Supporting Online Material. Crystal structure of a shark. single domain antibody V region. in complex with lysozyme

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1 Supporting Online Material Crystal structure of a shark single domain antibody V region in complex with lysozyme Robyn L. Stanfield 1@, Helen Dooley 3@, Martin F. Flajnik 3, and Ian A. Wilson 1,2 Dept. of Molecular Biology 1 and the Skaggs Institute for Chemical Biology 2, The Scripps Research Institute, N. Torrey Pines Road, La Jolla, CA 92037, USA, and Dept. of Microbiology and Immunology, University of Maryland at Baltimore 3, Baltimore, MD , authors contributed equally to this work Materials and Methods Protein preparation The HEL-specific HEL-5A7 IgNAR V region was selected from an immune nurse shark phagedisplayed library as previously described (1). This clone was expressed into the E. coli periplasm without affinity tags and protein was purified via lysozyme-affinity chromatography. The IgNAR V region-hel complex was formed by addition of hen egg white lysozyme (Sigma L-7651) to HEL-5A7, and subsequent purification of the complex on a Superdex 75 16/60 column, and concentration to ~3.5 mg/ml. The complex was stored at 4 C. 1

2 Crystallization Crystals were grown from a wide range of different molecular weight polyethylene glycol (PEG) precipitants; very thin plates were obtained from low molecular weight PEGs ( ), and thick plates or rods from mid- to high-molecular weight PEGs ( ,000). All crystals were grown in sitting drops containing 1µl protein and 1µl well solution. To verify that the crystals contained the complex, several crystals were dissolved in water and analyzed by Maldi-TOF mass spectrometry, which showed two peaks with molecular mass of 14,312 Da (HEL) and 11,972 Da (IgNAR V). Analysis of several crystals revealed two different, but closely-related crystal forms, in space group P , and with unit cell dimensions of 74.0Å, 109.8Å, 29.3Å (Form 1) or 75.8Å, 113.8Å, 58.4Å (Form 2) that grew under very similar conditions. Data were collected from single crystals grown either from a well solution containing 15% PEG 10,000, 0.1M imidazole HCl, ph 7.0 (Form 1), or 15% PEG 10,000, 0.1M imidazole malate, ph 6.0 (Form 2). The crystals were cryoprotected with well solution augmented with 25% ethylene glycol. Data collection Data were collected at Advanced Light Source Beamline at 100K, using an ADSC Q215 CCD detector and a wavelength of 1.116Å (Table S1). Molecular replacement The structure of crystal form 1 was determined by MR using the program EPMR (2), with data between 15-4Å resolution. While an unambiguous solution was readily obtained for HEL (with 2

3 coordinates from PDB 1LKS (1.1Å); CC=0.34 and R=0.54), many different IgNAR V region models (including over 400 different V L, V H, C L, and C H domains, 11 camel and llama V H H domains, and 23 other Ig-type domains) did not give a clear MR solution. A FASTA comparison of the IgNAR V sequence against all protein sequences for structures in the PDB showed the highest identity (36.5%) with the first 85 amino acids from the light chain in PDB file 1QLE (3), an Fv fragment in complex with cytochrome C. Accordingly, a model was prepared including residues L1-L88 (Kabat numbering) of 1QLE. With partial structure factors from the correctlypositioned HEL, and a minimum bump distance of 20Å (to discard solutions with bad packing), the correct orientation and translation were found for the truncated 1QLE model resulting in a CC of 0.39 and R of Model building and refinement The molecular replacement model was then refined with the simulated annealing protocol in CNS (4), using all data to 2.0Å resolution, resulting in R cryst and R free values of 43.5% and 47.8%. Alternating cycles of model building with Tom/Frodo (5) on a Silicon Graphics, and CNS refinement, while gradually extending the resolution to 1.45Å, allowed completion of the IgNAR Type II V region model. Refinement was concluded with several cycles of REFMAC (6) (without the TLS option, as this did not significantly improve the refinement statistics), to final R cryst and R free values of 18.8% and 21.2% for all measured data to 1.45Å resolution (Table S1). HEL residues 1-129, IgNAR V region residues 2-113, 189 waters, 1 chloride ion, and 5 ethylene glycol molecules are included in the deposited structure. 91.8% of the residues are in the most favored region of the Ramachandran plot (Table S1). The structure of crystal form 2 was determined by molecular replacement with the refined coordinates of crystal form 1 as the initial 3

4 model. Refinement with REFMAC (without TLS) gave final R cryst and R free values of 19.4% and 22.1% for all data to 1.7Å resolution (Table S1). The three independent copies of the shark antibody complex are very similar so, unless otherwise noted, discussion will focus on the higher resolution structure. The final models include residues of HEL (chain identifier L) and residues of the IgNAR V (chain identifier N; sequential numbering has been used as the IgNAR has several unusual insertions and deletions that the standard Kabat numbering system will not accommodate). Representative electron density is shown in Fig. S1, a summary of the data collection and refinement statistics in Table S1, and a comparison of the CDR1 loops from IgNAR and two camelid V H H in Fig. S2. Type II model A model of the IgNAR Type II domain was constructed using the Swiss-PDB viewer (7) using the Type I IgNAR structure as the initial template (Fig. S3). The CDR3 region was modeled based on the CDR3 from the camel V H H domain AMD10. Additional energy minimization was carried out with CNS (4). The 81 non-cdr3 residues have a sequence identity of 89% between the Type I and Type II domains; however, no sequence homology is found in the 21 residue CDR3 region. The Type II IgNAR has non-canonical cysteines at positions 29 (CDR1) and 93 (CDR3) which were modeled to form a disulfide bond. Positive and Negative selection While the HV2 mutation rate is lower in Type II than in Type I clones, HV2 in both Type I and II IgNAR V is a hotspot for amino acid insertions and deletions where, in one instance, 5 residues are inserted (8). Other positively-selected residues N62, N63 and N64 occur within 4

5 another loop (between strands D and E) adjacent to CDR1, in a region homologous to hypervariable region 4 (HV4) of T cell receptors (Fig. 3c), and so could also conceivably participate in antigen recognition. Highly conserved residues under negative selection included Tyr N37, located between the two CDR3-constraining disulfide bonds where it forms hydrogen bonds with main-chain Asp N93 O, Asp N93 N, and a water molecule. Also under negative selection are the charged and solvent-exposed residues Asp N26, Arg N38, Lys N39, Glu N47 and Glu N76. The conservation of these charged residues is probably necessary to maintain protein solubility, and may be especially important under the high salt, high urea conditions that IgNAR V regions encounter in vivo. Residues Gly N52, Gly N53, Arg N54 and Tyr N55 are also highly conserved in the V regions of all cartilaginous fish species found to have IgNAR to date (9). These residues form a type II' turn leading into strand D, with Arg N54 forming a charged interaction with Asp N77, and Tyr N55 filling a cavity between residues Ile N49 and the Arg N54. While the IgNAR conformation for N52-N53 differs from that seen in mouse and human Ig domains, N54 and N55 have structurally-equivalent residues (L61,L62 or H66,H67), the Arg N54 -Asp N77 interaction and the Tyr N55 -Ile N49 -Arg N54 trio are also highly conserved at equivalent positions in human and mouse V κ and V H domains. Structure Analysis RMS deviations were calculated with ProFit (10). Buried molecular surface areas were calculated with the program MS (11) with a 1.7Å probe radius and standard van der Waals radii (12). Hydrogen bonds were evaluated using the program HBPLUS (13) and van der Waals contacts were assigned with the program Contacsym (14). Structural comparisons with all structures in the PDB were facilitated with the programs Dali ( (15) 5

6 and SSM ( (16). The shape complementarity index was calculated with the program SC (17). Figures Figures 1-4, S2-S4 and Figure S1 were calculated with Molscript (18) and Bobscript (19), respectively. Surfaces in Figure 1 were calculated with Harmony (20) and surfaces in Figure S4 were calculated with Grasp (21). All figures were rendered with Raster3D (22). PDB coordinates used in figures are 1LSZ (HEL D52S mutant in complex with oligosaccharide (23)), 1A6V (IgG V κ domain from Fv B1-8 (24)),1KJ2 (TCR V α domain from TCR KB5-C20 (25)),1GGB (IgG C L domain from Fab 50.1 (26)), 1DBB (IgG V H domain from Fab DB3 (27)), 1JTT (Camel V H H cab-lys3 (28)), 1KXV (camel V H H AMD10 (29)), 1BZQ (camel V H H cab-rn05 (30)) and 1HZH (human IgG b12 (31). SOM References 1. H. Dooley, M. F. Flajnik, A. J. Porter, Mol. Immunol. 40, 25 (2003). 2. C. R. Kissinger, D. K. Gehlhaar, D. B. Fogel, Acta Crystallogr. D55, 484 (1999). 3. A. Harrenga, H. Michel, J. Biol. Chem. 274, (1999). 4. A. T. Brünger et al., Acta Crystallogr. D54, 905 (1998). 5. T. A. Jones, in Computational chemistry D. Sayre, Ed. (Clarendon Press, Oxford, 1982) pp CCP4, Acta Crystallogr. D50, 760 (1994). 7. N. Guex, M. C. Peitsch, Electrophoresis 18, 2714 (1997). 8. M. Diaz, J. Velez, M. Singh, J. Cerny, M. F. Flajnik, Int. Immunol. 11, 825 (1999). 6

7 9. M. F. Flajnik, unpublished observation. 10. A. C. R. Martin, SciTech Software (London, 1996). 11. M. L. Connolly, J. Mol. Graph. 11, 139 (1993). 12. B. R. Gelin, M. Karplus, Biochemistry 18, 1256 (1979). 13. I. K. McDonald, J. M. Thornton, J. Mol. Biol. 238, 777 (1994). 14. S. Sheriff et al., Proc. Natl. Acad. Sci. U.S.A. 84, 8075 (1987). 15. L. Holm, C. Sander, J. Mol. Biol. 233, 123 (1993). 16. E. Krissinel, K. Henrick, in 5th International Conference on Molecular Structural Biology, A. J. Kungl, P. J. Kungl, Eds. (Vienna, 2003) p M. C. Lawrence, P. M. Colman, J. Mol. Biol. 234, 946 (1993). 18. P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991). 19. R. M. Esnouf, Acta Crystallogr. D55, 938 (1999). 20. B. S. Duncan, A. J. Olson, Biopolymers 33, 219 (1993). 21. A. Nicholls, K. A. Sharp, B. Honig, Proteins Struct. Funct. Genet. 11, 281 (1991). 22. E. A. Merritt, D. J. Bacon, Meth. Enzymol. 277, 505 (1997). 23. A. T. Hadfield et al., J. Mol. Biol. 243, 856 (1994). 24. T. Simon, K. Rajewsky, Protein Eng. 5, 229 (1992). 25. J. B. Reiser et al., Immunity 16, 345 (2002). 26. J. M. Rini et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6325 (1993). 27. J. H. Arevalo, E. A. Stura, M. J. Taussig, I. A. Wilson, J. Mol. Biol. 231, 103 (1993). 28. K. Decanniere et al., J. Mol. Biol. 313, 473 (2001). 29. A. Desmyter et al., J. Biol. Chem. 277, (2002). 30. K. Decanniere et al., Structure Fold. Des. 7, 361 (1999). 7

8 31. E. O. Saphire et al., Science 293, 1155 (2001). 8

9 Table S1. Summary of crystallographic data. Data collection Form 1 Form 2 Wavelength (Å) Resolution (Å) ( ) ( ) 1 Space group; a,b,c (Å) P ; 74.02, , P ; 75.79,113.81,58.44 # of observations (2774) (12331) 1 # of unique reflections (1131) (2730) Completeness (%) 93.1 (52.6) (97.7) 3 R sym (%) 5.4 (39.4) 9.0 (49.7) Average I/σ 37.9 (2.0) 14.1 (2.3) Refinement statistics (all refl. > 0.0 σf ) Resolution (Å) ( ) ( ) # reflections (working set) # reflections (test set) R cryst (%) 19.6 (30.8) 19.4 (22.3) 5 R free (%) 22.5 (36.4) 22.1 (28.2) # of IgNAR atoms # of HEL atoms # of waters Average B-values (Å 2 ) IgNAR 20 12, 14 HEL 16 12, 10 Waters Wilson B-value (Å 2 ) 17 ( Å) 12 ( Å) Ramachandran Plot (%) Most favored Additionally allowed Generously allowed Disallowed R.m.s. deviations Bond lengths (Å) Angles ( ) Numbers in parentheses are for the highest resolution shell of data. 2 The data in the Å shell are 97.3% complete, with completeness falling off only in the very highest resolution shell. 3 R sym = Σ hkl I- <I> / Σ hkl I 4 R cryst = Σ hkl F o -F c / Σ hkl F o 5 R free is the same as R cryst, but for 5% of the data excluded from the refinement 9

10 6 Residue Ser N41 is in a distorted type II turn in crystal form 1 (i+1 residue N41 φ=2, ψ=-120 ; i+2 residue N42 φ=-84, ψ=20 ), but in a type II turn in both molecules in crystal form 2. The electron density is weak in both crystal forms, indicating mobility of this loop. 10

11 Table S2. van der Waals contacts between IgNAR HEL-5A7 V and HEL. Contacts were evaluated with Contacsym (14). IgNAR V residue HEL contact residue Tyr N29 Gly N32 Ser N33 Gly N86 Val N87 Ala N88 Gly N89 Tyr N91 Asp N93 Ala N95 Leu N96 Arg N100 Tyr N101 Gly L102, Asn L103, Asn L106 Asp L101 Asp L101 Asp L101 Trp L63, Leu L75, Asp L101 Trp L63, Leu L75, Lys L97, Asp L101 Asp L101 Trp L62, Arg L73, Leu L75 Trp L62, Arg L73 Arg L61, Trp L62 Trp L62 Asp L52, Asn L103, Ala L107, Val L109, Arg L112 Asn L59, Trp L62, Trp L63, Ile L98, Asn L103, Ala L107 11

12 Table S3. Buried surface areas for HEL-antibody interactions. Buried surface areas were calculated with the program MS (11) with a 1.7Å probe radius and standard van der Waals radii (12). The D1.3, D44.1, HuLys11, and HyHEL antibodies are all murine Fab or F v fragments, while the cab-hul6 and cab-lys3 are camelid V H H domains. Antibody name Buried surface area (Å 2 ) PDB code HEL Antibody HEL-5A7 IgNAR V SQ2 D FDL D MLC HuLys BVK HyHEL HFL HyHEL HFM HyHEL DQJ HyHEL NDM HyHEL NDG cab-hul OP9 cab-lys JTO 12

13 Table S4. Hydrogen bonds and salt bridge interactions between IgNAR HEL-5A7 V region and HEL. Hydrogen bonds were evaluated with HBPLUS (13). IgNAR V HEL Distance (Å) Ser N33 N (CDR1) Asp L101 Oδ Ser N33 Oγ (CDR1) Asp L101 Oδ Gly N89 N (CDR3) Asp L101 Oδ Gly N90 N (CDR3) Asp L101 Oδ Tyr N91 OH (CDR3) Arg L73 Nε 3.13 Asp N93 Oδ2 (CDR3) Arg L73 Nε 3.22 Asp N93 Oδ2 (CDR3) Arg L73 NH Arg N100 NH1 (CDR3) Asp L52 Oδ Arg N100 NH2 (CDR3) Asp L52 Oδ Arg N100 O (CDR3) Asn L103 Nδ Arg N100 O (CDR3) Arg L112 NH

14 SOM Figure Legends Figure S1. Stereoview of typical electron density from the Type I IgNAR V domain crystal structure (crystal form 1). The 2Fo-Fc electron density (green) for CDR1 is contoured at 2σ. Figure S2. CDR1 loops from IgNAR V, cab-rn05 (1BZQ), and cab-lys3 (1JTT). The IgNAR CDR 1 shares the canonical Type 4 conformation found in cab-rn05 and cab-lys3. The IgNAR CDR1 has root mean square (RMS) deviations from the cab-lys3 and cab-rn05 CDR1s of 1.2Å and 1.5Å respectively, for 15 Ca atoms (IgNAR residues N22-N36). Figure S3. Model of the IgNAR Type II V domain. (a) IgNAR Type I structure (b) model of IgNAR Type II (c) Camel V H H AMD10 structure (1KXV). The IgNAR Type II V domain was modeled using the germline Type II sequence and the IgNAR Type I V domain structure as a guide. The position of the single disulfide (Cys29-Cys93) would constrain CDR3 and extend it as for the AMD10 CDR3 (Cys30-Cys106). Interestingly, at Kabat position H93, both the AMD10 (residue 96) and the IgNAR Type II V model (residue N84) have a Lys, while this position is a Gly in IgNAR Type I or other camelid structures (Gly N84 in IgNAR). This residue is covered by the folded-over CDR3 in IgNAR Type I (see Fig. S3a) and most camelid domains, but when exposed in AMD10 (and probably IgNAR Type II V s), is a more hydrophilic lysine. Figure S4. Waters at the HEL-IgNAR V region interface. The IgNAR V and HEL surfaces have been opened by rotating one subunit by 180 around a vertical axis. Waters that are totally buried in the interface are shown in red, while the waters at the borders of the interface are shown in cyan. 14

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