Supplementary Material

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1 Supplementary Material HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal Anne-Kathrin Anders, Melissa J. Call, Monika-Sarah E. D. Schulze, Kevin D. Fowler, David A. Schuert, Nilufer P. Seth, Eric J. Sunderg & Kai W. Wucherpfennig 1

2 a 2 DR2 WT Protein injection Buffer injection 2 DR2 S53D Fc4 DM WT Fc3 DM Mut2 Fc2 SA 1 5 Fc4 DM WT Fc3 DM Mut2 Fc2 SA Supplementary Figure 1. Minimal ackground inding y a HLA-DR mutant compared to the streptavidin surface. In SPR experiments, the sensor detects refractive index changes when only uffer or protein in uffer is injected. Therefore, it is a widely accepted convention in Biacore studies to sutract the response in a control flow cell from the experimental flow cell. Such a control also addresses non-specific inding. We went one step further to rigorously control for non-specific inding: we designed a HLA-DM mutant that closely resemled the iophysical properties of wild-type HLA- DM ut which had no catalytic activity and did not ind to HLA-DR peptide complexes (HLA-DM Mut2). The HLA-DM Mut2 control surface (flow cell 3, Fc3) did not show increased ackground inding compared to the streptavidin surface (flow cell 2, Fc2) in all experiments that were performed. The two graphs compare results from HLA-DR2 WT (a) and the HLA-DR2 S53D mutant () for three surfaces: HLA-DM WT, HLA-DM Mut2 and streptavidin (SA). HLA-DR2 inding curves were very similar when HLA-DM Mut2 and streptavidin surfaces were compared. Data are representative of more than five (a) and of two () independent experiments, respectively. m m 2

3 a μl/min 25 μl/min 5 μl/min Supplementary Figure 2. Asence of significant reinding of HLA-DR to HLA-DM (mass transport). (a) HLA-DR2 CLIP complexes (2 μm) were run through HLA-DM WT and HLA-DM Mut2 flow cells (1 of immoilized protein) for 5 minutes at the indicated flow rates in a citratephosphate uffer with a ph of 5.35 at 3 C. This was followed y injection of uffer (stage 2) and 2 μm CLIP (stage 3). Binding in the HLA-DM Mut2 flow cell was sutracted from measurements in the HLA-DM WT flow cell. Data are representative of two independent experiments as shown and of more than three independent experiments under similar conditions. 3

4 a Antiody injection 2 αdnp (1:2) αdnp (1:5) αdnp (1:1) Control (1:1) c 1 75 Antiody injection 5 25 αdnp Control Supplementary Figure 3. The complex of HLA-DR and HLA-DM in stage 2 is devoid of peptide. (a) Specific inding of the DNP antiody to a DNP-laeled peptide ound to HLA-DR2. HLA- DR2 CLIP (control, no DNP group on peptide) or HLA-DR2 MBP(85-99)-DNP (5 of each protein) were immoilized on a streptavidin chip. DNP or control antiody (at the indicated dilutions) were injected for 2 min (25 C, 15 μl/min), followed y injection of uffer (citratephosphate uffer, ph 6). Binding in the HLA-DR2 CLIP (no DNP) control flow cell was sutracted from inding in the two experimental flow cells. () The stale complex of HLA-DM and HLA-DR in stage 2 is devoid of peptide. HLA-DR2 CLIP(87-11)-DNP (5 μm) was injected for 1 minutes (25 C, 15 μl/min), followed y injection of uffer and a 2 minute injection of antiody (DNP or control antiody at 2 μg/ml) in citrate-phosphate uffer (ph6). The SPR assay was carried out in consecutive flow cells with immoilized HLA-DM WT or HLA-DM Mut2 (5 of each protein) in citrate-phosphate uffer (ph 5.35). Binding in the HLA-DM Mut2 flow cell was sutracted from measurements in the HLA-DM WT flow cell. Data are representative of two 4

5 independent experiments. (c) The DNP antiody detects the DNP group of a covalently linked peptide. HLA-DR1 HA 6 P 2 -P 11 complex (.5 μm) with a C-terminal DNP group on the linked peptide was injected (as in ), followed y injection of DNP or control antiody in stage 2. 5

6 a DR2-CLIP DR2-MBP ΔFP (mp) DR2-CLIP +DM DR2-MBP +DM DR2-CLIP DR2-MBP Time (min) Supplementary Figure 4. Comparison of the kinetics of HLA-DM inding and HLA-DM catalysis for low-affinity versus high-affinity HLA-DR peptide complexes. (a) Comparison of HLA-DM inding y low-affinity HLA-DR2 CLIP and high-affinity HLA-DR2 MBP complexes. HLA-DR2 complexes preloaded with the indicated peptides (1 μm) were injected for 1 minutes (stage 1), followed y injection of uffer (stage 2) and 5 μm MBP (stage 3). The SPR assay was carried out in consecutive flow cells with 5 of immoilized HLA-DM WT and HLA-DM Mut2 in a citrate-phosphate uffer (ph 5.35) at 37 C at a flow rate of 15 μl/min. Measurements from the HLA-DM Mut2 flow cell were sutracted from data otained from the HLA-DM WT flow cell. HLA-DM inding y HLA-DR2 MBP was 8.7-fold slower compared to HLA-DR2 CLIP, ased on measurement of the initial rates. () Comparison of HLA-DM susceptiility of HLA-DR2 CLIP and HLA-DR2 MBP complexes. Binding of 2 nm Alexa488-laeled MBP to HLA-DR2 (2 nm) preloaded with the indicated peptides was measured in the presence or asence of 1 nm HLA-DM in 5 mm citrate-phosphate uffer (ph 5.3) containing 15 mm NaCl. The initial rate of HLA-DM-catalyzed peptide inding was 11.8-fold slower when HLA-DR2 MBP rather than HLA-DR2 CLIP was used as the input protein in the reaction. Data are representative of two independent experiments as shown (a, ) and of more than three under similar conditions (a) (FP assays were performed in triplicates). 6

7 a Elution c DR1-P 6 Cys no peptide HA6 P2-P11 51 kda 39 kda DRα-HA 6 P 2 -P 11 DRα 28 kda DRβ 14 kda Supplementary Figure 5. Production of homogenous disulfide-linked HLA-DR1 peptide complexes. (a-c) Purification of disulfide-linked complexes of HLA-DR1 P 6 Cys and HA 6 P 2 -P 11 peptide. (a) Removal of excess free DNP-laeled peptide following peptide loading and disulfide ond formation. HLA-DR1 P 6 Cys molecules were separated from DNP-laeled HA 6 P 2 -P 11 peptide y gel filtration (Superose 12). The lue trace (28nm) represents protein asorance, the red 7

8 trace (35 nm) DNP asorance. The peak etween 11-15ml contains the HLA-DR peptide complex, the later peak the free peptide. () Affinity purification of disulfide-linked HLA-DR1 HA 6 P 2 -P 11 complexes y DNP affinity chromatography from other HLA-DR species. Protein and peptide are traced as descried in (a). The earlier peak represents the flow-through, the later peak the peptide-loaded complex (elution with CAPS uffer, ph 11.5). (c) Analysis of disulfide linkage of HA 6 P 2 -P 11 peptide to the HLA-DRα chain y non-reducing SDS PAGE. Data are representative of at least four independent experiments. 8

9 Elution Elution a c DR1-HA DR1-HA 6 DR1-HA 6 P 2 -P 11 IP: W4F.5B L F1 W4F.5B L F1 W4F.5B L F1 98 kda 62 kda 49 kda 38 kda 28 kda IB: 17 kda 1 DR antiserum 2 α-rait HRP d ΔFP (mp) DR1-HA P 2 -P 11 DR1-HA 6 + DTT P 2 -P 11 no DTT DR1-CLIP low + DTT DR1-CLIP low no DTT 9

10 Supplementary Figure 6. Biochemical characterization of HLA-DR1 molecules with covalently linked full length or partial peptides. (a,) Affinity purification of disulfide-linked HLA-DR1 HA 6 (a) and HLA-DR1 HA 6 P 2 -P 11 () complexes y DNP affinity chromatography from other HLA-DR species. The lue trace (28nm) represents protein asorance, the red trace (35 nm) DNP asorance. The earlier peak represents the flow through, the later peak the peptide-loaded complex (elution with CAPS uffer, ph 11.5). The scales of 28 nm and 35 nm readings were adjusted ased on prior experiments with affinity purified, fully loaded HLA-DR peptide complexes such that identical peak heights reflected full occupancy of the peptide inding groove y DNP-laeled peptide. Based on these measurements, the peptide inding groove of oth HLA-DR1 HA 6 (a) and HLA- DR1 HA 6 P 2 -P 11 () was fully occupied. (c) Immunoprecipitation of covalent HLA-DR1-peptide complexes y conformation-sensitive antiodies to HLA-DRα and HLA-DRβ chains. HLA-DR1- HA peptide complexes (without covalent linkage) as well as HLA-DR1 HA 6 and HLA-DR1 HA 6 P 2 -P 11 complexes were immunoprecipitated with a control ma (W4F.5B), ma L243 (to HLA-DRα chain) or ma 9.3.F1 (to HLA-DRβ chain). The Western lot was laeled with a polyclonal anti-dr serum, followed y a polyclonal HRP-laeled rait IgG antiserum. (d) Lowaffinity HA 6 P 2 -P 11 peptide exchanges quickly from HLA-DR1 upon reduction of the disulfide ond. Binding of Alexa488-laeled HA (65 nm) to the indicated HLA-DR1 complexes (2 nm) was followed y FP with or without prior reduction of the disulfide ond etween the peptide and the HLA-DRα helix. Equilirium inding data are shown for reactions in which either the HLA-DR1 HA 6 P 2 -P 11 complex or the HLA-DR1 CLIP low complex (positive control) were used as input proteins. Data are representative of at least two independent experiments (FP assay performed in triplicates). 1

11 a C 3 C 25 C 2 C C 3 C 25 C 2 C Supplementary Figure 7. HLA-DM inding to HLA-DR loaded with full-length peptide is highly temperature dependent. (a,) Temperature profiles for HLA-DM inding y HLA-DR molecules loaded with CLIP peptide (HLA-DR2 CLIP, a) or a low-affinity CLIP variant (HLA-DR1 CLIP low, ). (a,) HLA-DR2 CLIP complexes (.5 μm, a) and HLA-DR1 CLIP low complexes (1 μm, ) were run over the HLA-DM WT and HLA-DM Mut2 flow cells (5 of immoilized proteins) at a flow rate of 15 μl/min in a citrate-phosphate uffer with a ph of 5.35 at the indicated temperatures. This was followed y injection of uffer and injection of 2 μm CLIP (a) or 5 μm HA (). Binding in the HLA-DM Mut2 flow cell was sutracted from measurements in the HLA-DM WT flow cell. Data are representative of two independent experiments shown (a, ) and of at least four independent experiments under similar conditions (a). 11

12 a k a (M -1 s -1 ) k d (s -1 ) c Temperature ( C) Temperature ( C) K D (M) Temperature ( C) Supplementary Figure 8. Kinetic parameters for HLA-DM inding to HLA-DR1 HA 6 P 2 -P 11 complex. (a-c) HLA-DR1 HA 6 P 2 -P 11 complexes (.1 to 5 μm) were injected into HLA-DM WT and HLA- DM Mut2 flow cells at temperatures ranging from 2 to 37 C. The datasets were fitted as in Fig. 7c and k a, k d and K d values were plotted against the corresponding temperatures. Data are representative of two independent experiments. 12

13 a ph 4.6 ph 5. ph 5.5 FP (mp) 25 2 ph 5.6 ph 5.11 ph Time (min) Supplementary Figure 9. Slower dissociation of HLA-DM HLA-DR complex at lower ph. (a) Low ph increases HLA-DM inding to HLA-DR CLIP and slows dissociation of HLA-DM HLA-DR complexes. HLA-DR2 CLIP complexes (5 μm) were run over the HLA-DM flow cell (5 of immoilized HLA-DM) and the SA flow cell for 1 minutes at a flow rate of 15 μl/min in a citrate-phosphate uffer with the indicated ph at 3 C, followed y injection of uffer (stage 2) and CLIP at a concentration of 2 μm (stage 3). Binding in the SA flow cell was sutracted from the measurements in the HLA-DM WT flow cell. () ph dependent peptide inding to HLA-DR2 molecules after photolysis of ound peptide. HLA-DR2 MBP complexes preloaded with UV-cleavale MBP peptide were UV irradiated for 1 minutes in 1 mm Tris ph 7. on ice to generate empty HLA-DR2 molecules. Binding of Alexa-488 laeled MBP (3 nm) to empty HLA-DR2 (15 nm) was measured directly following UV-cleavage using FP at the indicated ph. Data are representative of at least two independent experiments (FP assay performed in triplicates). 13

14 Stage 1 Stage 2 Stage 3 peptide DRα DM 5 High affinity peptide inding; DM dissociation high affinity peptide DRβ DR with tightly ound peptide; no DM recognition Partial peptide dissociation DM recognition Stale DM-DR complex low affinity peptide 6 Editing - transient occupancy Supplementary Figure 1. Model of the interaction of HLA-DM with HLA-DR. HLA-DR molecules with a fully ound peptide (1) do not interact with HLA-DM. Partial peptide dissociation at the N-terminus (2) enales HLA-DM inding (3). HLA-DM catalyzes peptide removal and forms a long-lived complex with HLA-DR in the asence of peptide (4). Binding of a high-affinity peptide to this complex will most likely result in its dissociation (5), while the HLA- DM HLA-DR complex is more likely to remain intact upon inding of a low-affinity peptide (6). Low-affinity peptide can e removed y the catalytic action of HLA-DM (editing) and the complex of empty HLA-DR and HLA-DM (4) is then again availale for peptide inding. The conformational change in HLA-DR molecules upon release of the N-terminal peptide segment is indicated here y a change in color, compared to HLA-DR molecules with tightly ound peptides. For all individual molecular steps, the corresponding stages of SPR experiments are indicated (stages 1-3). 14