PDBsum entry 2z4q

Immune system

PDB id: 2z4q

Contents

Protein chains

219 a.a. *

218 a.a. *

Metals

_CL

_CD ×2

Waters ×240

* Residue conservation analysis

PDB id:

2z4q

Name:

Immune system

Title:

Crystal structure of a murine antibody fab 528

Structure:

Anti egfr antibody fab, light chain. Chain: a. Anti egfr antibody fab, heavy chain. Chain: b

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Strain: balb/c. Other_details: this protein was generated by papain digestion of igg from hybridoma.. From hybridoma.

Resolution:

2.30Å R-factor: 0.191 R-free: 0.228

Authors:

T.Nakanishi,K.Tsumoto,R.Asano,H.Kondo,I.Kumagai

Key ref:

K.Makabe et al. (2008). Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528. J Biol Chem, 283, 1156-1166. PubMed id: 17947238 DOI: 10.1074/jbc.M706190200

Date:

22-Jun-07 Release date: 30-Oct-07

Protein chain

No UniProt id for this chain

Struc: 219 a.a.

Protein chain

No UniProt id for this chain

Struc: 218 a.a.

DOI no: 10.1074/jbc.M706190200

J Biol Chem 283:1156-1166 (2008)

PubMed id: 17947238

Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528.

K.Makabe, T.Nakanishi, K.Tsumoto, Y.Tanaka, H.Kondo, M.Umetsu, Y.Sone, R.Asano, I.Kumagai.

ABSTRACT

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Selected figure(s)

Figure 5.
FIGURE 5. Overall structures of the Fv portions of m528Fab and h528Fv. The structure of h528Fv, for which the C coordinates of the VL chain are superimposed on the C coordinates of m528Fv, is superimposed on the structure of m528Fv (gray). Blue, heavy chain of h528Fv; green, light chain of h528Fv; gray, Fv portion of m528Fab.

Figure 7.
FIGURE 7. Local structures of the VH-VL interfaces. Blue, heavy chain of h528Fv; green, light chain of h528Fv; gray, m528Fab. Residue numbers are according to Kabat et al. (53). The parentheses indicate m528Fab residues.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 1156-1166) copyright 2008.

Figures were selected by the author.