| Autor: |
Yang B; Institute for Physical Chemistry, Department of Chemistry, University of Basel, 4058 Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4056 Basel, Switzerland., Gomes DEB; Department of Physics, Auburn University, Auburn, Alabama 36849, United States., Liu Z; Institute for Physical Chemistry, Department of Chemistry, University of Basel, 4058 Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4056 Basel, Switzerland.; Present address: Department of Bionanoscience, Delft University of Technology, 2629HZ Delft, the Netherlands., Santos MS; Institute for Physical Chemistry, Department of Chemistry, University of Basel, 4058 Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4056 Basel, Switzerland., Li J; Institute for Physical Chemistry, Department of Chemistry, University of Basel, 4058 Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4056 Basel, Switzerland., Bernardi RC; Department of Physics, Auburn University, Auburn, Alabama 36849, United States., Nash MA; Institute for Physical Chemistry, Department of Chemistry, University of Basel, 4058 Basel, Switzerland.; Department of Biosystems Science and Engineering, ETH Zurich, 4056 Basel, Switzerland. |
| Abstrakt: |
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. |
