Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

Abstract

Background: Low molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes.

Methods: Different hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential.

Results: The simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL.

Conclusions: The best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

Fig. 1

Amino acid variability within various regions of the isolated sequences. Variability within a VH, b VL regions of the pre-selection sequences. Diversity at each amino acid position was classified into five groups. The total CDR or FW region variability was determined as a percentage of the overall diversity of positions within the selected regions (CDR or FW)

Fig. 2

The effect of amino acids within CDR H2 on the predicted positioning of CDRs H3 and L3. Amino acid positions H53 (red), H58 (yellow), and H59 (magentas) in clones a HSL1, b HSL2, c SQA A5, d SQA B3, e POR B11, f POR A7, g COP H3, and h COP A8. Position H53 has a profound effect on the orientation of CDR H3 (green), whilst positions H58 and H59 influence CDR L3 (blue) orientation. These homology images of the post-selection clones were constructed utilizing SWISS-MODEL workplace. The structures were viewed by PyMOL 1.3 (academic version)

Fig. 3

Side-chains orientations of potential amino acids within CDR H2. Site-specific interactions of amino acid positions H53, H58, and H59. a SQA A5 (red), SQA B3 (green), and SQA F1 (blue). b POR A7 (red), POR D11 (green), and POR B11 (blue). c COP E12 (red), COP H3 (green), and COP A8 (blue). d HSL 1 (red), HSL 2 (green), and HSL 4 (blue). These homology images of the post-selection clones were constructed utilizing SWISS-MODEL workplace. The structures were viewed by PyMOL 1.3 (academic version)

Fig. 4

Structural, docking, and surface-mapped electrostatic potential of antibodies. The homology structures of the post-selection clones were determined by SWISS-MODEL workplace for clones a HSL1, b SQA A5, c POR B11, d COP H3. Docking analyses were conducted with AutoDock Vina 1.5.6. The structures were viewed by PyMOL 1.3 (academic version). Measurements of electrostatic energies were calculated using Python Molecule Viewer (PMV) Version 1.5.6. The produced energy was mapped with medium surface quality and at a 1 Å distance from the surface. The map color was coded as white: 0 kT/e, Blue: 10.2 kT/e, Red: −10.2 kT/e