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bioRxiv · 10.1101/2024.05.21.595133

Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection

Yang, B.·Gomes, D. E. B.·Liu, Z.·Santos, M. S.·Li, J.·Bernardi, R. C.·Nash, M.
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Abstract

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibodys N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to [~] 190 pN under loading rates of [~]104-105 pN/sec, representing a [~]4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

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Yang, B., Gomes, D. E. B., Liu, Z., Santos, M. S., Li, J., Bernardi, R. C., Nash, M.. 2024-05-21. Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection. https://doi.org/10.1101/2024.05.21.595133

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