PDBsum entry 1cz8

Immune system

PDB id: 1cz8

Contents

Protein chains

94 a.a. *

213 a.a. *

218 a.a. *

Ligands

SO4 ×2

Waters ×419

* Residue conservation analysis

PDB id:

1cz8

Name:

Immune system

Title:

Vascular endothelial growth factor in complex with an affinity matured antibody

Structure:

Vascular endothelial growth factor a. Chain: v, w. Fragment: receptor binding fragment. Synonym: vegf-a, vascular permeability factor, vpf. Engineered: yes. Light chain of neutralizing antibody. Chain: l, x. Synonym: ranibizumab light chain. Engineered: yes.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: vegfa, vegf. Expressed in: escherichia coli. Expression_system_taxid: 562. Mus musculus. House mouse. Organism_taxid: 10090.

Biol. unit:

Hexamer (from PQS)

Resolution:

2.40Å R-factor: 0.208 R-free: 0.267

Authors:

Y.Chen,C.Wiesmann,G.Fuh,B.Li,H.W.Christinger,P.Mckay,A.M.De Vos, H.B.Lowman

Key ref:

Y.Chen 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. PubMed id: 10543973 DOI: 10.1006/jmbi.1999.3192

Date:

01-Sep-99 Release date: 20-Mar-00

Protein chains

P15692  (VEGFA_HUMAN) -  Vascular endothelial growth factor A, long form from Homo sapiens

Seq: 395 a.a.

Struc: 94 a.a.

Protein chains

No UniProt id for this chain

Struc: 213 a.a.

Protein chains

No UniProt id for this chain

Struc: 218 a.a.

DOI no: 10.1006/jmbi.1999.3192

J Mol Biol 293:865-881 (1999)

PubMed id: 10543973

Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.

Y.Chen, C.Wiesmann, G.Fuh, B.Li, H.W.Christinger, P.McKay, A.M.de Vos, H.B.Lowman.

ABSTRACT

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.

Selected figure(s)

Figure 2.
Figure 2. Radiolabeled VEGF binding assay. [ 125 I]VEGF was equilibrated (23 °C) with serial dilutions of unlabeled VEGF and (a) Fab-12 or (c) Y0317. Fabs were captured with an anti-Fab antibody-coated immunosorbant plate. Scatchard analysis (Munson & Rodbard, 1980) with a 1:1 binding model was used to calculate Kd of (b) 433 (±116) pM for Fab-12 and (d) 19.8(±4.3) pM for Y0317.

Figure 4.
Figure 4. Structure of the affinity-improved Y0317 Fab in complex with VEGF. A superposition of the structure (Muller et al., 1998a) of wild-type humanized antibody Fab-12 (gray) in complex with VEGF (gray) is shown with that of Fab Y0317 (green) in complex with VEGF (yellow). (a) Overall view of the complex, including one Fab molecule bound to one dimer of VEGF (a second Fab molecule is bound at left in the crystal) shows that the binding site for both antibody variants centers on the ``80's loop'' of VEGF. (b) A view of the four CDR changes between Fab-12 and Y0317 Fab shows that the new D28 and T100a side-chains do not directly contact antigen. However, H31 and Y97 form new contacts. (c) Interactions of H97 and an associated, buried water molecule in the Fab-12 complex, compared with those of Y97 in the Y0317 complex.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 293, 865-881) copyright 1999.

Figures were selected by an automated process.