Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition - PubMed (original) (raw)

Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition

Frederic A Fellouse et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Antigen-binding fragments (Fabs) with synthetic antigen-binding sites were isolated from phage-displayed libraries with restricted complementarity-determining region (CDR) diversity. Libraries were constructed such that solvent-accessible CDR positions were randomized with a degenerate codon that encoded for only four amino acids (tyrosine, alanine, aspartate, and serine). Nonetheless, high-affinity Fabs (K(d) = 2-10 nM) were isolated against human vascular endothelial growth factor (hVEGF), and the crystal structures were determined for two distinct Fab-hVEGF complexes. The structures revealed that antigen recognition was mediated primarily by tyrosine side chains, which accounted for 71% of the Fab surface area that became buried upon binding to hVEGF. In contrast, aspartate residues within the CDRs were almost entirely excluded from the binding interface. Alanine and serine residues did not make many direct contacts with antigen, but they allowed for space and conformational flexibility and thus played an auxiliary role in facilitating productive contacts between tyrosine and antigen. Tyrosine side chains were capable of mediating most of the contacts necessary for high-affinity antigen recognition, and, thus, it seems likely that the overabundance of tyrosine in natural antigen-binding sites is a consequence of the side chain being particularly well suited for making productive contacts with antigen. The findings shed light on the basic principles governing the evolution of natural immune repertoires and should also aid the development of improved synthetic antibody libraries.

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Figures

Fig. 1.

Fig. 1.

CDR sequences of anti-VEGF Fabs. Residues in gray were not randomized in the libraries. The numbering is shown for the parental Fab4D5 according to the nomenclature of Kabat et al. (1). (A) Heavy-chain sequences selected from a naive library in which solvent-accessible CDR residues were randomized with a KMT degenerate codon. In CDR-H3, loops of variable length were inserted between residues 94 and 100b. (B) Light-chain sequences of high-affinity Fabs obtained by recombining selected heavy chains with a light chain CDR library. Gln-90 in the light chain of YADS1 was deleted by spontaneous mutation.

Fig. 2.

Fig. 2.

The complex of hVEGF with YADS1 (A) and YADS2 (B). The main chain of the hVEGF homodimer is depicted as a gray ribbon, and residues that differ in comparison with mVEGF are colored red. Gly-88 is shown as a red sphere. The hVEGF homodimer binds to two symmetry-related Fabs, but only one Fab molecule is shown and is depicted as a molecular surface. Tyrosines at randomized positions are colored orange, whereas all other amino acid types at randomized positions are colored blue. Residues at positions that were not randomized in the libraries are colored white. These and other structural figures were derived from the crystal structure coordinates and were generated by using

pymol

(DeLano Scientific, San Carlos, CA).

Fig. 3.

Fig. 3.

The structural epitope for binding to YADS1 (A) or YADS2 (B) mapped on the molecular surface of hVEGF. The structural epitope consists of hVEGF residues that make contact with one or more residues of the Fab, with “contact” defined as a distance <4.1 Å. Residues that contact the heavy or light chain are colored green or yellow, respectively. The dashed line outlines the structural epitope for binding to Flt-1D2, as determined from a previously described x-ray structure (PDB code 1FLT) (34).

Fig. 4.

Fig. 4.

Composition of the antigen-binding sites. The CDRs of YADS1 and YADS2 contained a total of 66 aa located at positions that were randomized in the libraries. Among these 66 residues, the graph shows the percentage (y axis) that each of the amino acid types allowed in the library (x axis) represented in terms of (i) the overall abundance (black bars), (ii) the residues that contact hVEGF as defined in Fig. 3 (gray bars), and (iii) the surface area buried upon binding to hVEGF (white bars).

Fig. 5.

Fig. 5.

The CDR side chains of YADS1 (A) and YADS2 (B) that contact hVEGF. The structural epitope for binding to the Fab (see Fig. 3) was mapped onto the molecular surface of hVEGF, and residues that made contacts with tyrosines or other residue types are colored orange or blue, respectively. The side chains at CDR positions that were randomized in the libraries are shown. Side chains that do not make contact with hVEGF are colored white. Tyrosine side chains that make contacts with hVEGF are colored red, whereas all other contacting side chains are colored blue. The hVEGF molecules are shown in the same orientation in both panels.

Fig. 6.

Fig. 6.

The atomic composition of hVEGF–ligand interfaces. Each pair of circles represents the surface area buried upon complexation of ligand (Upper) with hVEGF (Lower). The colors indicate the proportion of the buried surface area composed of carbon (green), oxygen (red), nitrogen (blue), or sulfur (yellow).

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