Contribution of light chain residues to high affinity binding in an HIV-1 antibody explored by combinatorial scanning mutagenesis

Abstract

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.

Figure 1

(A) The crystal structure of the D5 Fab bound to 5-Helix reported by Luftig et al. (reference 22). Side chains involved in the interaction are shown in blue (heavy chain), red (light chain), or yellow (5-Helix). The critical HCDR2 is boxed. (B) Sequences of the HCDR2 region of V_H1-69, D5, CR6261, and 412D. Residues that differ from the germline are shown in gray. Viral targets for each antibody are indicated in parentheses. (C) Role of HCDR2 in recognition D5 (blue), CR6261 (magenta), and 412D (green). (Antigens shown in gray.) In all three antibodies, F54 contacts surface-exposed hydrophobic residues on the antigen.

Figure 2

Results from polyclonal phage ELISA of populations from unselected library (R0), and output phage from rounds 1 and 2 of the selection against 5-Helix (R1 and R2, respectively). A specific binding signal was observed in the R2 population. Phage titers for all three populations were ~2 × 10¹⁰ infectious units/mL.

Figure 3

Combinatorial alanine scanning results mapped onto the crystal structure of D5. Residues in the V_L domain are colored according to ΔΔG_Ala-WT: red, ≥ 1.0 kcal/mol; orange, 0.4 – 1.0 kcal/mol; green, 0 – 0.4 kcal/mol; cyan, ≤ 0 kcal/mol. Residues previously shown by Luftig et al. to be important for binding in the V_H domain are colored blue.

Figure 4

Interlocking interaction between opposing hydrophobes. The pocket on the D5 combining site, into which W571 of 5-Helix inserts, is lined by hotspot residues Y94, P95, and L96. Protruding residues from HCDR2 of D5 (F54 and T56) are shown in blue.

Figure 5

(A) Location of Y30 on the overall concave surface of the D5 combining site. (B) Results from phage ELISA with particles displaying WT D5 or a Y30A point mutant as a function of phage titer. The OD(450nm) at each phage concentration was corrected for background binding by subtracting the binding signal against wells coated with 5-Helix from wells coated with BSA (‘corrected’ OD(450nm)).

Figure 6

ΔΔG_Ala-WT and ΔΔG_mut-WT values obtained from combinatorial alanine scanning and ‘Scan 2’libraries. The positions are labeled with WT residue identity preceding the position number, and the substitution of the ‘Scan 2’ library following the number. A ‘cut-off’ of 1 kcal/mol (dashed line) was used to identify hotspot residues.