- Email: [email protected]
Molecular Recognition by a Binary Code
doi:10.1016/j.jmb.2005.03.041
J. Mol. Biol. (2005) 348, 1153–1162
Molecular Recognition by a Binary Code Frederic A. Fellouse, Bing Li, Deanne M. Compaan, Andrew A. Peden Sarah G. Hymowitz and Sachdev S. Sidhu* Department of Protein Engineering, Genentech Inc 1 DNA Way, South San Francisco, CA 94080, USA
Functional antibodies were obtained from a library of antigen-binding sites generated by a binary code restricted to tyrosine and serine. An antibody raised against human vascular endothelial growth factor recognized the antigen with high affinity (KDZ60 nM) and high specificity in cell-based assays. The crystal structure of another antigen binding fragment in complex with its antigen (human death receptor DR5) revealed the structural basis for this minimalist mode of molecular recognition. Natural antigen-binding sites are enriched for tyrosine and serine, and we show that these amino acid residues are intrinsically well suited for molecular recognition. Furthermore, these results demonstrate that molecular recognition can evolve from even the simplest chemical diversity. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: phage display; protein engineering; combinatorial mutagenesis; antibody library; vascular endothelial growth factor
Introduction The natural immune repertoire can generate antibodies that recognize essentially any antigen with high specificity and affinity. Antigen recognition is mediated by six complementarity determining regions (CDRs)1,2 that present a large surface for contact with antigen.3,4 CDR sequences are hypervariable, but it has become clear that the overall composition of functional CDRs is biased in favor of certain amino acid types.5–11 In particular, tyrosine and serine residues are highly abundant, and tyrosine residues are involved in a disproportionate number of antigen contacts.12–14 The overabundance of tyrosine and serine residues may be a coincidental consequence of biases that exist in the sequences of antibody genes.5,15–18 Alternatively, it is possible that these amino acid residues are particularly well suited for molecular recognition, and as a result, the immune system has evolved under selective pressure for the
Abbreviations used: CDR, complementarity determining region; CDR-Hn (where nZ1,2 or 3), heavy chain CDR 1,2 or 3; CDR-L3, light chain CDR3; ECD, extracellular domain; ELISA, enzyme-linked immunosorbent assay; Fab, antigen-binding fragment; hDR5, human death receptor DR5; hVEGF, human vascular endothelial growth factor; GFP, green fluorescent protein. E-mail address of the corresponding author: [email protected]
enrichment of tyrosine and serine in functional antigen-binding sites.5,12,19,20 We previously addressed this question by generating synthetic antibody libraries with solvent accessible CDR positions randomized with a degenerate codon that encodes for only four amino acid residues (tyrosine, serine, alanine and aspartate).20 Surprisingly, we were able to generate high affinity antibodies against human vascular endothelial growth factor (hVEGF), an angiogenic hormone that has been implicated in tumorogenesis.21,22 Structural analyses suggested that it may be possible to simplify the code for antigen recognition even further, as most of the contacts with antigen were mediated by tyrosine sidechains, while aspartate was almost entirely excluded from the binding sites. We now show that a binary code, restricted to only tyrosine and serine, is sufficient to mediate antigen recognition with high affinity and specificity. Thus, it appears that these amino acid residues are particularly well suited for molecular recognition, and furthermore, naı¨ve antigen recognition can evolve from even the simplest chemical diversity.
Results Antibodies from a binary code We constructed two libraries of phage-displayed antigen-binding fragments (Fabs) by randomizing
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
1154 CDR positions within a humanized Fab framework with a binary degenerate codon that encodes for equal proportions of tyrosine and serine. Solventaccessible positions in the first and second CDRs of the heavy chain (CDR-H1 and CDR-H2) were randomized, and random loops of variable lengths were inserted in place of seven residues in the third heavy chain CDR (CDR-H3). The libraries differed in that library A had a fixed light chain while the third CDR of the light chain (CDR-L3) was randomized in library B. Each library contained w1010 unique members, and thus, the actual library diversities were comparable to the maximum number of unique sequences encoded by each library design (w4!109). The libraries were used to generate Fabs against a panel of six protein antigens. Specific binding clones were identified by phage enzyme-linked immunosorbent assay (ELISA) screening, and were defined as Fab-phage that bound to the cognate antigen but did not exhibit detectable binding to seven other proteins. Specific Fabs were isolated against all six antigens and DNA sequencing revealed diverse CDR sequences that contained only tyrosine and serine at the randomized sites (Figure 1). Competitive phage ELISAs were used to estimate affinities as IC50 values. Fabs exhibited high affinity for four of the antigens (IC50!100 nM),
Molecular Recognition by a Binary Code
moderate affinity for insulin (IC50Z1.4 mM) and low affinity for maltose binding protein (IC50O5 mM). Thus, the binary chemical diversity was sufficient to mediate specific recognition of all six antigens, and for four of the antigens the binding interactions were of high affinity. Fabs against two of the antigens (hVEGF and human death receptor DR5 (hDR5)) were chosen for further functional and structural characterization. Functional characterization of anti-hVEGF Fabs DNA sequencing of 184 hVEGF-binding clones revealed 63 unique sequences, and some of these are shown in Figure 2(a). Most of the clones were from library B but some clones were also isolated from library A. Interestingly, the clones from library B exhibit homology within the selected CDR-L3 and CDR-H3 sequences. In contrast, the CDR-H3 sequences from library A exhibit homology amongst themselves but are very different from the sequences from library B. Thus, it appears that the nature of the CDR-L3 sequence influenced the selection of CDR-H3 sequences, and as a result, two distinct classes of anti-hVEGF antibodies arose from the different libraries. The clones were screened by competitive phage ELISA and exhibited IC50 values
Figure 1. Sequences and specificity profiles of synthetic Fabs. (a) CDR sequences in the randomized regions. The CDRL3 sequence is not shown for Fabs selected from library A in which the light chain was not randomized. Tyrosine and serine residues are shown in yellow or red, respectively. Residues in grey are buried residues that were not randomized in the libraries. The numbering is according to the nomenclature of Kabat et al.1 (b) Fab-phage selected for binding to a particular antigen (cognate antigen, x-axis) were assayed for binding to various immobilized proteins by phage ELISA and bound phage were detected spectrophotometrically (optical density at 450 nm, y-axis). Affinities for cognate antigen were estimated as IC50 values by competitive phage ELISA.37,38 The following proteins were used: neutravidin (NAV, red bar), maltose binding protein (MBP, orange bar), Erbin PDZ domain (PDZ, yellow bar), insulin (green bar), human vascular endothelial growth factor (hVEGF, blue bar), human death receptor 5 (hDR5, pink bar), glutathione-Stransferase (purple bar), human immunoglobulin G (black bar), bovine serum albumin (white bar).
Molecular Recognition by a Binary Code
1155
Figure 2. Affinity and specificity of anti-hVEGF Fabs. (a) CDR sequences of anti-hVEGF Fabs. The numbering is according to the nomenclature of Kabat et al.1 and only positions that were randomized in the libraries are shown. Clones 1 through 7 were isolated from library B, while clones 8 through 14 were isolated from library A. Dashes indicate gaps in the alignment. (b) Kinetic analysis of Fabs binding to immobilized hVEGF. The surface plasmon resonance traces are shown for Fab-YSv1 at four different concentrations (63 nM, 125 nM, 250 nM and 500 nM). Experimental data are represented by open circles and the global fits are represented by continuous lines. Kinetic parameters were determined for Fabs YSv1, YSv2 and YSv3. (c) Immunofluorescence staining with FabYSv1 (red) performed on A673 cells expressing murine VEGFGFP (green). VEGF-GFP and FabYSv1 staining co-localize in the extracellular space formed between cell-to-cell contacts (merge, yellow). The Fab-YSv1 staining was completely abolished by pre-incubation of the antibody with excess recombinant hVEGF (C VEGF panels). The scale bar represents 20 mm. (d) Immunoprecipitations performed on media collected from metabolically labeled A673 cells. Fab-YSv1 and monoclonal antibody A4.6.1 show comparable specificity, as evidenced by identical patterns of bands for precipitated hVEGF isoforms. Anti-GFP polyclonal antibody was used as a negative control.
ranging from approximately 50 nM to greater than 5 mM. The three anti-hVEGF clones with the highest estimated affinities (top three sequences in Figure 2(a)) were purified as free Fab proteins. The binding kinetics of the purified Fabs (designated YSv1, YSv2 and YSv3) were studied by surface plasmon resonance (Figure 2(b)). Fab-YSv1 exhibited the highest affinity for hVEGF (KdZ60 nM), while the other two Fabs bound approximately fivefold less tightly due to faster off rates. It is notable that the sequences of Fab-YSv1 and Fab-YSv2 differ in only three positions, and thus, these three differences account for the improved affinity of Fab-YSv1 in comparison with Fab-YSv2. We next investigated the specificity of Fab-YSv1 by using the protein to visualize VEGF in
mammalian cells transfected with a gene encoding for VEGF fused to green fluorescent protein (GFP). The immunohistochemical staining with Fab-YSv1 precisely overlapped with the fluorescence signal from the VEGF-GFP fusion (Figure 2(c)). Furthermore, the signal was completely blocked by incubating Fab-YSv1 with hVEGF prior to staining. We also conducted immunoprecipitations of endogenous hVEGF and compared the performance of Fab-YSv1 to that of a highly specific, natural anti-hVEGF monoclonal antibody (A4.6.1).23 Both antibodies immunoprecipitated an identical set of bands (Figure 2(d)) that likely represent hVEGF variants generated by alternative mRNA splicing.23 Taken together, these results show that Fab-YSv1 binds to hVEGF with high affinity and specificity comparable to that of a natural antibody, even in the complex cellular milieu.
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Molecular Recognition by a Binary Code
Structural characterization of an anti-hDR5 Fab
Table 1. Data collection and refinement statistics for the Fab-YSd1:hDR5-ECD complex
To gain insights into the structural basis for antigen recognition by the binary code, we studied a Fab that was selected for binding to the extracellular domain of hDR5 (hDR5-ECD), a cellsurface receptor that mediates apoptotic cell death.24 Nine unique anti-hDR5 clones were obtained, and the Fab that exhibited the highest estimated affinity by competitive phage ELISA (YSd1; Figure 1(a)) also bound to hDR5 with high affinity in surface plasmon resonance experiments (KdZ34 nM). The crystal structure of Fab-YSd1 in complex with the hDR5-ECD was solved and refined to ˚ resolution (Figure 3(a) and Table 1). While 3.35 A the structure is only of modest resolution, the quality of the structure is quite good with 98.9%
A. Diffraction data Space group ˚) Unit cell constants (A ˚) Resolution (A Rsymb hI/sIi No. of reflections No. of unique reflections Completeness (%) B. Refinement ˚) Resolution (A No. of reflections Rwork, Rfreec No. of residues No. of non-H atoms ˚) rmsd bond length (A rmsd angles (deg.) ˚ 2) rmsd B (bonded atoms) (A Ramachandran plot (%)d
P3221 aZ147, cZ145 50–3.35 (3.47–3.35)a 0.12 (0.34)a 8.3 (4.5)a 149,117 26,539 99.7 (99.8)a 30–3.35 26,003 0.224, 0.280 (0.232, 0.281) 1056 8,026 0.012 1.3 2.3 83.4, 15.5, 0.7, 0.4
a
Numbers in parentheses refer to the highest resolution shell. Rsym Z SjIK hIij=SI, where hIi is the average intensity of symmetry related observations of a unique reflection. c Rwork Z SjFo K Fc j=SFo , where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is the R-factor for a randomly selected 10% of the reflections excluded from all refinement. d Percentage of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of a Ramachandran plot. b
Figure 3. The structure of Fab-YSd1 bound to the hDR5ECD. (a) The complex of the hDR5-ECD with Fab-YSd1. The hDR5-ECD is depicted as a white molecular surface. The main-chain of Fab-YSd1 is shown as sticks; the light and heavy chains are shown in green and blue, respectively, and CDR-H3 is shown in yellow. (b) Experimental density for CDR-H3. The FoKFc omit map contoured at 2.75s shows unbiased electron density (blue) for CDR-H3. Phase bias was removed by omitting CDR-H3 prior to refinement. The coordinates of the final refined model for CDR-H3 are shown as sticks, colored according to atom type (carbon, yellow; oxygen, red; nitrogen, blue), with side-chains rendered as sticks. The hDR5-ECD is shown as a white molecular surface. Despite the relatively modest resolution of the structure ˚ ), the electron density maps are of high quality and (3.35 A allow for accurate determination of backbone and sidechain positions.
of residues in the most favored or additionally allowed parts of the Ramachandran plot. The data quality was limited by the availability of single crystals. All crystals showed evidence of more than one lattice, and thus, to collect a high quality data set, it was necessary to screen many crystals and to then find the most single portion of the best crystal. Nonetheless, the maps were of excellent clarity and the initial maps calculated with phases from the molecular replacement solution showed continuous density for CDR-H3 with side-chain density for many of the tyrosine residues (Figure 3(b)). The presence of two copies of the Fab:antigen complex in the asymmetric unit permitted the use of NCS restraints which aided the refinement process. Additionally, tyrosine is a large side-chain with a distinctive shape, and thus, it can be positioned ˚. accurately even in maps at a resolution of 3.35 A Other than the unusual CDR-H3 conformation, the structure of Fab-YSd1 is similar to that of the humanized Fab-4D5 variant that was used as the scaffold for the antibody library20,25 (Figure 4(a)), although the elbow angles differ due to differences in crystal packing. Overall, the structure shows that Fab-YSd1 interacts with antigen in a normal manner, but tyrosine residues form most of the contact surface (Figure 4(b) and (c)). Antigen ˚ 2 on the binding results in the burial of 460 A 2 ˚ on the light chain. Tyrosine heavy chain and 270 A and serine residues within CDRs H2, H3 and L3 account for 58% and 21% of the buried surface area, respectively. The remainder of the buried surface area (21%) is contributed by CDR-L1, which was not randomized and lies on the periphery of the
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Molecular Recognition by a Binary Code
interface (Figure 4(b)). On the antigen side of the interface, the buried surface area is contributed by a variety of amino acid types, but aromatic residues only account for a small fraction (14%) of the structural epitope. Thus, tyrosine plays a dominant role in antigen recognition by Fab-YSd1, but it does so by utilizing interactions with a diverse array of functional groups on the antigen, and interactions with aromatic side-chains do not play a major role. The very long CDR-H3 of Fab-YSd1 protrudes from the framework and makes extensive contact with hDR5, contributing 39% of the surface area ˚ 2). Structures of buried upon antigen binding (288 A antibodies with long CDR-H3 loops have been reported,26–28 but Fab-YSd1 is unique in that CDRH3 contains a “biphasic” helix with tyrosine and serine residues clustered on opposite faces (Figure 4(d)). The tyrosine face is buried against the surface of hDR5, and as a result, 99% of the CDR-H3 accessible surface buried upon binding to hDR5 is contributed by tyrosine, and serine residues likely play structural roles. In this regard, CDR-H3 exemplifies how even the most minimalist binary diversity can give rise to a structural solution that mediates antigen recognition with high affinity and specificity. Fab-YSd1 binds to the N-terminal portion of hDR5 and occludes the smaller of two binding sites for the natural hDR5 ligand Apo2L/TRAIL29 (Figure 5(a)). Intriguingly, one of the Apo2L/ TRAIL residues that contributes the most binding energy to the interaction with hDR5 is Tyr216, as substitution of this residue by alanine causes a 320fold loss in biological activity and a ninefold loss in affinity for hDR5.30 Tyr216 binds in a hydrophobic pocket in cysteine-rich domain 2 of hDR5 29 (Figure 5(b)), resulting in a snug fit that buries greater than 90% of the side-chain. Although FabYSd1 uses many tyrosine residues to bind to hDR5, none of them precisely mimics this interaction. Tyr56 of the heavy chain occupies a similar position as Tyr216 of Apo2L/TRAIL, but it lies along the antigen surface and only 60% of the side-chain is buried (Figure 5(b)). This structure is also the first for a tissue necrosis factor receptor (TNFR) family member bound to an antibody fragment, and as such, it provides insight into how an antibody recognizes antigens of this type. Overall, the structure of hDR5 bound to FabYSd1 is similar to that of hDR5 bound to Apo2L/ TRAIL.29 Cysteine-rich domains 1 and 2 (residues 22–86) from both structures superimpose on each other with a pair-wise root-mean-square deviation ˚ 2 on Ca atoms. This is the (rmsd) of less than 0.30 A same level of structural conservation observed amongst independent structures of hDR5 bound to Apo2L/TRAIL. 29 As in the Apo2L:hDR5 complex structure, which contains three crystallographically independent copies of hDR5, the orientation of cysteine-rich domain 3 in the two independent copies of hDR5 bound to Fab-YSd1 varies somewhat with respect to cysteine-rich domains 1 and 2, although variations between the
hDR5 molecules in the Fab-YSd1 and Apo2L/ TRAIL complexes are more extreme than those within either group. Thus, in both complexes, it appears that cysteine-rich domains 1 and 2 behave as a rigid body, while in contrast the third domain exhibits flexibility with respect to the rest of hDR5. Cysteine-rich domain 3 does not make contact with Fab-YSd1, but it makes extensive contact with Apo2L/TRAIL (Figure 5(a)). In both copies of the Fab:antigen complex contained within the asymmetric unit, cysteine-rich domain 3 is less well defined than it is in the structure of hDR5 bound to Apo2L/TRAIL. In particular, residues 91–104 exhibit poor density in the complex with Fab-YSd1, but this region is well-ordered when bound to Apo2L/TRAIL, and taken together, these differences suggest that the loop is not well-ordered in the absence of Apo2L/TRAIL. The membrane proximal end of cysteine-rich domain 3, including the last disulfide, is also poorly ordered in the complex with Fab-YSd1. Disorder is not uncommon in crystal structures of members of the TNFR family, as for example, the entire final cysteine-rich domain of TNFR1 is disordered in the complex with lymphotoxin.31
Discussion Despite extreme restrictions on chemical diversity, we isolated synthetic Fabs with affinities comparable to those obtained from naı¨ve libraries designed with high chemical diversity. Furthermore, the affinities compare favorably with the affinities of antibodies from the naı¨ve immune response, which are often in the micromolar range prior to affinity maturation through somatic mutation mechanisms. 16 Our results provide insight into how the naı¨ve immune repertoire can evolve high affinity antibodies against a seemingly endless array of antigens. It appears that the selection of antibodies with affinities and specificities sufficient for initial selection from a naı¨ve pool is a relatively simple process that may require only a few productive binding contacts. Thus, the limited diversity of the naı¨ve immune repertoire32 is sufficient to generate initial binding clones that can then attain improved function through subsequent cycles of affinity maturation. We focused our study on protein antigens, but it will be interesting to see if the limited diversity approach will be successful against other antigen classes (e.g. small molecules or carbohydrates). With the exception of CDR-H3, we also restricted CDR conformational diversity, as we did not randomize buried residues which influence CDR main-chain conformations.33 Thus, it may be possible to obtain further improvements in binding by targeting these regions and framework positions that are known to modulate affinity.25,34 The binary binding surfaces represent a minimal benchmark for antigen recognition into which additional diversity and conformational flexibility can be
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Molecular Recognition by a Binary Code
Figure 4 (legend opposite)
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Molecular Recognition by a Binary Code
introduced to study the details of affinity maturation. In particular, these systems may be very amenable to computer modeling studies, since both serine and tyrosine have few rotamers, and the simplicity of the system should simplify the computational process. Our results suggest that the high abundance of tyrosine and serine in antibody combining sites reflects an innate capacity for these amino acid residues to mediate antigen recognition. Both residues are hydrophilic and thus can be exposed on surfaces without promoting non-specific aggregation, but at the same time, both can also be accommodated readily at protein–protein interfaces. Because of its small size and neutral nature, the serine side-chain is unlikely to cause unfavorable steric hindrance or electrostatic repulsions. On the other hand, the large tyrosine side-chain contributes substantial surface area for contact, and yet, it has a unique chemical nature which allows it to participate in favorable binding interactions with many different types of functional groups.5,9,12,20 We hypothesize that the large tyrosine side-chain acts as a functional group that makes key binding contacts with antigen, while the small serine side-chain serves as an auxiliary group that provides space and flexibility for tyrosine to establish optimal binding contacts. Due to their complementary nature, it appears that tyrosine and serine may be particularly well suited to work together in antigen recognition. Much effort has been expended on the development of large, highly diverse libraries that can be used to obtain antagonists and agonists of biological processes.35–38 However, our results demonstrate that it is possible, and perhaps even advantageous, to restrict chemical diversity to small subsets of functional groups that are particularly well suited for mediating molecular recognition.
Materials and Methods Library construction and sorting A phagemid designed to display bivalent, humanized Fab-4D5 on the surface of M13 bacteriophage was used to
construct libraries, as described.20,39 Oligonucleotidedirected mutagenesis was used to replace CDR positions with TMT degenerate codons, (MZA/C in equal proportions). The positions chosen for randomization in CDR-L3, CDR-H1 and CDR-H2 are shown in Figure 1. In CDR-H3, positions 95 through 100a were replaced with random loops of all possible lengths ranging from 7 to 20 residues (library A) or 7 to 15 residues (library B). Phage from the libraries were cycled through rounds of binding selection with antigen immobilized on 96 well Maxisorp immunoplates (NUNC) as the capture target, as described. After five rounds of selection, phage were produced from individual clones grown in a 96 well format and the culture supernatants were used in phage ELISAs to detect specific binding clones. Competitive phage ELISA A modified phage ELISA37,38 was used to estimate the binding affinities of Fabs. Phage ELISAs were carried out on plates coated with antigen, as described above. Phage displaying antibody fragments were serially diluted in phosphate-buffered saline (PBS), 0.5% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20, and binding was measured to determine a phage concentration giving w50% of the signal at saturation. A fixed, sub-saturating concentration of phage was pre-incubated for two hours with serial dilutions of antigen and then transferred to assay plates coated with antigen. After 15 minutes incubation, the plates were washed with PBS, 0.05% Tween 20 and incubated 30 minutes with horseradish peroxidase/anti-M13 antibody conjugate (1:5000 dilution) (Pharmacia). The plates were washed, developed with TMB substrate (Kirkegaard and Perry Laboratories), quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm. The binding affinities of were estimated as IC 50 values defined as the concentration of antigen that blocked 50% of the phage binding to the immobilized antigen. Protein purification and affinity analysis Fab proteins were purified from Escherichia coli as described.40 Binding kinetics were determined by surface plasmon resonance using a BIAcoree-3000 with hVEGF immobilized on CM5 chips at w500 response units, as described.41 Serial dilutions of Fab proteins were injected, and binding responses were corrected by subtraction of responses on a blank flow cell. For kinetic analysis, a 1:1 Langmuir model of global fittings of kon and koff was used.
Figure 4. Antigen binding site of Fab-YSd1 and the binding interface with hDR5-ECD. (a) Stereo view of the superimposition of the variable domains of Fab-YSd1 and humanized Fab-4D5 (1N8Z).46 The Fab variable domains are represented as a cartoon. CDR-H3 of Fab-YSd1 is colored yellow; the rest of the Fab-YSd1 heavy chain is in blue and the light chain is in green. Humanized Fab-4D5 heavy and light chains are colored cyan and red, respectively. (b) Stereo view ˚ ). The CDR loops are labeled and are of CDR loops that contain residues in contact with the hDR5-ECD (closer than 4.5 A shown as transparent cartoons. CDR side-chains that make contact with the hDR5-ECD are rendered as sticks and are colored as follows: tyrosine, blue; serine, green; all other amino acids, white. A fragment of the hDR5-ECD is shown as a molecular surface; residues in contact with the heavy or light chains are colored yellow or red, respectively, and residues in contact with both are colored orange. (c) The amino acid composition of the interface between Fab-YSd1 and the hDR5-ECD. Each circle represents the surface area on Fab-YSd1 or the hDR5-ECD that becomes buried upon complex formation. The colors indicate the proportion of the buried surface area contributed by each amino acid type, and all amino acid types that contribute more than 1% of the total buried surface area on each molecule are shown. (d) Contacts between CDR-H3 and the hDR5-ECD. The hDR5-ECD is shown as a molecular surface and residues that contact CDRH3 are colored pink. The CDR-H3 main-chain is depicted as a yellow cartoon; tyrosine and serine side-chains are rendered as sticks and colored blue or green, respectively.
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Molecular Recognition by a Binary Code
The Kd values were determined from the ratios of kon and koff. Immunohistochemistry Human A673 cells expressing murine VEGF-GFP were stained and imaged, as described.42 In the plus VEGF panel, Fab-YSv1 was pre-incubated for five minutes with a fivefold excess of recombinant hVEGF before being incubated with the cells. Immunoprecipitation A673 cells were metabolically labeled and immunoprecipitations were performed from the media, as described,23 using 15 mg of anti-GFP polyclonal antibody (Clontech), Fab-YSv1 or monoclonal antibody A4.6.1. The immune complexes were eluted by boiling and resolved by SDS-PAGE on a 14% (w/v) acrylamide gel under reducing conditions. The gel was dried and then exposed to a phosphoimager plate overnight. Crystallization, structure determination and refinement DNA coding for the hDR5-ECD (residues 1–130) with an N-terminal His-tag was cloned into the baculovirus transfer vector pAcGP67-B (Pharmingen). Following transfection and viral amplification in Sf9 cells, protein was expressed and secreted from Hi5 cells grown at 27 8C. The culture medium was supplemented with 50 mM Tris (pH 8.0), 1 mM NiCl2, 5 mM CaCl2, and 1 mM phenylmethylsulfonyl fluoride. The pH was adjusted to pH 7.6 and the medium was filtered prior to loading on a NiNTA column (Qiagen). After elution with imidazole, the His-tag was removed by digestion with thrombin followed by purification on a Superdex-75 sizing column. Fab-YSd1 was expressed in E. coli and purified over a Protein G column.40 Fab-containing fractions were further purified by passage over a S-Sepharose column, followed by a Superdex-200 sizing column. Fab–receptor complex was made by mixing Fab-YSd1 with an excess of the hDR5-ECD domain, followed by purification over a Superdex-200 sizing column. The complex-containing fractions were pooled and concentrated to 11 mg/ml in 150 mM NaCl, 20 mM bis-Tris (pH 6.5) and used for crystallization trials. Crystals grew at 19 8C from hanging drops containing an equal volume of protein and well solution consisting of 20% polyethylene glycol (PEG) 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate (pH 6.2–6.6). Crystals were transferred to mother liquor containing the well solution with 20% PEG 300, prior to cryo-cooling by immersion in liquid nitrogen. The resulting crystals were visibly nonsingle and it was necessary to screen many crystals to find one that was mostly single. ˚ data set was collected at beamline 19BM at the A 3.35 A APS. Data processing was done with HKL.43 Calculation of the Matthew’s coefficient indicated that the asymmetric
Figure 5. Comparison of the interactions of hDR5 with Fab-YSd1 and Apo2L/TRAIL. (a) Mapping of the structural binding epitopes of hDR5 for Fab-YSd1 or Apo2L/TRAIL. The hDR5-ECD is shown as a molecular surface. The unshared portions of the epitopes for binding to Fab-YSd1 or Apo2L/TRAIL are colored yellow or blue, respectively, and the shared portion is in green.
(b) Comparison of Tyr56 of Fab-YSd1 and Tyr216 of Apo2L/TRAIL. The hDR5-ECD is shown as a molecular surface and colored as in (a). Tyr56 of Fab-YSd1 and Tyr216 of Apo2L/TRAIL are rendered as sticks and colored cyan or pink, respectively, as are the a-carbon traces of the surrounding loops. Structures were drawn with PyMOL (DeLano Scientific, San Carlos, CA).
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unit of the crystals contained two Fab:antigen complexes. The structure was solved in space group P3221 by molecular replacement with the program Phaser44 using the structures of two Fab-4D5 variants as search models.25 The resulting maps showed clear density for the hDR5ECD, which was placed manually, as well as for the Fab CDRs. Refinement was performed with the program REFMAC545 and included TLS refinement and NCS restraints imposed separately on the variable and constant domains of Fab-YSd1, as well as on the hDR5ECD.
8. 9.
10.
Protein Data Bank accession numbers The coordinates and structure factors for the FabYSd1:hDR5-ECD complex have been deposited in the RCSB Protein Data Bank (PDB:1ZA3).
11. 12.
Acknowledgements We are grateful to the Genentech Fermentation, DNA Synthesis and DNA Sequencing groups. We thank M. Franklin, A. Cochran, A. de Vos and H. Lowman for helpful discussions and H. Darbon for his support. We also thank Stephan L. Ginell, PhD of beamline 19BM at the APS (Advanced Photon Source). Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source, was supported by the US Department of Energy, Office of Energy Research, under contract no. W-31-109-ENG-38.
13. 14. 15.
16. 17.
18.
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combining sites: unusual structural features that may confer on these sites an enhanced capacity for binding ligands. Proteins: Struct. Funct. Genet. 7, 112–124. Lea, S. & Stuart, D. (1995). Analysis of antigenic surfaces of proteins. FASEB J. 9, 87–93. Ivanov, I., Link, J., Ippolito, G. C. & Schroeder, H. W., Jr (2002). Constraints on the hydropathicity and sequence composition of HCDR3 across evolution. In The Antibodies (Zanetti, M. & Capra, J., eds), pp. 43– 67, Taylor & Francis, London. Collis, A. V., Brouwer, A. P. & Martin, A. C. (2003). Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen. J. Mol. Biol. 325, 337–354. Lo Conte, L., Chothia, C. & Janin, J. (1999). The atomic structure of protein–protein recognition sites. J. Mol. Biol. 285, 2177–2198. Mian, I. S., Bradwell, A. R. & Olson, A. J. (1991). Structure, function and properties of antibody binding sites. J. Mol. Biol. 217, 133–151. Padlan, E. A. (1994). Anatomy of the antibody molecule. Mol. Immunol. 31, 169–217. Davies, D. R. & Cohen, G. H. (1996). Interactions of protein antigens with antibodies. Proc. Natl Acad. Sci. USA, 93, 7–12. Padlan, E. A. (1997). Does base composition help predispose the complementarity-determining regions of antibodies to hypermutation? Mol. Immunol. 34, 765–770. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature, 302, 575–581. Wilson, P. C., Wilson, K., Liu, Y. J., Banchereau, J., Pascual, V. & Capra, J. D. (2000). Receptor revision of immunoglobulin heavy chain variable region genes in normal human B lymphocytes. J. Expt. Med. 191, 1881–1894. Bassing, C. H., Swat, W. & Alt, F. W. (2002). The mechanism and regulation of chromosomal V(D)J recombination. Cell, 109, S45–S55. Villar, H. O. & Kauvar, L. M. (1994). Amino acid preferences at protein binding sites. FEBS Letters, 349, 125–130. Fellouse, F. A., Wiesmann, C. & Sidhu, S. S. (2005). Synthetic antibodies from a four amino acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl Acad. Sci. USA, 101, 12467–12472. Ferrara, N. (2001). Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. Cell Physiol. 280, C1358–C1366. Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med. 1, 27–31. Kim, K. J., Li, B., Houck, K., Winer, J. & Ferrara, N. (1992). The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors, 7, 53–64. Ashkenazi, A. & Dixit, V. M. (1998). Death receptors: signaling and modulation. Science, 281, 1305–1308. Eigenbrot, C., Randal, M., Presta, L., Carter, P. & Kossiakoff, A. A. (1993). X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling. J. Mol. Biol. 229, 969–995. Saphire, E. O., Parren, P. W. H. I., Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M. et al. (2001).
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Crystal structure of a neutralizing human IgG against HIV-1: a template for vaccine design. Science, 293, 1155–1159. Zwick, M. B., Komori, H. K., Stanfield, R. L., Church, S., Wang, M., Parren, P. W. H. I. et al. (2004). The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J. Virol. 78, 3155–3161. Darbha, R., Phogat, S., Labrijn, A. F., Shu, Y., Gu, Y., Andrykovitch, M. et al. (2004). Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope. Biochemistry, 43, 1410–1417. Hymowitz, S. G., Christinger, H. W., Fuh, G., Ultsch, M., O’Connell, M., Kelley, R. F. et al. (1999). Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol. Cell, 4, 563–571. Hymowitz, S. G., O’Connell, M. P., Ultsch, M. H., Hurst, A., Totpal, K., Ashkenazi, A. et al. (2000). A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry, 39, 633–640. Banner, D. W., D’Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C. et al. (1993). Crystal structure of the soluble human 55 kd TNF receptorhuman TNF beta complex: implications for TNF receptor activation. Cell, 73, 431–445. Silverstein, A. M. (2003). Splitting the difference: the germline-somatic mutation debate on generating antibody diversity. Nature Immunol. 4, 829–833. Al-Lazikani, B., Lesk, A. M. & Chothia, C. (1997). Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 273, 927–948. Chothia, C., Lesk, A. M., Gherardi, E., Tomlinson, I. M., Walter, G., Marks, J. D. et al. (1992). Structural repertoire of the human VH segments. J. Mol. Biol. 227, 799–817. Schaffitzel, C., Hanes, J., Jermutus, L. & Pluckthun, A. (1999). Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J. Immunol. Methods, 231, 119–135. Roberts, R. W. & Szostak, J. W. (1997). RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA, 94, 12297–12302.
37. Sidhu, S. S., Lowman, H. B., Cunningham, B. C. & Wells, J. A. (2000). Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363. 38. Deshayes, K., Schaffer, M. L., Skelton, N. J., Nakamura, G. R., Kadkhodayan, S. & Sidhu, S. S. (2002). Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function. Chem. Biol. 9, 495–505. 39. Lee, C. V., Liang, W.-C., Dennis, M. S., Eigenbrot, C., Sidhu, S. S. & Fuh, G. (2004). High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093. 40. Muller, Y. A., Chen, Y., Christinger, H. W., Li, B., Cunningham, B. C., Lowman, H. B. & de Vos, A. M. (1998). VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the com˚ resolution and mutational analysis of the plex at 2.4 A interface. Structure, 6, 1153–1167. 41. Chen, Y., Wiesmann, C., Fuh, G., Li, B., Christinger, H. W., McKay, P. et al. (1999). Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. J. Mol. Biol. 293, 865–881. 42. Peden, A. A., Oorschot, V., Hesser, B. A., Austin, C. D., Scheller, R. H. & Klumperman, J. (2004). Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins. J. Cell Biol. 164, 1065–1076. 43. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Macromol. Crystallog. Pt A, 276, 307–326. 44. Storoni, L. C., McCoy, A. J. & Read, R. J. (2004). Likelihood-enhanced fast rotation functions. Acta Crystallog. sect. D, 60, 432–438. 45. CCP4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallog. sect. D, 50, 760–763. 46. Cho, H.-S., Mason, K., Ramyar, K. X., Stanley, A. M., Gabelli, S. B., Denney, D. W. J. & Leahy, D. J. (2003). Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature, 421, 756–760.
Edited by I. Wilson (Received 22 December 2004; received in revised form 10 March 2005; accepted 14 March 2005)
J. Mol. Biol. (2005) 348, 1153–1162
Molecular Recognition by a Binary Code Frederic A. Fellouse, Bing Li, Deanne M. Compaan, Andrew A. Peden Sarah G. Hymowitz and Sachdev S. Sidhu* Department of Protein Engineering, Genentech Inc 1 DNA Way, South San Francisco, CA 94080, USA
Functional antibodies were obtained from a library of antigen-binding sites generated by a binary code restricted to tyrosine and serine. An antibody raised against human vascular endothelial growth factor recognized the antigen with high affinity (KDZ60 nM) and high specificity in cell-based assays. The crystal structure of another antigen binding fragment in complex with its antigen (human death receptor DR5) revealed the structural basis for this minimalist mode of molecular recognition. Natural antigen-binding sites are enriched for tyrosine and serine, and we show that these amino acid residues are intrinsically well suited for molecular recognition. Furthermore, these results demonstrate that molecular recognition can evolve from even the simplest chemical diversity. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: phage display; protein engineering; combinatorial mutagenesis; antibody library; vascular endothelial growth factor
Introduction The natural immune repertoire can generate antibodies that recognize essentially any antigen with high specificity and affinity. Antigen recognition is mediated by six complementarity determining regions (CDRs)1,2 that present a large surface for contact with antigen.3,4 CDR sequences are hypervariable, but it has become clear that the overall composition of functional CDRs is biased in favor of certain amino acid types.5–11 In particular, tyrosine and serine residues are highly abundant, and tyrosine residues are involved in a disproportionate number of antigen contacts.12–14 The overabundance of tyrosine and serine residues may be a coincidental consequence of biases that exist in the sequences of antibody genes.5,15–18 Alternatively, it is possible that these amino acid residues are particularly well suited for molecular recognition, and as a result, the immune system has evolved under selective pressure for the
Abbreviations used: CDR, complementarity determining region; CDR-Hn (where nZ1,2 or 3), heavy chain CDR 1,2 or 3; CDR-L3, light chain CDR3; ECD, extracellular domain; ELISA, enzyme-linked immunosorbent assay; Fab, antigen-binding fragment; hDR5, human death receptor DR5; hVEGF, human vascular endothelial growth factor; GFP, green fluorescent protein. E-mail address of the corresponding author: [email protected]
enrichment of tyrosine and serine in functional antigen-binding sites.5,12,19,20 We previously addressed this question by generating synthetic antibody libraries with solvent accessible CDR positions randomized with a degenerate codon that encodes for only four amino acid residues (tyrosine, serine, alanine and aspartate).20 Surprisingly, we were able to generate high affinity antibodies against human vascular endothelial growth factor (hVEGF), an angiogenic hormone that has been implicated in tumorogenesis.21,22 Structural analyses suggested that it may be possible to simplify the code for antigen recognition even further, as most of the contacts with antigen were mediated by tyrosine sidechains, while aspartate was almost entirely excluded from the binding sites. We now show that a binary code, restricted to only tyrosine and serine, is sufficient to mediate antigen recognition with high affinity and specificity. Thus, it appears that these amino acid residues are particularly well suited for molecular recognition, and furthermore, naı¨ve antigen recognition can evolve from even the simplest chemical diversity.
Results Antibodies from a binary code We constructed two libraries of phage-displayed antigen-binding fragments (Fabs) by randomizing
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
1154 CDR positions within a humanized Fab framework with a binary degenerate codon that encodes for equal proportions of tyrosine and serine. Solventaccessible positions in the first and second CDRs of the heavy chain (CDR-H1 and CDR-H2) were randomized, and random loops of variable lengths were inserted in place of seven residues in the third heavy chain CDR (CDR-H3). The libraries differed in that library A had a fixed light chain while the third CDR of the light chain (CDR-L3) was randomized in library B. Each library contained w1010 unique members, and thus, the actual library diversities were comparable to the maximum number of unique sequences encoded by each library design (w4!109). The libraries were used to generate Fabs against a panel of six protein antigens. Specific binding clones were identified by phage enzyme-linked immunosorbent assay (ELISA) screening, and were defined as Fab-phage that bound to the cognate antigen but did not exhibit detectable binding to seven other proteins. Specific Fabs were isolated against all six antigens and DNA sequencing revealed diverse CDR sequences that contained only tyrosine and serine at the randomized sites (Figure 1). Competitive phage ELISAs were used to estimate affinities as IC50 values. Fabs exhibited high affinity for four of the antigens (IC50!100 nM),
Molecular Recognition by a Binary Code
moderate affinity for insulin (IC50Z1.4 mM) and low affinity for maltose binding protein (IC50O5 mM). Thus, the binary chemical diversity was sufficient to mediate specific recognition of all six antigens, and for four of the antigens the binding interactions were of high affinity. Fabs against two of the antigens (hVEGF and human death receptor DR5 (hDR5)) were chosen for further functional and structural characterization. Functional characterization of anti-hVEGF Fabs DNA sequencing of 184 hVEGF-binding clones revealed 63 unique sequences, and some of these are shown in Figure 2(a). Most of the clones were from library B but some clones were also isolated from library A. Interestingly, the clones from library B exhibit homology within the selected CDR-L3 and CDR-H3 sequences. In contrast, the CDR-H3 sequences from library A exhibit homology amongst themselves but are very different from the sequences from library B. Thus, it appears that the nature of the CDR-L3 sequence influenced the selection of CDR-H3 sequences, and as a result, two distinct classes of anti-hVEGF antibodies arose from the different libraries. The clones were screened by competitive phage ELISA and exhibited IC50 values
Figure 1. Sequences and specificity profiles of synthetic Fabs. (a) CDR sequences in the randomized regions. The CDRL3 sequence is not shown for Fabs selected from library A in which the light chain was not randomized. Tyrosine and serine residues are shown in yellow or red, respectively. Residues in grey are buried residues that were not randomized in the libraries. The numbering is according to the nomenclature of Kabat et al.1 (b) Fab-phage selected for binding to a particular antigen (cognate antigen, x-axis) were assayed for binding to various immobilized proteins by phage ELISA and bound phage were detected spectrophotometrically (optical density at 450 nm, y-axis). Affinities for cognate antigen were estimated as IC50 values by competitive phage ELISA.37,38 The following proteins were used: neutravidin (NAV, red bar), maltose binding protein (MBP, orange bar), Erbin PDZ domain (PDZ, yellow bar), insulin (green bar), human vascular endothelial growth factor (hVEGF, blue bar), human death receptor 5 (hDR5, pink bar), glutathione-Stransferase (purple bar), human immunoglobulin G (black bar), bovine serum albumin (white bar).
Molecular Recognition by a Binary Code
1155
Figure 2. Affinity and specificity of anti-hVEGF Fabs. (a) CDR sequences of anti-hVEGF Fabs. The numbering is according to the nomenclature of Kabat et al.1 and only positions that were randomized in the libraries are shown. Clones 1 through 7 were isolated from library B, while clones 8 through 14 were isolated from library A. Dashes indicate gaps in the alignment. (b) Kinetic analysis of Fabs binding to immobilized hVEGF. The surface plasmon resonance traces are shown for Fab-YSv1 at four different concentrations (63 nM, 125 nM, 250 nM and 500 nM). Experimental data are represented by open circles and the global fits are represented by continuous lines. Kinetic parameters were determined for Fabs YSv1, YSv2 and YSv3. (c) Immunofluorescence staining with FabYSv1 (red) performed on A673 cells expressing murine VEGFGFP (green). VEGF-GFP and FabYSv1 staining co-localize in the extracellular space formed between cell-to-cell contacts (merge, yellow). The Fab-YSv1 staining was completely abolished by pre-incubation of the antibody with excess recombinant hVEGF (C VEGF panels). The scale bar represents 20 mm. (d) Immunoprecipitations performed on media collected from metabolically labeled A673 cells. Fab-YSv1 and monoclonal antibody A4.6.1 show comparable specificity, as evidenced by identical patterns of bands for precipitated hVEGF isoforms. Anti-GFP polyclonal antibody was used as a negative control.
ranging from approximately 50 nM to greater than 5 mM. The three anti-hVEGF clones with the highest estimated affinities (top three sequences in Figure 2(a)) were purified as free Fab proteins. The binding kinetics of the purified Fabs (designated YSv1, YSv2 and YSv3) were studied by surface plasmon resonance (Figure 2(b)). Fab-YSv1 exhibited the highest affinity for hVEGF (KdZ60 nM), while the other two Fabs bound approximately fivefold less tightly due to faster off rates. It is notable that the sequences of Fab-YSv1 and Fab-YSv2 differ in only three positions, and thus, these three differences account for the improved affinity of Fab-YSv1 in comparison with Fab-YSv2. We next investigated the specificity of Fab-YSv1 by using the protein to visualize VEGF in
mammalian cells transfected with a gene encoding for VEGF fused to green fluorescent protein (GFP). The immunohistochemical staining with Fab-YSv1 precisely overlapped with the fluorescence signal from the VEGF-GFP fusion (Figure 2(c)). Furthermore, the signal was completely blocked by incubating Fab-YSv1 with hVEGF prior to staining. We also conducted immunoprecipitations of endogenous hVEGF and compared the performance of Fab-YSv1 to that of a highly specific, natural anti-hVEGF monoclonal antibody (A4.6.1).23 Both antibodies immunoprecipitated an identical set of bands (Figure 2(d)) that likely represent hVEGF variants generated by alternative mRNA splicing.23 Taken together, these results show that Fab-YSv1 binds to hVEGF with high affinity and specificity comparable to that of a natural antibody, even in the complex cellular milieu.
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Structural characterization of an anti-hDR5 Fab
Table 1. Data collection and refinement statistics for the Fab-YSd1:hDR5-ECD complex
To gain insights into the structural basis for antigen recognition by the binary code, we studied a Fab that was selected for binding to the extracellular domain of hDR5 (hDR5-ECD), a cellsurface receptor that mediates apoptotic cell death.24 Nine unique anti-hDR5 clones were obtained, and the Fab that exhibited the highest estimated affinity by competitive phage ELISA (YSd1; Figure 1(a)) also bound to hDR5 with high affinity in surface plasmon resonance experiments (KdZ34 nM). The crystal structure of Fab-YSd1 in complex with the hDR5-ECD was solved and refined to ˚ resolution (Figure 3(a) and Table 1). While 3.35 A the structure is only of modest resolution, the quality of the structure is quite good with 98.9%
A. Diffraction data Space group ˚) Unit cell constants (A ˚) Resolution (A Rsymb hI/sIi No. of reflections No. of unique reflections Completeness (%) B. Refinement ˚) Resolution (A No. of reflections Rwork, Rfreec No. of residues No. of non-H atoms ˚) rmsd bond length (A rmsd angles (deg.) ˚ 2) rmsd B (bonded atoms) (A Ramachandran plot (%)d
P3221 aZ147, cZ145 50–3.35 (3.47–3.35)a 0.12 (0.34)a 8.3 (4.5)a 149,117 26,539 99.7 (99.8)a 30–3.35 26,003 0.224, 0.280 (0.232, 0.281) 1056 8,026 0.012 1.3 2.3 83.4, 15.5, 0.7, 0.4
a
Numbers in parentheses refer to the highest resolution shell. Rsym Z SjIK hIij=SI, where hIi is the average intensity of symmetry related observations of a unique reflection. c Rwork Z SjFo K Fc j=SFo , where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is the R-factor for a randomly selected 10% of the reflections excluded from all refinement. d Percentage of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of a Ramachandran plot. b
Figure 3. The structure of Fab-YSd1 bound to the hDR5ECD. (a) The complex of the hDR5-ECD with Fab-YSd1. The hDR5-ECD is depicted as a white molecular surface. The main-chain of Fab-YSd1 is shown as sticks; the light and heavy chains are shown in green and blue, respectively, and CDR-H3 is shown in yellow. (b) Experimental density for CDR-H3. The FoKFc omit map contoured at 2.75s shows unbiased electron density (blue) for CDR-H3. Phase bias was removed by omitting CDR-H3 prior to refinement. The coordinates of the final refined model for CDR-H3 are shown as sticks, colored according to atom type (carbon, yellow; oxygen, red; nitrogen, blue), with side-chains rendered as sticks. The hDR5-ECD is shown as a white molecular surface. Despite the relatively modest resolution of the structure ˚ ), the electron density maps are of high quality and (3.35 A allow for accurate determination of backbone and sidechain positions.
of residues in the most favored or additionally allowed parts of the Ramachandran plot. The data quality was limited by the availability of single crystals. All crystals showed evidence of more than one lattice, and thus, to collect a high quality data set, it was necessary to screen many crystals and to then find the most single portion of the best crystal. Nonetheless, the maps were of excellent clarity and the initial maps calculated with phases from the molecular replacement solution showed continuous density for CDR-H3 with side-chain density for many of the tyrosine residues (Figure 3(b)). The presence of two copies of the Fab:antigen complex in the asymmetric unit permitted the use of NCS restraints which aided the refinement process. Additionally, tyrosine is a large side-chain with a distinctive shape, and thus, it can be positioned ˚. accurately even in maps at a resolution of 3.35 A Other than the unusual CDR-H3 conformation, the structure of Fab-YSd1 is similar to that of the humanized Fab-4D5 variant that was used as the scaffold for the antibody library20,25 (Figure 4(a)), although the elbow angles differ due to differences in crystal packing. Overall, the structure shows that Fab-YSd1 interacts with antigen in a normal manner, but tyrosine residues form most of the contact surface (Figure 4(b) and (c)). Antigen ˚ 2 on the binding results in the burial of 460 A 2 ˚ on the light chain. Tyrosine heavy chain and 270 A and serine residues within CDRs H2, H3 and L3 account for 58% and 21% of the buried surface area, respectively. The remainder of the buried surface area (21%) is contributed by CDR-L1, which was not randomized and lies on the periphery of the
1157
Molecular Recognition by a Binary Code
interface (Figure 4(b)). On the antigen side of the interface, the buried surface area is contributed by a variety of amino acid types, but aromatic residues only account for a small fraction (14%) of the structural epitope. Thus, tyrosine plays a dominant role in antigen recognition by Fab-YSd1, but it does so by utilizing interactions with a diverse array of functional groups on the antigen, and interactions with aromatic side-chains do not play a major role. The very long CDR-H3 of Fab-YSd1 protrudes from the framework and makes extensive contact with hDR5, contributing 39% of the surface area ˚ 2). Structures of buried upon antigen binding (288 A antibodies with long CDR-H3 loops have been reported,26–28 but Fab-YSd1 is unique in that CDRH3 contains a “biphasic” helix with tyrosine and serine residues clustered on opposite faces (Figure 4(d)). The tyrosine face is buried against the surface of hDR5, and as a result, 99% of the CDR-H3 accessible surface buried upon binding to hDR5 is contributed by tyrosine, and serine residues likely play structural roles. In this regard, CDR-H3 exemplifies how even the most minimalist binary diversity can give rise to a structural solution that mediates antigen recognition with high affinity and specificity. Fab-YSd1 binds to the N-terminal portion of hDR5 and occludes the smaller of two binding sites for the natural hDR5 ligand Apo2L/TRAIL29 (Figure 5(a)). Intriguingly, one of the Apo2L/ TRAIL residues that contributes the most binding energy to the interaction with hDR5 is Tyr216, as substitution of this residue by alanine causes a 320fold loss in biological activity and a ninefold loss in affinity for hDR5.30 Tyr216 binds in a hydrophobic pocket in cysteine-rich domain 2 of hDR5 29 (Figure 5(b)), resulting in a snug fit that buries greater than 90% of the side-chain. Although FabYSd1 uses many tyrosine residues to bind to hDR5, none of them precisely mimics this interaction. Tyr56 of the heavy chain occupies a similar position as Tyr216 of Apo2L/TRAIL, but it lies along the antigen surface and only 60% of the side-chain is buried (Figure 5(b)). This structure is also the first for a tissue necrosis factor receptor (TNFR) family member bound to an antibody fragment, and as such, it provides insight into how an antibody recognizes antigens of this type. Overall, the structure of hDR5 bound to FabYSd1 is similar to that of hDR5 bound to Apo2L/ TRAIL.29 Cysteine-rich domains 1 and 2 (residues 22–86) from both structures superimpose on each other with a pair-wise root-mean-square deviation ˚ 2 on Ca atoms. This is the (rmsd) of less than 0.30 A same level of structural conservation observed amongst independent structures of hDR5 bound to Apo2L/TRAIL. 29 As in the Apo2L:hDR5 complex structure, which contains three crystallographically independent copies of hDR5, the orientation of cysteine-rich domain 3 in the two independent copies of hDR5 bound to Fab-YSd1 varies somewhat with respect to cysteine-rich domains 1 and 2, although variations between the
hDR5 molecules in the Fab-YSd1 and Apo2L/ TRAIL complexes are more extreme than those within either group. Thus, in both complexes, it appears that cysteine-rich domains 1 and 2 behave as a rigid body, while in contrast the third domain exhibits flexibility with respect to the rest of hDR5. Cysteine-rich domain 3 does not make contact with Fab-YSd1, but it makes extensive contact with Apo2L/TRAIL (Figure 5(a)). In both copies of the Fab:antigen complex contained within the asymmetric unit, cysteine-rich domain 3 is less well defined than it is in the structure of hDR5 bound to Apo2L/TRAIL. In particular, residues 91–104 exhibit poor density in the complex with Fab-YSd1, but this region is well-ordered when bound to Apo2L/TRAIL, and taken together, these differences suggest that the loop is not well-ordered in the absence of Apo2L/TRAIL. The membrane proximal end of cysteine-rich domain 3, including the last disulfide, is also poorly ordered in the complex with Fab-YSd1. Disorder is not uncommon in crystal structures of members of the TNFR family, as for example, the entire final cysteine-rich domain of TNFR1 is disordered in the complex with lymphotoxin.31
Discussion Despite extreme restrictions on chemical diversity, we isolated synthetic Fabs with affinities comparable to those obtained from naı¨ve libraries designed with high chemical diversity. Furthermore, the affinities compare favorably with the affinities of antibodies from the naı¨ve immune response, which are often in the micromolar range prior to affinity maturation through somatic mutation mechanisms. 16 Our results provide insight into how the naı¨ve immune repertoire can evolve high affinity antibodies against a seemingly endless array of antigens. It appears that the selection of antibodies with affinities and specificities sufficient for initial selection from a naı¨ve pool is a relatively simple process that may require only a few productive binding contacts. Thus, the limited diversity of the naı¨ve immune repertoire32 is sufficient to generate initial binding clones that can then attain improved function through subsequent cycles of affinity maturation. We focused our study on protein antigens, but it will be interesting to see if the limited diversity approach will be successful against other antigen classes (e.g. small molecules or carbohydrates). With the exception of CDR-H3, we also restricted CDR conformational diversity, as we did not randomize buried residues which influence CDR main-chain conformations.33 Thus, it may be possible to obtain further improvements in binding by targeting these regions and framework positions that are known to modulate affinity.25,34 The binary binding surfaces represent a minimal benchmark for antigen recognition into which additional diversity and conformational flexibility can be
1158
Molecular Recognition by a Binary Code
Figure 4 (legend opposite)
1159
Molecular Recognition by a Binary Code
introduced to study the details of affinity maturation. In particular, these systems may be very amenable to computer modeling studies, since both serine and tyrosine have few rotamers, and the simplicity of the system should simplify the computational process. Our results suggest that the high abundance of tyrosine and serine in antibody combining sites reflects an innate capacity for these amino acid residues to mediate antigen recognition. Both residues are hydrophilic and thus can be exposed on surfaces without promoting non-specific aggregation, but at the same time, both can also be accommodated readily at protein–protein interfaces. Because of its small size and neutral nature, the serine side-chain is unlikely to cause unfavorable steric hindrance or electrostatic repulsions. On the other hand, the large tyrosine side-chain contributes substantial surface area for contact, and yet, it has a unique chemical nature which allows it to participate in favorable binding interactions with many different types of functional groups.5,9,12,20 We hypothesize that the large tyrosine side-chain acts as a functional group that makes key binding contacts with antigen, while the small serine side-chain serves as an auxiliary group that provides space and flexibility for tyrosine to establish optimal binding contacts. Due to their complementary nature, it appears that tyrosine and serine may be particularly well suited to work together in antigen recognition. Much effort has been expended on the development of large, highly diverse libraries that can be used to obtain antagonists and agonists of biological processes.35–38 However, our results demonstrate that it is possible, and perhaps even advantageous, to restrict chemical diversity to small subsets of functional groups that are particularly well suited for mediating molecular recognition.
Materials and Methods Library construction and sorting A phagemid designed to display bivalent, humanized Fab-4D5 on the surface of M13 bacteriophage was used to
construct libraries, as described.20,39 Oligonucleotidedirected mutagenesis was used to replace CDR positions with TMT degenerate codons, (MZA/C in equal proportions). The positions chosen for randomization in CDR-L3, CDR-H1 and CDR-H2 are shown in Figure 1. In CDR-H3, positions 95 through 100a were replaced with random loops of all possible lengths ranging from 7 to 20 residues (library A) or 7 to 15 residues (library B). Phage from the libraries were cycled through rounds of binding selection with antigen immobilized on 96 well Maxisorp immunoplates (NUNC) as the capture target, as described. After five rounds of selection, phage were produced from individual clones grown in a 96 well format and the culture supernatants were used in phage ELISAs to detect specific binding clones. Competitive phage ELISA A modified phage ELISA37,38 was used to estimate the binding affinities of Fabs. Phage ELISAs were carried out on plates coated with antigen, as described above. Phage displaying antibody fragments were serially diluted in phosphate-buffered saline (PBS), 0.5% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20, and binding was measured to determine a phage concentration giving w50% of the signal at saturation. A fixed, sub-saturating concentration of phage was pre-incubated for two hours with serial dilutions of antigen and then transferred to assay plates coated with antigen. After 15 minutes incubation, the plates were washed with PBS, 0.05% Tween 20 and incubated 30 minutes with horseradish peroxidase/anti-M13 antibody conjugate (1:5000 dilution) (Pharmacia). The plates were washed, developed with TMB substrate (Kirkegaard and Perry Laboratories), quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm. The binding affinities of were estimated as IC 50 values defined as the concentration of antigen that blocked 50% of the phage binding to the immobilized antigen. Protein purification and affinity analysis Fab proteins were purified from Escherichia coli as described.40 Binding kinetics were determined by surface plasmon resonance using a BIAcoree-3000 with hVEGF immobilized on CM5 chips at w500 response units, as described.41 Serial dilutions of Fab proteins were injected, and binding responses were corrected by subtraction of responses on a blank flow cell. For kinetic analysis, a 1:1 Langmuir model of global fittings of kon and koff was used.
Figure 4. Antigen binding site of Fab-YSd1 and the binding interface with hDR5-ECD. (a) Stereo view of the superimposition of the variable domains of Fab-YSd1 and humanized Fab-4D5 (1N8Z).46 The Fab variable domains are represented as a cartoon. CDR-H3 of Fab-YSd1 is colored yellow; the rest of the Fab-YSd1 heavy chain is in blue and the light chain is in green. Humanized Fab-4D5 heavy and light chains are colored cyan and red, respectively. (b) Stereo view ˚ ). The CDR loops are labeled and are of CDR loops that contain residues in contact with the hDR5-ECD (closer than 4.5 A shown as transparent cartoons. CDR side-chains that make contact with the hDR5-ECD are rendered as sticks and are colored as follows: tyrosine, blue; serine, green; all other amino acids, white. A fragment of the hDR5-ECD is shown as a molecular surface; residues in contact with the heavy or light chains are colored yellow or red, respectively, and residues in contact with both are colored orange. (c) The amino acid composition of the interface between Fab-YSd1 and the hDR5-ECD. Each circle represents the surface area on Fab-YSd1 or the hDR5-ECD that becomes buried upon complex formation. The colors indicate the proportion of the buried surface area contributed by each amino acid type, and all amino acid types that contribute more than 1% of the total buried surface area on each molecule are shown. (d) Contacts between CDR-H3 and the hDR5-ECD. The hDR5-ECD is shown as a molecular surface and residues that contact CDRH3 are colored pink. The CDR-H3 main-chain is depicted as a yellow cartoon; tyrosine and serine side-chains are rendered as sticks and colored blue or green, respectively.
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The Kd values were determined from the ratios of kon and koff. Immunohistochemistry Human A673 cells expressing murine VEGF-GFP were stained and imaged, as described.42 In the plus VEGF panel, Fab-YSv1 was pre-incubated for five minutes with a fivefold excess of recombinant hVEGF before being incubated with the cells. Immunoprecipitation A673 cells were metabolically labeled and immunoprecipitations were performed from the media, as described,23 using 15 mg of anti-GFP polyclonal antibody (Clontech), Fab-YSv1 or monoclonal antibody A4.6.1. The immune complexes were eluted by boiling and resolved by SDS-PAGE on a 14% (w/v) acrylamide gel under reducing conditions. The gel was dried and then exposed to a phosphoimager plate overnight. Crystallization, structure determination and refinement DNA coding for the hDR5-ECD (residues 1–130) with an N-terminal His-tag was cloned into the baculovirus transfer vector pAcGP67-B (Pharmingen). Following transfection and viral amplification in Sf9 cells, protein was expressed and secreted from Hi5 cells grown at 27 8C. The culture medium was supplemented with 50 mM Tris (pH 8.0), 1 mM NiCl2, 5 mM CaCl2, and 1 mM phenylmethylsulfonyl fluoride. The pH was adjusted to pH 7.6 and the medium was filtered prior to loading on a NiNTA column (Qiagen). After elution with imidazole, the His-tag was removed by digestion with thrombin followed by purification on a Superdex-75 sizing column. Fab-YSd1 was expressed in E. coli and purified over a Protein G column.40 Fab-containing fractions were further purified by passage over a S-Sepharose column, followed by a Superdex-200 sizing column. Fab–receptor complex was made by mixing Fab-YSd1 with an excess of the hDR5-ECD domain, followed by purification over a Superdex-200 sizing column. The complex-containing fractions were pooled and concentrated to 11 mg/ml in 150 mM NaCl, 20 mM bis-Tris (pH 6.5) and used for crystallization trials. Crystals grew at 19 8C from hanging drops containing an equal volume of protein and well solution consisting of 20% polyethylene glycol (PEG) 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate (pH 6.2–6.6). Crystals were transferred to mother liquor containing the well solution with 20% PEG 300, prior to cryo-cooling by immersion in liquid nitrogen. The resulting crystals were visibly nonsingle and it was necessary to screen many crystals to find one that was mostly single. ˚ data set was collected at beamline 19BM at the A 3.35 A APS. Data processing was done with HKL.43 Calculation of the Matthew’s coefficient indicated that the asymmetric
Figure 5. Comparison of the interactions of hDR5 with Fab-YSd1 and Apo2L/TRAIL. (a) Mapping of the structural binding epitopes of hDR5 for Fab-YSd1 or Apo2L/TRAIL. The hDR5-ECD is shown as a molecular surface. The unshared portions of the epitopes for binding to Fab-YSd1 or Apo2L/TRAIL are colored yellow or blue, respectively, and the shared portion is in green.
(b) Comparison of Tyr56 of Fab-YSd1 and Tyr216 of Apo2L/TRAIL. The hDR5-ECD is shown as a molecular surface and colored as in (a). Tyr56 of Fab-YSd1 and Tyr216 of Apo2L/TRAIL are rendered as sticks and colored cyan or pink, respectively, as are the a-carbon traces of the surrounding loops. Structures were drawn with PyMOL (DeLano Scientific, San Carlos, CA).
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unit of the crystals contained two Fab:antigen complexes. The structure was solved in space group P3221 by molecular replacement with the program Phaser44 using the structures of two Fab-4D5 variants as search models.25 The resulting maps showed clear density for the hDR5ECD, which was placed manually, as well as for the Fab CDRs. Refinement was performed with the program REFMAC545 and included TLS refinement and NCS restraints imposed separately on the variable and constant domains of Fab-YSd1, as well as on the hDR5ECD.
8. 9.
10.
Protein Data Bank accession numbers The coordinates and structure factors for the FabYSd1:hDR5-ECD complex have been deposited in the RCSB Protein Data Bank (PDB:1ZA3).
11. 12.
Acknowledgements We are grateful to the Genentech Fermentation, DNA Synthesis and DNA Sequencing groups. We thank M. Franklin, A. Cochran, A. de Vos and H. Lowman for helpful discussions and H. Darbon for his support. We also thank Stephan L. Ginell, PhD of beamline 19BM at the APS (Advanced Photon Source). Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source, was supported by the US Department of Energy, Office of Energy Research, under contract no. W-31-109-ENG-38.
13. 14. 15.
16. 17.
18.
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Edited by I. Wilson (Received 22 December 2004; received in revised form 10 March 2005; accepted 14 March 2005)