
==== Front
bioRxiv
BIORXIV
bioRxiv
Cold Spring Harbor Laboratory

10.1101/2024.05.21.595133
preprint
1
Article
Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection
Yang Byeongseon http://orcid.org/0000-0003-0660-5193

Gomes Diego E. B. http://orcid.org/0000-0003-4855-040X

Liu Zhaowei http://orcid.org/0000-0001-8214-8882

Santos Mariana Sá http://orcid.org/0000-0002-9227-7941

Li Jiajun http://orcid.org/0009-0001-8806-5735

Bernardi Rafael C. http://orcid.org/0000-0003-0758-2026

Nash Michael A. http://orcid.org/0000-0003-3842-1567

21 5 2024
2024.05.21.595133http://biorxiv.org/lookup/doi/10.1101/2024.05.21.595133
nihpp-2024.05.21.595133.pdf
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 Affibody’s 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 ∼10 4 -10 5 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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pmc
