Supporting Information for the article entitled UV-induced ligand exchange in MHC class I protein crystals by Patrick H.N. Celie 1, Mireille Toebes 2, Boris Rodenko 3, Huib Ovaa 3, Anastassis Perrakis 1,* and Ton N.M. Schumacher 2,* The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 1 Division of Biochemistry, 2 Division of Immunology, 3 Division of Cell Biology II, p.celie@nki.nl, m.toebes@nki.nl, b.rodenko@nki.nl, h.ovaa@nki.nl *Corresponding Authors: a.perrakis@nki.nl, t.schumacher@nki.nl )
Figure S1. Superposition of KILGJ 2 VFJ 1 V structures before and after UV exposure. (A) The structure of KILGJ 2 VFJ 1 V is shown as ribbons (light to dark blue for the heavy chain, green for the β2m subunit) and is superimposed on the HLA- A2.1:KILGJ 2 VFJ 1 V structure obtained after UV exposure (orange ribbons for both the heavyand β2m chains) with an rmsd value of 0.33 Ǻ between 375 Cα atoms of the heavy- and β2m chains. The intact and cleaved peptides are shown as sticks with yellow and orange carbons, respectively. Peptide oxygen atoms are in red, nitrogen atoms in blue. The orientation is identical to that shown in Figure 1A. (B) Same superposition as in (A) but now zoomed in on the peptides and the HLA α1 and α2 domains of the heavy chain.
Figure S2. Superposition of two HLA-A2.1 structures bound to peptide ILKEPVHGV. The structure of ILKEPVHGV obtained after peptide exchange is superimposed on the previously solved ILKEPVHGV crystal structure (pdb code 1HHJ) with an rmsd value of 0.29 Ǻ and the peptides are displayed as sticks (carbons of exchanged peptide in dark- cyan, oxygen atoms in red, nitrogen atoms in blue and peptide from 1HHJ in grey).
70 63 ± 0.1 60 50 48 ± 1.2 IC50 ( M) 40 30 20 17 ± 1.0 17 ± 2.1 20 ± 1.2 10 0 ILKEPVHGV (HIV-1 rt) KLTPLCVTL (HIV-1 env) NLVPMVATV (CMV) AMDSNTLEL (H5N1) NLVBMVATV (diol) Figure S3. Fluorescence polarization binding assays. IC 50 values of peptides used for in crystallo exchange were determined using a peptide exchange fluorescence polarization assay as described. 1 HLA A2.1(Se-Mβ2m)::Se-MILGJ 1 VFJ 2 V was used as conditional MHC class I. The HLA-A2-binding peptide FLPSDCFPSV was labeled with TMR-5-maleimide (Anaspec), purified by reverse-phase HPLC as described 1 and used as a tracer for fluorescence polarization (FP) experiments. Labeled peptide was standardized against a fluorescence intensity curve by using free TMR-5-maleimide as a standard. Data were analyzed by using GraphPad Prism software (GraphPad). (1) Bakker, A.H.; Hoppes, R.; Linnemann, C.;, Toebes, M.; Rodenko, B.; Berkers, C.R.; Hadrup, S.R.; van Esch, W.J.;Heemskerk, M, Ovaa H, Schumacher T.N. Proc Natl Acad Sci U S A 2008,1053825-3830.
Table S.1 Data collection and refinement statistics a IKEPVHGV KLTPLCVTL NLVPMVATV AMDSNTLEL UV cleaved Diffraction Space group P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 P2 1 Cell dimensions a, b, c (Å) 61.98, 81.80, 61.51, 86.52, 62.21, 61.99, 81.90, 62.20, 86.50, 62.73, 87.01, 62.03, 86.49, 80.47 62.38, 84.11, 80.04 79.71 80.00 82.34, 79.63 79.75 80.78 79.91 α, β, γ ( ), 90.77,, 90.34,,, 90.87,, 90.04,, 90.04,, 90.21,, 90.97, 90.70, Resolution (Å) 50.0 2.0 50.0 2.34 50.0 2.3 50.0 2.3 50.0 2.3 50.0 2.3 50.0 1.9 50.0 2.55 R sym or R merge 5.0 (22.5) b 5.9 (30.7) 9.3 (40.9) 8.8 (39.5) 11.2 (49.0) 5.8 (28.4) 7.1 (64.0) 8.0 (50.9) I / σi 9.8 (2.9) 9.0 (2.5) 6.1 (1.5) 6.3 (1.5) 5.0 (1.5) 8.6 (2.7) 7.6 (1.1) 5.8 (1.1) Completeness (%) 97.2 (98.1) 92.4 (92.7) 99.9 (100) 97.2 (98.1) 97.2 (98.1) 97.2 (98.1) 97.3 (96.1) 99.7 (99.7) Anomalous 90.5 (86.4) 98.2 (99.2) 83.3 (87.4) 87.5 (87.6) completeness (%)
Table S.1 Data collection and refinement statistics (continued) IKEPVHGV KLTPLCVTL NLVPMVATV AMDSNTLEL UV cleaved Anomalous 1.9 (1.9) 1.8 (1.8) 1.8 (1.8) 1.6 (1.6) redundancy Refinement Resolution (Å) 20.0 2.0 20.0 2.34 20.0 2.3 20.0 2.3 20.0 2.3 20.0 2.3 20.0 1.9 20.0 2.55 No. reflections 52219 33140 45961 35524 37902 38227 64885 26956 R work / R free 16.7 / 23.4 18.9 / 26.2 18.3 / 23.4 17.9 / 24.1 15.7 / 22.5 13.4 / 18.1 16.7 / 20.6 17.7 / 24.5 Twin Law -h, -k, l -h, -k, l -h, -k, l -h, -k, l Twin fraction 0.32 0.37 0.34 0.05 No. atoms Protein 6312 6213 6393 6269 6228 6305 6218 6168 Peptide 163 73 149 136 162 130 162 136
Table S.1 Data collection and refinement statistics (continued) IKEPVHGV KLTPLCVTL NLVPMVATV AMDSNTLEL UV cleaved No. atoms Ligand/ion 90 84 120 90 96 54 42 30 Water 776 240 425 320 449 373 321 141 Occupancy (Pept 1 / pept 2) c 92 / 100 85 / 70 84 / 79 93 / 94 99 / 97 R.m.s. deviations Bond lengths 0.009 0.004 0.003 0.004 0.005 0.005 0.006 0.006 (Å) Bond angles ( ) 1.002 0.623 0.571 0.644 0.742 0.617 0.919 1.126 a Single crystals were used to collect data for each of the structures. b Numbers between brackets represent the statistics of the highest resolution shell c Occupancy refinement of peptides (pept 1 and pept2) in both molecules within the asymmetric unit was performed using the Phenix software (Adams et al., 2002)
Table S2. Anomalous difference electron density levels (e - /Å 3 ) of selenium atoms Se-M residues β2m a Se-M residues peptide Crystal Structure Se-M B0 Se-M B99 Se-M D0 Se-M D99 Se-M E1 Se-M F1 Ratio b Se-M(pept.)/ Se-M(β2m) Exchange efficiency (%) c Se-MILGJ 1 VFJ 2 V n.a. n.a. n.a. n.a. 0.105 0.150 n.a. NLVPMVATV n.a. n.a. n.a. n.a. 0.017 d 0.010 d n.a. n.a. HLA-A2.1(Se-Mβ2m):Se-MILGJ 1 VFJ 2 V 0.073 0.080 0.098 0.137 0.108 0.151 1.34 ± 0.41 HLA-A2.1(Se-Mβ2m):AMDSNTLEL e 0.074 0.103 0.047 0.078 0.083 g 0.031 g 0.75 ± 0.40 44 ± 34 HLA-A2.1(Se-Mβ2m):AMDSNTLEL f 0.026 0.072 0.035 0.058 0.026 g 0.022 g 0.50 ± 0.20 63 ± 19 a There are two copies of HLA-A2.1-peptide molecules in the asymmetric unit b Signal ratio is calculated as (signal from Se in peptide chains E + F) / (signal from Se in β2m chains B + D) c Exchange efficiency is calculated as 100 % - ((signal ratio Se-M (exchanged structure))/ (signal ratio Se-M (structure with conditional ligand)) x 100%) d The structure of Se-MILGJ 1 VFJ 2 V is superimposed on the NLVPMVATV structure to determine the anomalous difference electron density levels of residual selenium atoms within the NLVPMVATV structure after exchange e Crystal structure solved at 2.3 Å f Crystal structure solved at 2.55 Å (see table S1) g The structure of HLA-A2.1(Se-Mβ2m):Se-MILGJ 1 VFJ 2 V is superimposed on the HLA-A2.1(Se-Mβ2m):AMDSNTLEL structures to determine the anomalous difference electron density levels of residual selenium atoms within the HLA-A2.1(Se-Mβ2m):AMDSNTLEL structures after exchange