Supplementary Information Viral immunoevasin targeting of a Natural Killer cell receptor family Richard Berry 1, Natasha Ng 1, Philippa M. Saunders 2, Julian P. Vivian 1, Jie Lin 2, Felix A. Deuss 1, Alexandra J. Corbett 2, Catherine A. Forbes 3, Jacqueline Widjaja 2, Lucy C. Sullivan 2, Adrian D. McAlister 1, Matthew A. Perugini 4, Melissa J. Call 5, Anthony A. Scalzo 3, Mariapia Degli-Esposti 3, Jerome D. Coudert 3, Travis Beddoe* 1, Andrew G. Brooks 2* & Jamie Rossjohn 1,6* 1 Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia 2 Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. 3 Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Western Australia 6009, Australia and Centre for Experimental Immunology, Lions Eye Institute, 2 Verdun Street, Nedlands, Western Australia 6009, Australia 4 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne Victoria 3086 Australia 5 Structural Biology Division, Walter and Eliza Hall Institute, University of Melbourne, 1G Royal Parade, Parkville Victoria 3052, Australia 6 Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff CF14 4XN, UK. * Correspondence should be addressed to J.R. (jamie.rossjohn@monash.edu)
crystallization drop crystal m157 kda Ly49H NKD- α3s marker a 37 m157 25 20 Ly49H NKD- α3s Ly49H NKD 15 b Final - Shots 2000-20120615; Run #4; Label B13 9284.9 100 Final - Shots 2000-20120615; Run #4; Label B15 7591.4 1887.2 100 457.8 90 90 7817.3 80 80 7663.1 7873.8 70 70 60 60 50 6179.8 40 50 18522.6 18644.3 6220.4 8020.0 14961.9 7454.1 7001.0 14762.8 14398.5 30 9627.7 20 17161.6 10 0 6002.0 16287.3 7969.9 40 6019.3 8343.0 30 20 15630.2 13274.0 18204.9 9732.911715.3 21336.4 10 11810.8 17619.6 23428.4 29237.2 Mass (m /z) 35046.0 0 6002.0 11810.8 17619.6 23428.4 Mass (m /z) 25429.4 29237.2 35046.0 c Supplementary Fig. 1. Ly49-m157 crystals contain both m157 and the entire Ly49H NKD-α3s construct. (a) SDS-PAGE analysis of purified proteins used for crystallization, washed crystals and the uncrystallized protein from the crystallization drop. Only the full length Ly49H NKD-α3s protein is incorporated in to the crystals. Note the proteins in the crystallization drop sample migrate abnormally due to the presence of PEG 4000. (b) Mass spectrometry analysis of the washed crystal (left) and crystallization drop (right). The predicted mass for the Ly49H NKD-α3s construct
based on amino acid sequence is 18,672 Da. (c) Packing of molecules in the Ly49Hm157 crystal. One molecule of m157 (pink) and two Ly49H stalks (blue) are coloured and all other symmetry related molecules are in grey. A large vacant space where the Ly49 NKD is expected to be located is circled.
a b NKD-α3s (E.coli) Stalk (HEK293) K D = 121.3 ± 14.5 µm NB c 600 Response (RU) 400 200 0-200 20 40 60 80 100 Time (sec) Supplementary Fig. 2. E. coli refolded Ly49H NKD- 3s used for crystallization is correctly folded. (a) Circular dichroism spectra of Ly49H B6 (solid line) and Ly49C B6 (dashed line) NKD- 3s constructs. Both Ly49 s exhibit a spectrum with a double minimum at 208 and 222 nm, indicative of a primarily -helical structure. (b) SPR sensograms (top) and equilibrium binding curves (bottom) of E.coli produced Ly49C NKD- 3s and HEK293T produced Ly49C stalk to H-2K b. K D = equilibrium
dissociation constant, NB = no binding. Data are representative of one experiment performed in duplicate with error bars representing s.e.m. of the duplicates. (c) Binding of E.coli produced Ly49H NKD- 3s to the NKD specific mab 3D10. Approximately 4000 response units of 3D10 antibody was coupled to a CM5 sensor chip (GE Healthcare) and 30 g/ml Ly49H (in the presence of 0.5M NaCl to prevent non-specific binding) was used as the analyte.
a b c Q120 Y174 I116 T127 W123 I97 E119 Y115 Y174 M175 S178 M175 Y115 E119 Y174 Y115 M175 W123 E119 to NKD Q120 to NKD Supplementary Fig. 3. Electron density at the Ly49-m157 interface. (a) Unbiased electron density for the Ly49 stalk following molecular replacement with m157. The 2Fo-Fc map (blue) and Fo-Fc map (green) are contoured at 1σ and 3σ respectively. (b) Omit map of the Ly49 stalk contoured at 3σ. (c) Final refined 2Fo-Fc maps representing electron density at the Ly49 (blue) and m157 (pink) interface contoured at 1σ.
a b c d identity to Smith (%) G1F N5 N1 W8211 K17E G4 0 20 40 60 80 100 Smith N5 N1 W8211 K17E G4 0 20 40 60 80 100 Identity to G1F (%) G1F MVLVLLVKLLLIVIVSERALTIFNFNPNPDDMYIVNPDDLQLTFTMEFEVTVTRDGFHKRTISVDNGRPVVVWDGGDKDPKICKI N5 MVLVLLVKLLLIVIVSERALTIFNFNPNPDDMYIVNPDDLQLTFTMEFEVTVTRDGFHKRTISVDNGRPVVVWDGGDKDPKICKI smith MVIVPLVKLLLIVFISERAVTIFNPDP--DDTYIVNMDDFQFTFTMEFEVTVTRGGVHKRTISVDNGRPVVVWDVGDRDPKICKI N1 MVLVLLVKLLLIVVVSERALTIFNPNPDPDNTYIVNLDDSQFTFTMEFEVIVTTSGFHKRTISVDNGRPVVVWDVGDKDPKICKI W8211 MVLIPLVKLLLIVFVSERAVTILNPNPDLDDAYSVNMDHFQFTFTMEFEVTVTRSGFHKRTISVNNGRPVVVWDVGDKNPKICKI K17E MVLIPLVKLLLIVFVSERAVTILNPNRDPDDAHLVNLDDSQFTFTMEFEVTVTRSGLHKRTISVDNGRPVVVWDVGDKNPKICKI G4 MLLVPLIKFLVVGVLPRVVAIFNPNPNPNPVVIHVDSGDRRIEFIMEFEVTVTRSGFQKCTISFNNGRPVVVWE--DEGPKVCKL G1F CPAVNSINTENIFLDIQKMRLNNLLAQGLWDIQRICVRYVCLFLGFDVVCDVYHTTDRVRAAYTRQTGKIDIQGSGTFSTSDAKG N5 CPAVNSINTENIFLGIQKMRLNNLLAQGLWDTQRICVRYVCLFLGFDVFCDVYHTTDTVRVSYIKQTGKINIQGSGTFPPSDAKE Smith CPDVSSTDIEYVFLDIQKMRLNNLLTQSLWDTQRICVRYACLFLGFDVICDVYHTTDTVRVAYTGQTGKINIQGSGKFSTSDAKE N1 CPSVGSINTEYLFLDIQKMRLSNLLAQSLWGTQRICVRYACLFSRFNVLCDVYHTTDTVRVTYTYQTGKINIQGSGTFTTSDAKG W8211 CPAVSSINTEYVFLDIQKMRLSNLLVQGLWETQRICVRYVCLFLGFNVLCDVYHTTDTVRVAYIHQTRKINIQGSGTFPISDAKE K17E CPAVSSINIEYLFLDIQKMRLSNLLAQSLRDTQRICVRYACLFSRFNVLCDVYHTTDTVRVTYTYQTGKINIQGSGSFPTSDAKE G4 CPPLSSENGEGLFLNMQLTYLQQLLNK-LNDTQRICIRYMCLFWIFGVGCNVYHTTDTVRVSYTLGTGNTNIQGPGTFQTSDAKG G1F IGTYMIESNVREIKNKWRPTVQKLKQLGYMNETEVEFWYNTTGLTTCVVTSRSNVPFTVELSLNTNSSAIVTEESTVDCQTVTVK N5 IGTYMIKSNVREIKKTWRPTVEKLKQLGYMNETEVEFWYNTTGLTTCVVTSRSNVPFTVELSLNTNNSAIVTDESTVDYQTVTLK Smith IGTYMIKSNVREIKNRWRSTVQKLKQLAYMNATEVEFWYNTTGLTTCVVTSRSNVPFTVELSLNTNSSAIVTDESTVDCQILTVK N1 IGTYMLRNRVQEIKNTWRPTVQQLKQLGYMNATEVEFWYNTTGLTTCVVTSRSNVPFTVELSLNTNSSAIVTDESTVDYQTVTLK W8211 FAIFMLTSNVQEIKKTWRPTVQQLKQLGYMNVTEVEFWYNTTGLTTCVVTSRSNAPFTVELSLNTNSSAIVTDESTVDYQTVTLK K17E FGTYMLRNRVQEIKNTWRSTVQKLKQLGYMNATEVEFWYNTTGLTTCVVTSRSNVPFTVELSLNTNSSAIVTEESTVDCQIVTVK G4 IGTTLLEERREEITKRWWPVIQELNHLGCMNETDVEFWYDMSGVTTCVVKSRSSMPFIVELSLSDNSSMTVTDESTVDCQIVTVK Ly49H B6 + + + - - - - G1F APGSHAQRCYVTSSLGWKGVVTPPSQYRTKRAPVNISSSKWTGIVNWKGSVNRSTASHLPNLPILILCVVFMRLVV N5 ASDSYPQRCYVTSSLGWKGVVTPPSQYRTKRAPVNISSSKWTGIVNWKGSVNRSTASHLPNLPILILCVVFMRLVV Smith APGSHAQRCYVTSSLGWKGVVTPPSQYRTKRVPVNISSSKWTGIVNWKGNVNRSTASHLPNLPILILCVVFMRLVV N1 VSDSYPQRCYVTSSLGWKGVVTPPSQYRTKRTPVNISSSKRTGIVNWRGNVNRSTDSYSPNLTILILCAVFMHLII W8211 VSDSYPQRCYVTSSLGWKGVVTPPSQYRTKRTPVNISSSKRTGIVNWRGNVNRSTDSYSPNLTILILCAVFMHLIV K17E APGSHAQRCYVTSSLGWKGVVTPPSQYRTKRVPVNISSSKWTGIVNWKGNVNRSTASHLPNLPILILCVVFMRLVV G4 APGSHAQRCYVTSSLGWKGVVTPPSHYRTKRVPVNISSSKWTGIVNWKGTANHFTASRSPNLTILVLCAVFMRLVV
Supplementary Fig. 4. m157 sequence variability. (a) Overlay of the ly49hm157 G1F complex structure with that of the unliganded m157 smith (PDB:2NYK). m157 G1F is in pink, m157 smith is in grey and Ly49 is in cyan (stalk 1) and blue (stalk2). Amino acid residues that would clash are shown as sticks. (b) Amino acid residues that differ between the Smith and G1F strains (dots) are mapped on to the structure of the Ly49H-m157 G1F complex. Residues that do not contact the Ly49H-α3s stalk are shown in red, those that do contact are shown in green. (c) Sequence alignment of m157 strains performed by CLUSTALW. Residues that form contacts with Ly49H in the Ly49H-m157 G1F structure are in red. The hypervariable region (residues 87-96) is highlighted with a blue background. The ability of each m157 strain to be recognized by Ly49H B6 is indicated. (d) Comparison of the amino acid sequence identity of a number of m157 strains to that of m157 Smith (left) and m157 G1F (right). Percentage identity of the full length protein (filled bars) and amino acid residues 87-96 (open bars) are shown.
Supplementary Fig. 5. Ly49 mutants are correctly folded. CD measurements were performed on the entire extracellular regions of Ly49C B6 (black) and Ly49A B6 (red) in addition to the following Ly49C B6 mutants: Y115A (purple), Q120A (pink), W123A (yellow), K128 (green) and the NKD alone (cyan).
Supplementary Fig. 6. The Ly49 backfolded conformation is incompatible with m157 binding. The structure of Ly49-L4 (PDB 3G8L) comprising the NKD and α3s stalk in the backfolded conformation (black ribbon) was superimposed on to the α3s stalk (cyan) of the Ly49H-m157 structure. The NKD of Ly49-L4 sterically clashes with both m157 molecules (pink and magenta surfaces).
Supplementary Table 1. Data collection and refinement statistics Data collection statistics Temperature (K) 100 X-ray source MX2 Australian Synchrotron Space group P3 1 12 Cell dimensions 87.87, 87.87, 133.37 90.0, 90.0, 120.0 Resolution (Å) 76-3.2 Total no. observations 43902 (6564) No. unique observations 9772 (1441) Multiplicity 4.5 (4.6) Data completeness (%) 98.4 (100) I/σ I 8.3 (1.9) R merge (%) 9.5 (91.4) R pim (%) 4.8 (45.4) Refinement statistics Non-hydrogen atoms Protein 2067 Water 0 Sugar 14 R factor (%) 22.5 R free (%) 25.9 r.m.s.d from ideality Bond lengths (Å) 0.009 Bond angles ( ) 1.07 Ramachandran plot Favoured regions (%) 93.57 Allowed regions (%) 6.43 Disallowed regions (%) 0 1 R merge = Σ hkl Σ j I hkl,j - < I hkl > / Σ hkl Σ j I hkl,j. 2 R factor = Σ hkl F o - F c /Σ hkl F o for all data excluding the 5% that comprised the R free used for cross-validation.
Supplementary Table 2. Contacts table between m157 G1F and Ly49H B6 NKD-α 3 S Ly49 stalk 1 m157 Type Glu111 C,CA,CB Asn93 CG,OD1 VDW Glu111 O Asn93 OD1 H-bond Glu111 C,O Ile97 CD1 VDW Leu112 N,CA,CD1 Ile97 CD1,CG1,CG2 VDW Tyr115 N,CA,CB Asn93 ND2 VDW Tyr115 CB Ile97 CD1 VDW Tyr115 OH Tyr174 CE1,CZ,OH VDW Tyr115 CZ,OH Met175 CG VDW Ly49 Stalk2 m157 Type Met110 CE Asn108 OD1 VDW Met110 CE Gln112 CG,CD,NE2 VDW Leu113 CD2 Arg105 CB VDW Leu113 CD1 Asn108 CB,CG,ND2 VDW Ile116 CG2 Gly172 N VDW Glu119 CB Tyr174 CE2,CZ,OH VDW Gln120 OE1 Gly172 N H-bond Gln120 OE1 Gly172 CA,C VDW Gln120 CD,OE1,NE2 Thr173 N,CA,C,CB VDW Gln120 OE1 Thr173 N H-bond Gln120 CD,OE1,NE2 Tyr174 N,CA,CB,CD2 VDW Gln120 OE1,NE2 Tyr174 N H-bond Gln120 NE2 Glu177 OE1 VDW Trp123 CB,CG,CD1 Tyr174 CA,CB VDW Trp123 CH2,CD1,NE1,CE2,CZ2 Glu177 C,O,CB,CD,OE1 VDW Typ123 NE1 Glu177 OE1 H-bond Trp123 CH2,CE3,CZ2,CZ3 Ser178 N,CA,OG VDW Atomic contacts determined with the CCP4i implementation of CONTACT with a cutoff of 4.5Å. Van der Waals interactions are defined as non-hydrogen bond contact distances of 4Å or less. Hydrogen bond interactions are defined as contact distances of 3.5Å or less. Only amino acid residues that are well defined in electron density are included.