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Supplementary Figure 1 Abacavir ([(1R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-1-cyclopent-2-enyl]methanol), and the structures of drugs (allopurinol and carbamazepine) associated with HLA class I-linked immunological hypersensitivity reactions in vivo.

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Supplementary Figure 2 (a) The ABC12.20.Jurkat cell line, or the control LC13.Jurkat cell line (expressing the irrelevant LC13 TCR1), were co-incubated for 7 hours with C1R-B*57:01 or C1R-B*57:11 that had been treated with abacavir or left untreated. Jurkat cells were cells were identified using GFP fluorescence and CD3 surface staining via flow cytometry. Activation was measured as the mean fluorescence intensity of staining for the early activation marker, CD69. Each bar represents the average MFI of triplicate samples with error bars of one standard deviation. The dashed line separates experiments performed with the ABC12.20.Jurkat (left) and the control cell line LC13.Jurkat (right). (b) Activation of abacavir-stimulated T cell lines with abacavir and peptide agonists. Two abacavir-responsive T cell lines were raised in vitro from two HLA-B*57:01+ healthy donors by co-culturing with PBMCs cells in the presence abacavir. These lines were used in a IFN-γ ELISPOT using C1R-B*57:01 and T2-B*57:01 target cells with or without abacavir pre-treatment. A mixture of five peptides (KSAPELKTGI, HSVEDTHVQI, RVAQLEQVYI, KTFEEKQGTEI and VTYPAKAKGTFI), identified from the surface of abacavir-treated C1R-HLA-B*57:01 cells, were also included along with anti-CD3 mAb positive controls. Wells were analysed in duplicate and spot forming units displayed on the Y-axis.

Reference 1) Macdonald WA, Chen ZJ, Gras S, Archbold JK, Tynan FE, et al. 2009. T Cell Allorecognition via Molecular Mimicry. Immunity 31: 897-908

a

b


SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature11147

Supplementary Figure 3. Mode of abacavir binding. (a) Crystal structure of the HLA-B*5701:RVAQ-abacavir complex shows close correlation to the HLA-B*5701:LTTK-abacavir complex (b)Abacavir binding within the HLA-B*57:01 Ag-binding cleft, comparing the docking mode in the crystal structure (green) and from the auto-docking procedure (magenta).

a b



Supplementary Figure 4. HLA-B*15:02 association with carbamazepine a) Co-purification of carbamazepine with immunoaffinity purified HLA-B*15:02. C1R cells (3x107) transfected with genes encoding either HLA-B*15:02, HLA-B*57:01 or HLA-B*58:01, either treated with carbamazepine or without treatment were lysed under mild conditions and the class I-peptide complexes isolated by immunoaffinity chromatography using immobilised W6/32 mAb. The lysates were then separated by RP-HPLC and fractions screened for the presence of carbamazepine by LC-MS. Extracted ion chromatograms (MH+=237.10224 with a 0.005 Da window) for carbamazepine are shown. There was substantial enrichment of carbamazepine from HLA B*15:02 eluates. The flat line traces represent untreated samples. b) Carbamazepine modifies selection of side chains within the central region of the bound peptides. The frequency of amino acids occurring at anchor (P2 and PΩ), P3, P4, P5 and P6 in nonamer peptides eluted from HLA-B*15:02 isolated from exclusively from untreated (clear bars) or exclusively from carbamazepine treated (solid bars) C1R transfectants. Sequence determination of up to ~1600 peptides was conducted using a high speed LC-MS/MS system (TripleToF 5600 (AB SCIEX)).



Supplementary Figure 4 cont’d. HLA-B*15:02-CBZ docking (c) Location of CBZ (cyan) within the central region of the Ag-binding cleft (d) Environment of residues surrounding CBZ (shown as stick format) CBZ sits sandwiched between three Tyrosine residues, stacking against the aromatic group of Tyr 99 at the floor of the groove and flanked by Tyr 9 and Tyr 159 on either side of the peptide-binding groove. (e) location of CBZ wrt the peptide (green), and the polymorphic position (156) that controls CBZ specificity. The docking of CBZ within this pocket has implications for allotype specificity with polymorphism at position 156. The B*1502 allotype differs from B*1501 at this position with a Leu 156 to Trp substitution. The presence of Trp 156 in B*1501 closes the D pocket and would sterically clash with the docked conformation of CBZ.

c d

e



Supplementary Figure 5. Mechanism of action of abacavir



Supplementary Table 1. Repertoire data for HLA-B*57:01, abacavir treated and related allomorphs HLA-B*57:01 9mers (n=1290)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

W

Strong (>20%)

S T

D F

Moderate (>10%)

K V I

A F

L P L I V

V L

T L

L Y

Increased (>5%)

L S A T R F H

V Y

K V A F R

K E V L D S T I

T K A E F

I Q P A T

R E V Q S L T A K

K E S N Q V R

HLA-B*57:01 10mers (n=794)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

W

Strong (>20%)

S T

F

Moderate (>10%)

K V

A L A

D P

D G

L L V R

L T

Increased (>5%)

I L R A H G T S

V K D I F S V R

E K S T

E L S A P V I

T V I A E S G D K

A I V G S N D K Q

Q E K L S T

S K V E Q

HLA-B*57:01 11mers (n=553)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

W

Strong (>20%)

T S

Moderate (>10%)

K V A

A V

S L

P D L A

L G A

L I

R L

L T

F

Increased (>5%)

R S I L T H

G A F I K D

S L E D A G K T

E V S P Q G

P S A G I K V E D T

L S I T V D Q

V K S G T E

Q V A S I

S A E Y Q V K

Y



HLA-B*57:01 – Abacavir treated 9mers (n=1131)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

Strong (>20%)

T S

D W

Moderate (>10%)

K V I

A F

P K

L V

V L

V

T K

F I L

Increased (>5%)

S L A F T H K

V Y

L A F K V I

E D L S V T

I T A K E

I Q P A T

E R T S Q L I P

L E S Q N H R V

M


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

W

Strong (>20%)

S T

Moderate (>10%)

K V R

A L I A

P D E

D G

L L G

V L T

I F L

Increased (>5%)

I L A G H T

V F D K V

K S T

E L V A S P

I T V E A G D S K

A V N I D S K

R E I Q A T K L S F

K S V E Q N


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

W

Strong (>20%)

T S

P

Moderate (>10%)

K R

V A

L S F I

D A E

G L

L V L T

F I

Increased (>5%)

V A L S I

A Y K

E S D L K G T V

L P G V S N Q

P G L K A I S D T E

A S I T V D E K

K I V G S E

L R Q I A S F

E S V Y K R Q

L



HLA-B*57:03 9mers (n=1533)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

F

Strong (>20%)

S T

W

Moderate (>10%)

K I L V

A L P K

L I V

L T K L

L

Increased (>5%)

A R S T

V A V D I F Y

E R D T L V I

E T F D

V I Q O T F K A S

E V P H F Q N I L T A S

S E Q R N

I

HLA-B*57:03 10mers (n=943)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

F

Strong (>20%)

S T

W

Moderate (>10%)

K R I V

A L A I

P E

D L V

G L

V T L

Increased (>5%)

L A H

V V F S

K D R S Q A T

E G L V A S P I

I T A G S E P K

A N V I K D

Q E N I L T A

S K E R Q H

L

HLA-B*57:03 11mers (n=803)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

F

Strong (>20%)

S T

P W

Moderate (>10%)

K R I

A S L F A

D A

L V

G A S

L L V

T L

L

Increased (>5%)

A V S L Q H

V I V

S E D K A G L V

S V E L P T N

I G P S A T D E

L D K T V P I

I V N S G K T D E Q

Q I T A S

Q S R E K F N Y

I




P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

F

Strong (>20%)

S T

W

Moderate (>10%)

K I L

A L D

P K

L I V

L T K L

L

Increased (>5%)

V R A S

V A V F I Y

E D R T L S V I

E T F

V I Q P T K A F

E V P H Q T L N A S Y F I

S Q E R N

I


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

F

Strong (>20%)

K S T

L W

Moderate (>10%)

R I V L

A A I

P K E

D E

L V I

L G

V Q

T L S

Increased (>5%)

A H

V V F S

D R S T Q

G L V A S P

A T G P S E

A V N K I Q D

E L N I T A

K E Q V H R

L


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

F

Strong (>20%)

K S T

P

W

Moderate (>10%)

R A S L A

D A

L I V

G A S

L L T L

L

Increased (>5%)

I A L V S Q H

V F I D V

E D A K S L T G V

E V S L T N

P G S T A D

L K D V P I T

I V S K G T N E D

V Q I T S A

S K Q R V E F N



HLA-B*58:01 9mers (n=1255)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

W

Strong (>20%)

K S F

Moderate (>10%)

L

A T

D V

P K

V L I

V L

E L

T L K

Increased (>5%)

R I V S A

V F

N L A I S T F

E D R T V L S A Q

T E D F A

Q I T N P

V H Q F P S A T Y

E S R Q N

Y L

HLA-B*58:01 10mers (n=672)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

S W

Strong (>20%)

Moderate (>10%)

K R L

T A

L V I

P

D E

L

L E V

L T

F

Increased (>5%)

I V H A

V A D F S N

K D E S A Q R T

G V L A S P I

D I V T E S K P G A

G E A N D I S V

Q L A S D

S K Q V N R

HLA-B*58:01 11mers (n=519)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

S W

Strong (>20%)

K P

Moderate (>10%)

R T A

L I F

D E A

G

L L Q T L

F

Increased (>5%)

L I A V S

V

V A S D Y

S E A D K G

S N V L P G T

S D E A L P I T K

A E S T G I V D P

G I N A S V D T E

S E L D V A

Q S K R V E




P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

W

Strong (>20%)

K S F

Moderate (>10%)

I L

T A

V

P K

V L

V L Q

E L

T K

Increased (>5%)

V R A S

V I

D N L A I S F T

E R D S L V

I T E D F A

I T H S N P

V Q H P F S A T Y

L E S Q R N

Y L


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

S W

Strong (>20%)

T

Moderate (>10%)

K R L

A L I V

P D

D E

L

L E V

T L S

F

Increased (>5%)

V I H A

V A F D

E K S R Q

G V L A S P

D I V T E S K P G A

G D N A E I S V Q

Q L S I D

K Q E N V


P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

Dominant (>40%)

S W

Strong (>20%)

K P

Moderate (>10%)

R T A

L F I

D S A E

D S

L L

Q L T

F

Increased (>5%)

L A I V S

V V S A D Y

S E D G K A

V L P N T

G E A L P T I V

E A I T S D G V

I V G N A S

S E L A V D

Q S R K V

Y



Supplementary Table2 Abacavir- HLA-B*57:01 occupancy and stability a) Estimation of abacavir occupancy from isolated HLA B*57:01 derived from three independent peptide elution experiments used to determine ligand sequences. Abacavir occupancy was calculated by comparing the ratio of abacavir peak area to beta-2-microglobulin during the first RP-HPLC separation of the complexes as shown in Fig 1b to refolded recombinant HLA-B*57:01-LTTK-abacavir(considered fully occupied by abacavir) b) Stabilisation of HLA B*5701-β-2M and abacavir-dependent peptides in absence and presence of abacavir 2a)

Sample Abacavir peak area (mAU*mL)

b2m peak area (mAU*mL) ratio

fraction of fully occupied

B*5701 abacavir rep 1 1.2127 78.9095 0.015368 0.19 B*5701 abacavir rep 2 2.68 90.7232 0.02954 0.37 B*5701 abacavir rep 3 2.4684 70.5058 0.03501 0.44

Rec. HLA-‐B*5701-‐LTTK-‐abacavir 9.8102 123.26588 0.079586 1.00

average occupancy 0.33

std dev 0.13 2b) Peptide-HLA B*5701 100%

unfold (°C) 100% unfold with ABC (°C)

Stabilizing effect of ABC

LTTKLTNTNI 53.2 ± 0.3 65.2 ± 0.8 Increase RVAQLEQVYI 62.5 ± 0.5 71.2 ± 0.8 Increase KVYVSDNIRI 63.0 ± 1.0 72.0 ± 1.0 Increase KTAGYPNVNI 68.0 ± 1.0 72.5 ± 0.5 Increase ISIHVGQAGVQI No refold 66.0 ± 1.0 Increase VTYPAKAKGTFI 62.0 ± 1.0 67.7 ± 0.3 Increase IALYLQQNW (control) 74.2 ± 0.3 75.0 ± 1.0 No change



Supplementary Table 3. Sequencing of T cell receptor CDR3 loops from abacavir-specific T cells

Donor TRAV CDR3!" TRAJ CD3! length TRBV CDR3# TRBJ CDR3# length 1 12-2 HRTNYGGSQGNL! 42 12 4-1/2/3 SSGQGGNSPL! 1-6 10

5 TPLYSGAGSYQL! 28 11 30 GGGTGTYEQ! 2-7 9 6 GGSYIP! 6 6 30 NGDTEA! 1-1 6

8-2 TELNTGGFKT! 9 10 5-5/6/7 SEKTGDSYYGY! 1-2 11 12-3 SADNYGQNF! 26 9 20-1 RGLAGGSVPGEL! 2-2 12

8 1-1 TPVYNFNKF! 21 9 28 TKGDFTDTQ! 2-1 9 3 RYSGAGSYQL! 28 10 28 TKGDFTDTQ! 2-3 9

1-1 RGDNARL! 31 7 29-1 ESDSYEQ! 2-7 7 12-2 NMEYGNKL! 47 8 18 SPSGGSYNEQ! 2-1 10 20 QGFGNVL! 35 7 28 RWVSGNYNEQ! 2-1 10

9 13-1 KPLDFGNEKL! 48 10 15 SRDWPRYEQ! 2-7 9 20 QAVHSTSYGKL! 52 11 7-8 SPGTVLYTGEL! 2-2 11 8-2 TRMDSSYKL! 12 9 29-1 RRDRNTEA! 1-1 8 8-2 TRKDSSYKL! 12 9 29-1 RRDRNTEA! 1-1 8



Supplementary Table 4 Data collection and refinement statistics B*57:01-LTTK-abacavir B*57:01-RVAQ-abacavir Data collection statistics

Temperature (K) 100 100 X-ray source MX2 Australian

synchrotron MX2 Australian

synchrotron Space group P21 P21 Cell Dimensions a = 64.3, b = 49.0, c = 70.9,

β = 99.5 a = 64.3, b = 49.0, c = 70.9,

β = 99.5 Resolution Å 70 – 1.90 (2.0 – 1.90) 60 – 1.60 (1.69 – 1.60) Total no. observations 193238 (6397) 225625 (32803) No. unique observations 34469 (1157) 57626 (8389) Multiplicity 5.6 (5.7) 3.9 (3.9) Data completeness (%) 99.6 (99.9) 99.6 (99.6) 1/σI 13.6 (5.2) 13.5 (3.4) Rmerge (%)1 11.3 (43.0) 6.5 (46.9)

Refinement statistics Non-hydrogen atoms 3588 3763

Protein 3172 3172 Water 395 570 Ligand 21 21 Rfactor (%)2 17.8 18.6 Rfree (%)2 21.7 21.7 r.m.s.d. from ideality

Bond lengths (Å) 0.005 0.006 Bond angles (°) 1.10 1.14 Dihedrals (°) 16.8 16.1

Ramachandran plot Favoured regions (%) 97.9 98.4 Allowed regions (%) 2.1 1.6

B-factors (Å2) Average main chain 16.7 16.8 Average side chain 23.1 22.2 Average water 30.4 32.6 Average ligand 11.7 12.9

1 Rmerge = ∑hkl ∑j |Ihkl,j - < Ihkl > | / ∑hkl ∑j Ihkl,j. 2 Rfactor = ∑hkl Fo| - |Fc /∑hkl|Fo| for all data excluding the 5% that comprised the Rfree used for cross-validation.



Supplementary Table 5a Repertoire data for HLA-B*15:02, and CBZ treated samples. HLA-B*15:02 8mers (n=47)

P1 P2 P3 P4 P5 P6 P7 P8

Dominant (>40%)

Y

Strong (>20%)

D P R Y

I V

V

Moderate (>10%)

F L

Q L F

L K R V

A T

H V S

E Q

M

Increased (>5%)

I E M N V Y

L M A D V

D E I A

S H

I R T A F N

K T S

F

HLA-B*15:02 9mers (n=1209)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

Y

Strong (>20%)

L F

Moderate (>10%)

D T F

V Q

Y R A

E P D

A

T V

S E A V T

M

Increased (>5%)

S H E M Y N

I M F P

D F L K

K L Q A

V P H T S E F L Q

V S L I G T D K R

S L Q E I R N A

Q K

L

HLA-B*15:02 10mers (n=241)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

L Y

Strong (>20%)

P F

Moderate (>10%)

T H S F

V Y R

D E

A G

P G

G E

T V E

T S A E

M

Increased (>5%)

E M Y

Q M I

F P A L D K

Q G

P F E H Y N

S T I K L A

A S Q R L T

A L S I N

V L Q



HLA-B*15:02 – Carbamazepine treated 8mers (n=50)

P1 P2 P3 P4 P5 P6 P7 P8

Dominant (>40%)

Y

Strong (>20%)

P Y K V I

E V

Moderate (>10%)

D F L

V L R

V L

A H

L M

Increased (>5%)

M I N P V Y

A F Q L S Y

F K

R A E P

H I R T F K S

T Q A L

HLA-B*15:02 – Carbamazepine treated 9mers (n=1179)

P1 P2 P3 P4 P5 P6 P7 P8 P9

Dominant (>40%)

Y

Strong (>20%)

L F

Moderate (>10%)

T D F

V Q

Y R A

E P D

V T L

S E A V Q

M

Increased (>5%)

H S M Y E N

I M P F

D L F K I

L K Q G A

A V S P H T E L Q F G

V I L S G T H K R

S E I Q A

T K

L

HLA-B*15:02– Carbamazepine treated 10mers (n=260)

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

Dominant (>40%)

L Y

Strong (>20%)

P F

Moderate (>10%)

F H T S

V Q

Y R

D E

A G

P G

E G

T V E

T A S E

Increased (>5%)

M Y D Q E

M I F

F P L A D I

S Q G

P E F S H V Y

S L T K I Q

S L A V R T F Q

A S I L Q

V L

M



Supplementary Table 5b. Summary of CBZ-induced repertoire shift Position specific changes in the amino acid usuage of HLA-B*15:02 ligands after treatment with abacavir. Noteworthy changes in prevalence of these amino acids are shown. No significant change was observed for the anchor residues at P2 Leu and P9 Tyr/Phe.

P1 P2 P3 P4 P5 P6 P7 P8 P9

Asp ! Leu remains dominant anchor residue

Ala " Gly " Ala " Gly " His " Ser " Tyr/Phe remain dominant anchor residues

Glu ! Asp ! Val ! Pro " Asp ! Thr ! Lys !

Gln " Cys ! Phe ! Val !

Ile !



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