Contribution of light chain residues to high affinity binding in an HIV-1 antibody explored by combinatorial scanning mutagenesis.
Detailed analysis of factors governing high affinity antibody-antigen interactions yields important insight into molecular recognition and facilitates the design of functional antibody libraries. Here we describe comprehensive mutagenesis of the light chain complementarity determining regions (CDRs) of HIV-1 antibody D5 (which binds its target, "5-Helix", with a reported K(D) of 50 pM). Combinatorial scanning mutagenesis libraries were prepared in which CDR residues on the D5 light chain were varied among WT side chain identity or alanine. Selection of these libraries against 5-Helix and then sequence analysis of the resulting population were used to quantify energetic consequences of mutation from wild-type to alanine (DeltaDeltaG(Ala-WT)) at each position. This analysis revealed several hotspot residues (DeltaDeltaG(Ala-WT) >or= 1 kcal/mol) that formed combining site features critical to the affinity of the interaction. Tolerance of D5 light chain residues to alternative mutations was explored with a second library. We found that light chain residues located at the center and at the periphery of the D5 combining site contribute to shape complementarity and electrostatic characteristics. Thus, the affinity of D5 for 5-Helix arises from extended interactions involving both the heavy and light chains of D5. These results provide significant insight for future antibody engineering efforts.
Da Silva
Gustavo F
GF
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Harrison
Joseph S
JS
Lai
Jonathan R
JR
D000915:Antibody Affinity; D020650:Combinatorial Chemistry Techniques; D015483:HIV Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D016296:Mutagenesis; D019151:Peptide Library; D015202:Protein Engineering; D055672:Static Electricity
D015483:HIV Antibodies; D007147:Immunoglobulin Light Chains; D019151:Peptide Library