP) belongs to the M1 subfamily of zinc-dependent metalopeptidases. This protein is a multifunctional aminopeptidase involved in regulation of innate immune responses and inflammation. Its best-characterised function is the final trimming of peptides for MHC class I presentation. ERAP1 exerts its activity towards peptides between 20-8 aa, with preference for peptides containing hydrophobic carboxy-terminal aminoacids, particularly leucine (Saric et al., 2002; Chang et al., 2005), playing a central role in immunodominance (York et al., 2006). ERAP1 has been reported to bind to cytokine receptors at the cell surface such as IFNR1 (Cui et al., 2002), IL6R1 (Cui et al., 2003) and IL1RII (decoy IL1 receptor) (Cui et al., 2003), promoting their ectodomain shedding. ERAP1 is additionally involved in regulating blood pressure through the inactivation of angiotensin II and conversion of kallidin to bradikinin in the kidney (Tsujimoto M and Hattori A., 2005). ERAP1 has also been reported to play other roles such as in angionesis (Miyashita et al., 2003), and several polymorphisms are associated to diseases with an autoimmune aetiology such as ankylosing spondylitis and inflammatory arthritis (Brown M., 2009; Harvey et al., 2009). Additionally, it has also been shown that ERAP1/2 imbalance and downregulation may favour tumour escape by avoiding immunological detection of transformed cancerous cells (Fruci et al., 2008).

Here we present the structure of full-length ERAP1 in the unliganded open conformation at 3.0Å resolution (3QNF).

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: BC030775

SGC Construct ID: ERAP1A-c200

GenBank GI number: gi|94818901

Vector: Vector: pFB-CT10HF-LIC. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CTTAAGAAGGAGATATACTATGGTGT
TTCTGCCCCTCAAATGGTCCCTTGCA
ACCATGTCATTTCTACTTTCCTCACT
GTTGGCTCTCTTAACTGTGTCCACTC
CTTCATGGTGTCAGAGCACTGAAGCA
TCTCCAAAACGTAGTGATGGGACACC
ATTTCCTTGGAATAAAATACGACTTC
CTGAGTACGTCATCCCAGTTCATTAT
GATCTCTTGATCCATGCAAACCTTAC
CACGCTGACCTTCTGGGGAACCACGA
AAGTAGAAATCACAGCCAGTCAGCCC
ACCAGCACCATCATCCTGCATAGTCA
CCACCTGCAGATATCTAGGGCCACCC
TCAGGAAGGGAGCTGGAGAGAGGCTA
TCGGAAGAACCCCTGCAGGTCCTGGA
ACACCCCCGTCAGGAGCAAATTGCAC
TGCTGGCTCCCGAGCCCCTCCTTGTC
GGGCTCCCGTACACAGTTGTCATTCA
CTATGCTGGCAATCTTTCGGAGACTT
TCCACGGATTTTACAAAAGCACCTAC
AGAACCAAGGAAGGGGAACTGAGGAT
ACTAGCATCAACACAATTTGAACCCA
CTGCAGCTAGAATGGCCTTTCCCTGC
TTTGATGAACCTGCCTTCAAAGCAAG
TTTCTCAATCAAAATTAGAAGAGAGC
CAAGGCACCTAGCCATCTCCAATATG
CCATTGGTGAAATCTGTGACTGTTGC
TGAAGGACTCATAGAAGACCATTTTG
ATGTCACTGTGAAGATGAGCACCTAT
CTGGTGGCCTTCATCATTTCAGATTT
TGAGTCTGTCAGCAAGATAACCAAGA
GTGGAGTCAAGGTTTCTGTTTATGCT
GTGCCAGACAAGATAAATCAAGCAGA
TTATGCACTGGATGCTGCGGTGACTC
TTCTAGAATTTTATGAGGATTATTTC
AGCATACCGTATCCCCTACCCAAACA
AGATCTTGCTGCTATTCCCGACTTTC
AGTCTGGTGCTATGGAAAACTGGGGA
CTGACAACATATAGAGAATCTGCTCT
GTTGTTTGATGCAGAAAAGTCTTCTG
CATCAAGTAAGCTTGGCATCACAATG
ACTGTGGCCCATGAACTGGCTCACCA
GTGGTTTGGGAACCTGGTCACTATGG
AATGGTGGAATGATCTTTGGCTAAAT
GAAGGATTTGCCAAATTTATGGAGTT
TGTGTCTGTCAGTGTGACCCATCCTG
AACTGAAAGTTGGAGATTATTTCTTT
GGCAAATGTTTTGACGCAATGGAGGT
AGATGCTTTAAATTCCTCACACCCTG
TGTCTACACCTGTGGAAAATCCTGCT
CAGATCCGGGAGATGTTTGATGATGT
TTCTTATGATAAGGGAGCTTGTATTC
TGAATATGCTAAGGGAGTATCTTAGT
GCTGACGCATTTAAAAGTGGTATTGT
ACAGTATCTCCAGAAGCATAGCTATA
AAAATACAAAAAACGAGGACCTGTGG
GATAGTATGGCAAGTATTTGCCCTAC
AGATGGTGTAAAAGGGATGGATGGCT
TTTGCTCTAGAAGTCAACATTCATCT
TCATCCTCACATTGGCATCAGGAAGG
GGTGGATGTGAAAACCATGATGAACA
CTTGGACACTGCAGAAGGGTTTTCCC
CTAATAACCATCACAGTGAGGGGGAG
GAATGTACACATGAAGCAAGAGCACT
ACATGAAGGGCTCTGACGGCGCCCCG
GACACTGGGTACCTGTGGCATGTTCC
ATTGACATTCATCACCAGCAAATCCG
ACATGGTCCATCGATTTTTGCTAAAA
ACAAAAACAGATGTGCTCATCCTCCC
AGAAGAGGTGGAATGGATCAAATTTA
ATGTGGGCATGAATGGCTATTACATT
GTGCATTACGAGGATGATGGATGGGA
CTCTTTGACTGGCCTTTTAAAAGGAA
CACACACAGCAGTCAGCAGTAATGAT
CGGGCGAGTCTCATTAACAATGCATT
TCAGCTCGTCAGCATTGGGAAGCTGT
CCATTGAAAAGGCCTTGGATTTATCC
CTGTACTTGAAACATGAAACTGAAAT
TATGCCCGTGTTTCAAGGTTTGAATG
AGCTGATTCCTATGTATAAGTTAATG
GAGAAAAGAGATATGAATGAAGTGGA
AACTCAATTCAAGGCCTTCCTCATCA
GGCTGCTAAGGGACCTCATTGATAAG
CAGACATGGACAGACGAGGGCTCAGT
CTCAGAGCGAATGCTGCGGAGTCAAC
TACTACTCCTCGCCTGTGTGCACAAC
TATCAGCCGTGCGTACAGAGGGCAGA
AGGCTATTTCAGAAAGTGGAAGGAAT
CCAATGGAAACTTGAGCCTGCCTGTC
GACGTGACCTTGGCAGTGTTTGCTGT
GGGGGCCCAGAGCACAGAAGGCTGGG
ATTTTCTTTATAGTAAATATCAGTTT
TCTTTGTCCAGTACTGAGAAAAGCCA
AATTGAATTTGCCCTCTGCAGAACCC
AAAATAAGGAAAAGCTTCAATGGCTA
CTAGATGAAAGCTTTAAGGGAGATAA
AATAAAAACTCAGGAGTTTCCACAAA
TTCTTACACTCATTGGCAGGAACCCA
GTAGGATACCCACTGGCCTGGCAATT
TCTGAGGAAAAACTGGAACAAACTTG
TACAAAAGTTTGAACTTGGCTCATCT
TCCATAGCCCACATGGTAATGGGTAC
AACAAATCAATTCTCCACAAGAACAC
GGCTTGAAGAGGTAAAAGGATTCTTC
AGCTCTTTGAAAGAAAATGGTTCTCA
GCTCCGTTGTGTCCAACAGACAATTG
AAACCATTGAAGAAAACATCGGTTGG
ATGGATAAGAATTTTGATAAAATCAG
AGTGTGGCTGCAAAGTGAAAAGCTTG
AACGTATGGCAGAGAACCTCTACTTC
CAATCGCACCATCATCACCATCACCA
TCACCACCATGATTACAAGGATGACG
ACGATAAGTGAGGATCC

Final protein sequence (Tag sequence in lowercase):
MVFLPLKWSLATMSFLLSSLLALLTV
STPSWCQSTEASPKRSDGTPFPWNKI
RLPEYVIPVHYDLLIHANLTTLTFWG
TTKVEITASQPTSTIILHSHHLQISR
ATLRKGAGERLSEEPLQVLEHPRQEQ
IALLAPEPLLVGLPYTVVIHYAGNLS
ETFHGFYKSTYRTKEGELRILASTQF
EPTAARMAFPCFDEPAFKASFSIKIR
REPRHLAISNMPLVKSVTVAEGLIED
HFDVTVKMSTYLVAFIISDFESVSKI
TKSGVKVSVYAVPDKINQADYALDAA
VTLLEFYEDYFSIPYPLPKQDLAAIP
DFQSGAMENWGLTTYRESALLFDAEK
SSASSKLGITMTVAHELAHQWFGNLV
TMEWWNDLWLNEGFAKFMEFVSVSVT
HPELKVGDYFFGKCFDAMEVDALNSS
HPVSTPVENPAQIREMFDDVSYDKGA
CILNMLREYLSADAFKSGIVQYLQKH
SYKNTKNEDLWDSMASICPTDGVKGM
DGFCSRSQHSSSSSHWHQEGVDVKTM
MNTWTLQKGFPLITITVRGRNVHMKQ
EHYMKGSDGAPDTGYLWHVPLTFITS
KSDMVHRFLLKTKTDVLILPEEVEWI
KFNVGMNGYYIVHYEDDGWDSLTGLL
KGTHTAVSSNDRASLINNAFQLVSIG
KLSIEKALDLSLYLKHETEIMPVFQG
LNELIPMYKLMEKRDMNEVETQFKAF
LIRLLRDLIDKQTWTDEGSVSERMLR
SQLLLLACVHNYQPCVQRAEGYFRKW
KESNGNLSLPVDVTLAVFAVGAQSTE
GWDFLYSKYQFSLSSTEKSQIEFALC
RTQNKEKLQWLLDESFKGDKIKTQEF
PQILTLIGRNPVGYPLAWQFLRKNWN
KLVQKFELGSSSIAHMVMGTTNQFST
RTRLEEVKGFFSSLKENGSQLRCVQQ
TIETIEENIGWMDKNFDKIRVWLQSE
KLERMaenlyfq^shhhhhhhhhhdy
kddddk

^ TEV cleavage site

Tags and additions: C-terminal, TEV cleavable decahistidine tag and Flag tag.

Host: Trichoplusia Ni (High five)

Growth medium, induction protocol: High five cells were grown in Insect Express medium at 27°C. Cells were infected at a density of 2x106/ml with recombinant baculovirus (virus stock P2; 1ml of virus stock/1l of cell culture) Culture was supplemented with FCS to final concentration 1%. 120 hours post-infection the cultures were collected and centrifuged for 30min at 2000rpm. Cellular pellets were discarded and supernatants were used as a source of a protein.

Column 1: Ni-affinity, Ni-sepharose - (GE Healthcare) purification in batch.

Column 1 Buffers:
Wash buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 10 mM Imidazole; 1 mM PMSF; 0.5 mM TCEP.
Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 250 mM Imidazole; 0.5 mM TCEP.

Column 1 Procedure: Supernatant was supplemented with Tris buffer pH 8.0 to final concentration 50 mM, NaCl to final concentration 300 mM, and NiSO₄ to final concentration 1 mM. Solution was supplemented with PMSF to final concentration 1 mM and 1 tablet of protease inhibitors per 1l of solution. A resin was added and incubation was performed in room temprature for 4 hours. Suspension was loaded on gravity column and after washing with 20 volumes of washing buffer, protein was eluted in 4 elution fractions (10ml each). Protein fractions were analysed by SDS-PAGE.

Column 2: Superdex S200 column, HiPrep 16/60 (Amersham)

GF Buffer: 10 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP.

Column 2 Procedure: Target protein containing fractions were concentrated using Amicon Ultra-15 concentrators with 30kDa cut-off, and purified on a gel filtration column (Superdex S200) on an Åkta Express system. Fractions containing protein were analysed by SDS-PAGE.

Protein concentration: Using Amicon Ultra-15 concentrators with 30kDa cutoff, the sample was concentrated to 17.1mg/ml. Concentrations were determined from the absorbance at 280nm using a NanoDrop spectrophotometer.

Mass spectrometry characterization: The calculated mass of the construct was 110554.5Da, and the observed mass (ESI-MS) was 111563Da, suggesting glycosylation of the protein.

Crystallisation: Crystals were grown by vapor diffusion at 20°C in 150nl sitting drops. The drops were prepared by mixing 100nl of protein solution and 50nl of precipitant consisting of 0.1 M HEPES pH 7.5; 25% PEG 3350. Crystals were flash-cooled in liquid nitrogen with 25% glycerol as cryoprotectant.

Data collection:
Resolution: 3Å.
X-ray source: Diamond Light Source beamline I03.

References

Saric T, Chang SC, Hattori A, York IA, Markant S, Rock KL, Tsujimoto M, Goldberg AL. (2002) An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims 12436109 to MHC class I-presented peptides. Nat Immunol. 3: 1169-76. PubMed 12436109
Chang SC, Momburg F, Bhutani N, Goldberg AL. (2005) The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a "molecular ruler" mechanism. Proc Natl Acad Sci U S A. 102: 17107-12. PubMed 16286653
York IA, Brehm MA, Zendzian S, Towne CF, Rock KL. (2006) Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance. PNAS. 103: 9202-7. PubMed 16754858
Cui X, Hawari F, Alsaaty S, Lawrence M, Combs CA, Geng W, Rouhani FN, Miskinis D, Levine SJ. (2002). Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding. J Clin Invest. 110: 515-26. PubMed 12189246
Cui X, Rouhani FN, Hawari F, Levine SJ. (2003) An aminopeptidase, ARTS-1, is required for interleukin-6 receptor shedding. J Biol Chem. 278: 28677-85. PubMed 12748171
Cui X, Rouhani FN, Hawari F, Levine SJ. 2003 Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding. J Immunol. 171: 6814-9. PubMed 14662887
Tsujimoto M, Hattori A. (2005) The oxytocinase subfamily of M1 aminopeptidases. Biochim Biophys Acta. 1751: 9-18. PubMed 16054015
Miyashita H, Yamazaki T, Akada T, Niizeki O, Ogawa M, Nishikawa S, Sato Y. (2002) A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis. Blood. 99: 3241-9. PubMed 11964289
Brown MA. (2009). Genetics and the pathogenesis of ankylosing spondylitis. Curr Opin Rheumatol. 21: 318-23. PubMed 19496308
Harvey D, Pointon JJ, Evans DM, Karaderi T, Farrar C, Appleton LH, Sturrock RD, Stone MA, Oppermann U, Brown MA, Wordsworth BP. (2009) Investigating the genetic association between ERAP1 and ankylosing spondylitis. Hum Mol Genet. 18: 4204-12. PubMed 19692350
Fruci D, Giacomini P, Nicotra MR, Forloni M, Fraioli R, Saveanu L, van Endert P, Natali PG. (2008) Altered expression of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in transformed non-lymphoid human tissues. J Cell Physiol. 216:742-9. PubMed 18393273