PDBsum entry 2eks

Immune system/hydrolase

PDB id: 2eks

Contents

Protein chains

107 a.a. *

113 a.a. *

129 a.a. *

Waters ×158

* Residue conservation analysis

PDB id:

2eks

Name:

Immune system/hydrolase

Title:

Crystal structure of humanized hyhel-10 fv-hen lysozyme complex

Structure:

Anti-lysozyme antibody fv region. Chain: a. Fragment: vl fragment. Engineered: yes. Anti-lysozyme antibody fv region. Chain: b. Fragment: vh fragment. Engineered: yes. LysozymE C.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562. Gallus gallus. Chicken. Organism_taxid: 9031

Resolution:

2.00Å R-factor: 0.221 R-free: 0.265

Authors:

T.Nakanishi,K.Tsumoto,A.Yokota,H.Kondo,I.Kumagai

Key ref:

T.Nakanishi et al. (2008). Critical contribution of VH-VL interaction to reshaping of an antibody: The case of humanization of anti-lysozyme antibody, HyHEL-10. Protein Sci, 17, 261-270. PubMed id: 18227432 DOI: 10.1110/ps.073156708

Date:

24-Mar-07 Release date: 25-Mar-08

Protein chain

No UniProt id for this chain

Struc: 107 a.a.

Protein chain

No UniProt id for this chain

Struc: 113 a.a.

Protein chain

P00698  (LYSC_CHICK) -  Lysozyme C from Gallus gallus

Seq: 147 a.a.

Struc: 129 a.a.

Enzyme reactions

• Enzyme class: Chain C: E.C.3.2.1.17 - lysozyme.
• Reaction: Hydrolysis of the 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglycan heteropolymers of the prokaryotes cell walls.

DOI no: 10.1110/ps.073156708

Protein Sci 17:261-270 (2008)

PubMed id: 18227432

Critical contribution of VH-VL interaction to reshaping of an antibody: The case of humanization of anti-lysozyme antibody, HyHEL-10.

T.Nakanishi, K.Tsumoto, A.Yokota, H.Kondo, I.Kumagai.

ABSTRACT

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.

Selected figure(s)

Figure 1.
Figure 1. Titration calorimetry of the interaction between the HyHEL-10 Fv fragment and HEL. (A, B) (Top) Typical calorimetric titration of hHyHEL-10 mutant Fv fragment (5 µM) with 50 µM HEL at pH 7.2 and 30°C; (bottom) integration plot of the data calculated from the raw data. The solid line corresponds to the best-fit curve obtained by least-squares deconvolution. (A) hHyHEL-10; (B) HQ39KW47Y mutant.

Figure 3.
Figure 3. Overall structure of the hHyHEL-10 Fv–HEL and mutant–HEL complexes. The structure of the three humanized Fv–HEL complexes, whose C coordinates of HEL are superimposed on the C coordinates of HEL complexed with mHyHEL-10, is superposed on the structure of Fv (gray). Red, hHyHEL-10 Fv–HEL complex; blue, HW47Y Fv–HEL complex; green, HQ39KW47Y Fv–HEL complex. Generated with PyMOL (DeLano Scientific).

The above figures are reprinted by permission from the Protein Society: Protein Sci (2008, 17, 261-270) copyright 2008.

Figures were selected by the author.