PDBsum entry 1l7i

Immune system

PDB id: 1l7i

Contents

Protein chains

214 a.a. *

222 a.a. *

Ligands

SO4 ×2

Waters ×392

* Residue conservation analysis

PDB id:

1l7i

Name:

Immune system

Title:

Crystal structure of the anti-erbb2 fab2c4

Structure:

Chimera of fab2c4: "humanized" murine monoclonal antibody. Chain: l. Fragment: light chain (residues 1-214). Engineered: yes. Other_details: the chimera consists of the human ig and the original murine sequence. Chimera of fab2c4: "humanized" murine monoclonal antibody. Chain: h. Fragment: heavy chain (residues 1-216).

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.197 R-free: 0.230

Authors:

F.F.Vajdos,C.W.Adams,T.N.Breece,L.G.Presta,A.M.De Vos,S.S.Sidhu

Key ref:

F.F.Vajdos et al. (2002). Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis. J Mol Biol, 320, 415-428. PubMed id: 12079396 DOI: 10.1016/S0022-2836(02)00264-4

Date:

15-Mar-02 Release date: 10-Jul-02

Protein chain

Q7Z3Y4  (Q7Z3Y4_HUMAN) -  Ig-like domain-containing protein from Homo sapiens

Seq: 236 a.a.

Struc: 214 a.a.*

Protein chain

No UniProt id for this chain

Struc: 222 a.a.

* PDB and UniProt seqs differ at 19 residue positions (black crosses)

DOI no: 10.1016/S0022-2836(02)00264-4

J Mol Biol 320:415-428 (2002)

PubMed id: 12079396

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.

F.F.Vajdos, C.W.Adams, T.N.Breece, L.G.Presta, A.M.de Vos, S.S.Sidhu.

ABSTRACT

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.

Selected figure(s)

Figure 4.
Figure 4. Mapping of the functional epitopes for binding of ErbB2-ECD onto the structure of Fab2C4. The functional epitopes defined by (a) shotgun alanine-scanning or (b) shotgun homolog-scanning are shown. Unscanned light or heavy chain residues are colored cyan or blue, respectively. Scanned residues are color-coded according to the magnitudes of F[wt/mut] values, as follows: red, >30; yellow, 10-30; grey, <10. Labeled residues with asterisks (*) indicate light chain residues. The solvent-exposed Cg2 group of hT33 is located directly over a hydrogen bond network involving hT33 and several buried side-chains (Figure 5). Data are also shown graphically in Figure 2 and were obtained from Table 3 and Table 4. The Fab2C4 structure is shown in CPK representation. This Figure and Figure 5 were generated using PyMOL (http://pymol.sourceforge.net).

Figure 5.
Figure 5. The buried hydrogen bond network in the heavy chain of Fab2C4. Side-chain atoms are shown as sticks colored as follows: carbon, gray; oxygen, red; nitrogen, blue. Main chains are shown as gray tubes and hydrogen bonds are shown as dashed magenta lines. The hydrogen bond network ties together residues from each of the three heavy chain CDRs, and with the exception of hT33, all the interacting side-chains are buried. The g-OH group of hT33 is anchored by the hydrogen bonds, resulting in the Cg2 group being solvent exposed at the base of a central depression in the Fab2C4 functional epitope (Figure 4).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 320, 415-428) copyright 2002.

Figures were selected by an automated process.