Structure and Dynamics of Stacking Interactions in an Antibody Binding Site

Abstract

For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab-MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of Phe^H98, which interacts with the chromophore via T-stacking, and the side chain of Trp^L91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.

Figure 1

Amino-acid sequences of Abs 10A6 and 5D11, and their predicted germline precursor (GL; only sequence corresponding to the identified V and J gene segments is shown), with differences indicated. CDRs as defined by Kabat are highlighted. Regions installed by cloning primers (and thus where differences do not correspond to somatic mutations) are underlined. The dash in the V_L sequence corresponds to a deletion according to Kabat numbering (Kabat numbering shown above).

Figure 2

Absorption spectra of MPTS in buffer (blue line) and bound to Abs 5D11 (black line) or 10A6 (red line).

Figure 3

Normalized transient absorption spectra after 0.2 ps (red), 1 ps (green) and 50 ps (black) delay time for (A) MPTS bound to Ab 5D11, (B) MPTS in buffer.

Figure 4

3PEPS decay for Ab 5D11 (circles) and 10A6 (triangles).

Figure 5

Crystal structure of 10A6-MPTS complex. (A) Swung-out and (B) swung-in conformations of Phe^H98 over MPTS. (C) H-bonds formed between Ab and Ag. (D) Sites of assigned or potential somatic mutations in 10A6 and/or 5D11 indicated on the 10A6 structure.