Adaptive mutations alter antibody structure and dynamics during affinity maturation

Abstract

While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.

Figure 1

Schematic representation of barrier crossings on a protein free-energy landscape (A) and peak shift decay (B) corresponding to elastic, anelastic, and plastic deformations. Coordinate Q represents a projection of all internal degrees of freedom of the system.

Figure 2

Structure of MPTS.

Figure 3

Amino acid sequence of the anti-MPTS Abs. CDRs are underlined. Kabat numbering is shown at the top. The consensus sequence is listed first with identical residues in the three the anti-MPTS Abs indicated with a dash.

Figure 4

Repertoire of proteins recognized by each anti-MPTS Ab as determined by ELISA. The 45 randomly selected proteins (see Table S2) are arranged according to the affinity with which they are bound by 6C8. Data were collected in triplicate and bars represent average absorbance normalized by Ab concentration.

Figure 5

Overlay of the 6C8 (blue) and 8B10 (red) crystal structures, and the 6C6 (grey) model structure based on the superposition of the constant regions.

Figure 6

3PEPS decays for Abs 6C8, 8B10, and 6C6 (symbols, data points; lines, best fit to data).

Figure 7

View of the MPTS binding site in the crystal structures of Ab 6C8 (A) and 8B10 (B) and the model structure of Ab 6C6 (C). The V_H and V_L are shown in orange and light blue, respectively, side-chains of somatic mutations are shown in green, and waters in panels B and C are shown as blue spheres. Each Ab structure is viewed from an optimal perspective to visualize its unique set of somatic mutations.