Supplemental Information. Structure of the Complex of Human Programmed. Death 1, PD-1, and Its Ligand PD-L1

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1 Structure, Volume 23 Supplemental Information Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1 Krzysztof M. Zak, Radoslaw Kitel, Sara Przetocka, Przemyslaw Golik, Katarzyna Guzik, Bogdan Musielak, Alexander Dömling, Grzegorz Dubin, and Tad A. Holak

2 Supplemental Information Structure of the Complex of Human Programmed Death-1 (PD-1) and Its Ligand PD-L1 Krzysztof M. Zak, Radoslaw Kitel, Sara Przetocka, Przemyslaw Golik, Katarzyna Guzik, Bogdan Musielak, Alexander Dömling, Grzegorz Dubin and Tad A. Holak TABLE OF CONTENTS Figures S1. Alignment of amino acid sequences of the extracellular domains of the human and murine PD-1 receptors. S2. Canonical designations of strands and loops within hpd-1 and hpd-l1 S3. The hpd-1/hpd-l1 interface differs significantly from that within mpd-1/hpd-l1 complex. S4. Example results of fragment evaluation by NMR. Tables S1. Tyrosine analogues evaluated using NMR assay for potential affinity towards Gly124 cleft of hpd-1 Supplemental Experimental Procedures Constructs Design and Validation Expression and Purification of Recombinant hpd-1 and hpd-l1 Crystallization of the hpd-1/hpd-l1 complex and apo-hpd-l1 Structure Determination and Refinement NMR Methods Supplemental References 1

3 Figure S1: Figure S1, related to Figure 1. Alignment of amino acid sequences of the extracellular domains of the human and murine PD-1 receptors. Secondary structure elements are indicated. The variable residues are highlighted red. Figure S2: Figure S2, related to Figure 1. Canonical designations of strands and loops within hpd-1 and hpd-l1. hpd-1 is colored blue; hpd-l1 molecule is depicted in green color. 2

4 Figure S3: Figure S3, related to Figure 3. The hpd-1/hpd-l1 interface differs significantly from that within mpd-1/hpd-l1 complex. (A-C) Top view of the Gly124 cleft ( L Tyr123-accommodating cavity) within PD-1 structures shown in surface representation: (A) human apo-pd-1, (B) human PD-1 complexed with hpd-l1, and (C) mouse PD-1 complexed with hpd-l1. te the induced surface rearrangement upon ligand binding (compare (A) and (B)) and the highlighted differences in amino acid constituting the top side of the cleft in hpd-1 and mpd-1 (compare (B) and (C)). (D-F) Cross sections through Gly124 cleft shown in panels A-C, respectively. (G-I) Selected differences within the interaction surface between the hpd-1/hpd-l1 and mpd-1/hpd-l1 complexes. (G) The human-human complex. hpd-1 and hpd-l1 are shown in blue and green, respectively. Hydrogen bonds are depicted as blue dashed lines. (H) Overlay of the structures shown in panels G and I. A pronounced difference between the structures within this region is clearly discernable. Tyrosine is present at position 68 in hpd-1 whereas asparagine residue is found at corresponding position in mpd-1. In the structure of fully human complex (h/h) the 3

5 intermolecular interaction is based on the π - π binding mediated by the sidechains of Tyr68 and LTyr123. An additional hydrogen bond connects the Tyr68 hydroxyl and the sidechain of L Asp122. In the mouse/human (m/h) complex the sidechain of Asn68 is involved in a hydrogen bond with the hydroxyl moiety of L Tyr123. The large cavity occupied by the aromatic ring of Tyr68 in the h/h complex remains empty in the m/h structure. Smaller changes in the sidechain disposition of Glu136 and L Arg125 are also observed. direct interaction between Asn68 and L Asp122 is observed in the m/h complex (compare Tyr68 interactions in h/h complex described above). Of further differences within the binding interface, valine is present at position 64 in human PD-1 whereas methionine is found in the mouse orthologue. The short sidechain of Val64 does not contribute to intermolecular interaction, but creates a small cavity where a water molecule mediates a hydrogen bond interaction between the sidechain of Asn66 and carbonyl oxygen of L Ala122. In the m/h complex the described cavity is partially filled with a bulky sidechain of Met64 which provides hydrophobic surface mediating a direct intermolecular interaction (mainly around the sidechain of Ala121). (I) The mouse-human complex. mpd-1 and hpd-l1 are shown in yellow and orange, respectively. Hydrogen bonds are depicted as orange dashed lines. 4

6 Table S1, related to Figure 3. Tyrosine analogues evaluated using NMR assay for potential affinity towards Gly124 cleft of hpd-1 Symbol Name and molecular weight (MW) Formula hpd-1 binding * E7P2 4-(aminomethyl)phenol MW 123 PHP-1077 S1 methyl 2 [2 (4 hydroxyphenyl) 2 formamidoaceta mido]acetate MW amino N (5 methyl 1,3,4 thiadiazol 2 yl) benzene 1 sulfonamide (sulfamethizole) MW 270 S2 PHP amino N (6 methoxypyrimidin 4 yl) Nmethylbenzene 1 sulfonamide MW 270 methyl 2 [2 (4 chlorophenyl) 2 formamidoacetamido]acetate MW 284 DN methyl 2 [2 (1H indol 5 yl) 2 formamidoacetamido]acetate MW 289 DN methyl 2 [2 (1H indazol 5 yl) 2 formamidoacetamido]acetate MW 290 TR187 4 (2 {[1 (1 butyl 1H 1,2,3,4 tetrazol 5 yl)b utyl]amino}ethyl)phenol MW 317 * for experimental details see section NMR methods 5

7 Supplemental Experimental Procedures Construct Design and Validation The expression construct used to obtain the recombinant human PD-1 encompassed the major part (residues ; UniProt Q15116) of the extracellular domain of the receptor (residues ; signal peptide 1-20). Cysteine at position 93 was replaced with the serine residue to aid protein stability. The extracellular domain of human PD-L1 (residues ; signal peptide 1-18; UniProt Q9NZQ7) is composed of two Ig like domains, only one of which (residues ) is known to mediate interaction with PD-1 (Lin et al., 2008). Only the interacting domain of PD-L1 was expressed to facilitate crystallization. Expression and Purification of Recombinant hpd-1 and hpd-l1 Genes encoding the interacting extracellular domains of human PD-1 (amino acids ) and human PD-L1 (amino acids ) were cloned into pet-24d and pet-21b, respectively. In the former construct Cys93 was exchanged to serine. Both proteins were expressed in E. coli BL21(DE3) in the form of inclusion bodies. Cells were cultured at 37 C in LB and induced with 1 mm IPTG at OD 600 of 1.0. Following induction the cells were incubated for 5 h at 30 o C (hpd-1) or 37 o C (hpd-l1). The cells were collected by centrifugation, re-suspended in PBS and lysed by sonication. Inclusion bodies were recovered by centrifugation ( x g for 15 minutes), washed 2 times with 50 mm Tris-HCl ph 8.0 containing 200 mm NaCl, 0.5% Triton X-100, 10 mm EDTA and 10 mm 2-mercaptoethanol followed by another wash with the same buffer without the detergent. The inclusion bodies were re-suspended in 50 mm Tris ph 8.0 containing 6M GuHCl, 200 mm NaCl and 10 mm 2-mercaptoethanol by stirring overnight. Solubilized fraction was clarified by centrifugation ( x g for 30 minutes). hpd-1 was refolded by drop-wise dilution in 0.1 M Tris ph 8.0 containing 0.4 M L-Arg hydrochloride, 2 mm EDTA, 5 mm cystamine and 0.5 mm cysteamine. hpd-l1 was refolded in a similar manner in 0.1 M Tris ph 8.0 containing 1 M L-Arg hydrochloride, 0.25 mm oxidized glutathione and 0.25 mm reduced glutathione. Each protein was dialyzed 3 times against 10 mm Tris ph 8.0 containing 20 mm NaCl, concentrated, and purified to homogeneity by gel filtration on Superdex 75 (GE Healthcare) in 10 mm Tris ph 8.0 containing 20 mm NaCl. The purity and protein folding were evaluated by SDS-PAGE and NMR, respectively. Gel filtration analysis of hpd-1 (residues ) and hpd-l1 (residues ) indicated that both constructs are monomeric in solution. NMR spectroscopy indicated that both constructs are characterized by an ordered tertiary structure (results not shown). Size exclusion chromatography 6

8 indicated that hpd-1 and hpd-l1 form a 1:1 complex in solution suggesting that the recombinant proteins reproduce the interaction of native hpd-1 and hpd-l1. Crystallization of the hpd-1/hpd-l1 complex and apo-hpd-l1 Purified hpd-1 and hpd-l1 were mixed in 1:1 molar ratio and the complex was purified by gel filtration on Superdex 75 column in 10 mm Tris ph 8.0 containing 20 mm NaCl. Complex containing fractions were pooled and concentrated to 3 mg/ml. Screening for crystallization conditions was performed using commercially available buffer sets in a sitting-drop vapor diffusion setup by mixing 1 µl of protein complex solution and 1 µl of buffer solution. Initially obtained crystals were optimized according to art. Diffraction-quality crystals were obtained at room temperature from 0.1 M BIS-Tris ph 5.5 containing 1.84 M ammonium sulfate. Apo-PD-L1 (residues ) was purified by gel filtration on Superdex 75 column in the same buffer as the complex. Protein was concentrated to 5 mg/ml and screening was performed using commercially available buffer sets in a sitting-drop vapor diffusion setup. Initially obtained conditions were optimized. Diffraction-quality crystals were obtained at room temperature from 1.80 M sodium formate. Structure Determination and Refinement Crystals were cryo-protected in 20% glycerol in the mother liquor and flash-cooled in liquid nitrogen. The diffraction data were collected at the Helmholtz Centrum 14.1 beamline at BESSY (Berlin, Germany; Mueller et al., 2015). The data were indexed and integrated using XDS (Krug et al., 2012; Kabsch, 2010) and scaled and merged using Scala (Evans, 2006). The initial phases were obtained by molecular replacement calculated using Phaser (McCoy et al., 2007) and hpd-1 and hpd-l1 models derived from PDB entries 3RRQ and 3BIK, respectively. The models were manually built in the resulting electron density maps using Coot (Emsley et al., 2010). Restrained refinement was performed using Phenix (Adams et al., 2010) and Refmac 5.0 (Murshudov, 2011). Five percent of the reflections were used for cross-validation analysis and the behavior of the R free was employed to monitor the refinement strategy. Water molecules were added using Coot and subsequently manually inspected. The final models were deposited in the Protein Data Bank under accession numbers 4ZQK and 5C3T. All molecular graphics were prepared with PyMOL ( NMR Methods 7

9 Uniform 15 N isotope labeling was achieved by expression of hpd-1 in the M9 minimal medium containing 15 NH 4 Cl as the sole nitrogen source. Final purification step consisted of gel filtration into the NMR compatible buffer (25 mm sodium phosphate containing 100 mm NaCl ph 6.4). 10% (v/v) of D 2 O was added to the samples to provide lock signal. All spectra were recorded at 300K using a Bruker Avance 600 MHz spectrometer. The 15 N-labeled PD-1 was titrated with each evaluated compound and 1 H- 15 N 2D-HMQC spectra were recorded prior and after each compound has been added (Figure S4). In each case the protein was over-titrated (5:1 compound to protein molar ratio) to detect weak interactions, if any. This method is based on monitoring of chemical shift changes in protein amide backbone resonances upon protein interaction with a small molecule (Shuker et al., 1996; Stoll et al., 2001). Figure S4: A B Figure S4, related to Figure 3. Example results of fragment evaluation by NMR. The 1 H- 15 N SOFAST HMQC spectra (Schanda et a., 2005) of 15 N apo-pd-1 (blue) and apo-pd-1 titrated with (A) TRL187 or (B) S1, both at 5:1 molar ratio (red). The lack of perturbations in positions of NMR signals upon compound addition indicates no interaction with the tested protein. 8

10 Supplemental References Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W. et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, Evans, P. R. (2006). Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62, Kabsch, W. (2010). XDS. Acta Crystallogr D Biol Crystallogr 66, Krug, M., Weiss, M.S., Heinemann, U., Mueller, U. (2012). XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Cryst. 45, McCoy A. J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J. Appl. Cryst. 40, Mueller, U., Förster, R., Hellmig, M., Huschmann, F. U., Kastner, A., Malecki, P., Pühringer, S., Röwer, M., Sparta, K., Steffien, M., et al. (2015). The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives. Eur. Phys. J. Plus 130, 141. Murshudov, G. N., Skubák, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A.A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol Crystallogr 67, Schanda, P., and Brutscher, B. (2005). Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J. Am. Chem. Soc. 127, Shuker, S.B., Hajduk, P.J., Meadows, R.P., and Fesik, S.W. (1996). Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, Stoll, R., Renner, C., Hansen, S., Palme, S., Klein, C., Belling, A., Zeslawski, W., Kamionka, M., Rehm, T., Muhlhahn, P., et al. (2001). Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53. Biochemistry 40,

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