PDBsum entry 1l7t

Immune system

PDB id: 1l7t

Contents

Protein chains

219 a.a. *

220 a.a. *

Waters ×291

* Residue conservation analysis

PDB id:

1l7t

Name:

Immune system

Title:

Crystal structure analysis of the anti-testosterone fab fragment

Structure:

Anti-testosterone (light chain). Chain: l. Fragment: fab77 fragment. Engineered: yes. Anti-testosterone (heavy chain). Chain: h. Fragment: fab77 fragment. Engineered: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.213 R-free: 0.275

Authors:

J.Valjakka,A.Hemminki,S.Niemi,H.Soderlund,K.Takkinen,J.Rouvinen

Key ref:

J.Valjakka et al. (2002). Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains. J Biol Chem, 277, 44021-44027. PubMed id: 12196551 DOI: 10.1074/jbc.M208392200

Date:

17-Mar-02 Release date: 02-Oct-02

Protein chain

Q65ZC0  (Q65ZC0_MOUSE) -  Kappa light chain C_region (Fragment) from Mus musculus

Seq: 219 a.a.

Struc: 219 a.a.*

Protein chain

Q91Z05  (Q91Z05_MOUSE) -  Ighg protein from Mus musculus

Seq: 473 a.a.

Struc: 220 a.a.*

* PDB and UniProt seqs differ at 82 residue positions (black crosses)

DOI no: 10.1074/jbc.M208392200

J Biol Chem 277:44021-44027 (2002)

PubMed id: 12196551

Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains.

J.Valjakka, A.Hemminki, S.Niemi, H.Söderlund, K.Takkinen, J.Rouvinen.

ABSTRACT

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

Selected figure(s)

Figure 6.
Fig. 6. Superposition of binding sites of the 77 Fab (top panel) and wt Fab (bottom panel) in the free and testosterone-bound form. The free structures are shown in pink, and testosterone complex is shown in black. Stereopairs were prepared with the Molscript program (29).

Figure 7.
Fig. 7. The binding site in the 77 Fab (A) and in the wt Fab (B) structures. The solvent-accessible surface was produced using the O program (21).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 44021-44027) copyright 2002.

Figures were selected by an automated process.