1L7T

Crystal Structure Analysis of the anti-testosterone Fab fragment

Summary for 1L7T

• Entry DOI: 10.2210/pdb1l7t/pdb
• Related: 1L7S
• Descriptor: anti-testosterone (light chain), anti-testosterone (heavy chain) (3 entities in total)
• Functional Keywords: anti-testosterone, fab fragment, affinity and specificity matured, immune system
• Biological source: Mus musculus (house mouse); More
• Cellular location: Cell membrane ; Single-pass membrane protein : P01869
• Total number of polymer chains: 2
• Total formula weight: 47735.61

Authors

Valjakka, J.,Hemminki, A.,Niemi, S.,Soderlund, H.,Takkinen, K.,Rouvinen, J. (deposition date: 2002-03-17, release date: 2002-10-02, Last modification date: 2024-11-20)

Primary citation

Valjakka, J.,Hemminki, A.,Niemi, S.,Soderlund, H.,Takkinen, K.,Rouvinen, J.
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, 2002

PubMed 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.

PubMed: 12196551
DOI: 10.1074/jbc.M208392200

Experimental method

X-RAY DIFFRACTION (2.1 Å)