PDBsum entry 1tzh

Immune system

PDB id: 1tzh

Contents

Protein chains

94 a.a. *

210 a.a. *

222 a.a. *

Waters ×110

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Synthetic antibodies from a four-Amino-Acid code: a dominant role for tyrosine in antigen recognition.

Authors

F.A.Fellouse, C.Wiesmann, S.S.Sidhu.

Ref.

Proc Natl Acad Sci U S A, 2004, 101, 12467-12472. [DOI no: 10.1073/pnas.0401786101]

PubMed id

15306681

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.

Figure 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-1[D2], as determined from a previously described x-ray structure (PDB code 1FLT [PDB] ) (34).

Figure 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.

Secondary reference #1

Title

Phage-Displayed antibody libraries of synthetic heavy chain complementarity determining regions.

Authors

S.S.Sidhu, B.Li, Y.Chen, F.A.Fellouse, C.Eigenbrot, G.Fuh.

Ref.

J Mol Biol, 2004, 338, 299-310. [DOI no: 10.1016/j.jmb.2004.02.050]

PubMed id

15066433

Figure 1.
Figure 1. Heavy chain CDR residues selected for diversification in phage-displayed libraries. (a) The backbones of the humanized 4D5 V[H] and V[L] domains are shown as grey or pink tubes, respectively. The side-chains of CDR residues included in the library diversification strategy are shown, and the residues are colored yellow (CDR-H1), green (CDR-H2) or blue (CDR-H3). b, CPK representation of a. Residues are numbered according to the nomenclature described by Kabat et al.[52.] This Figure was generated with Insight II (Accelrys, San Diego) using coordinates for the crystal structure of free Fv4D5 (PDB entry 1FVC). [16.]

Figure 5.
Figure 5. High-throughput phage competition ELISA for estimating affinities of anti-mVEGF scFvs. The x axis shows the clone number; clones 1-68 were from Lib-scFv[2] and clones 69-86 were from Lib-scFv. The y axis shows the ratio of the phage ELISA signal (A[450]) in the presence of 100 nM mVEGF (+VEGF) to that in the absence of solution-phase mVEGF ( -VEGF). Asterisks indicate clones that were chosen for further analysis. See Materials and Methods for further details.

The above figures are reproduced from the cited reference with permission from Elsevier