We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in k off by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.

Anticalin is a non-antibody protein scaffold with potential as an alternative to monoclonal antibodies for targeted drug delivery to cytotoxic T-lymphocyte antigen 4 (CTLA-4) positive T-cells. In this context, one limiting factor is the ability of the anticalin:CTLA-4 complex to resist mechanical forces exerted by local shear stress. Here, we used single-molecule AFM force spectroscopy (AFM-SMFS) to screen residues along the anticalin backbone and determine the optimal anchor point that maximizes binding strength of the anticalin:CTLA-4 complex. We used non-canonical amino acid incorporation by amber suppression combined with click chemistry to attach an Fgβ peptide at internal residues of the anticalin. We then used Fgβ as a handle to pick up and mechanically dissociate anticalin from CTLA-4 from eight different anchoring residues using an AFM cantilever tip. By quantifying the unbinding energy landscape for each pulling geometry, we found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2-3 orders of magnitude over a force range of 50-200 pN. Five of the six internal pulling points tested were significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1-10 nN/sec. Anisotropic network modelling, along with molecular dynamics (MD) simulations using the Gō-MARTINI approach explain the mechanism underlying the geometric dependency of mechanostability. These results suggest that optimization of attachment residue position for therapeutic and diagnostic cargo can provide large improvements in binding strength, allowing affinity maturation without requiring genetic mutation of binding interface residues.

ligand binding. We demonstrate the observed differences in the ligand modulation arise due to differences in binding site and binding mode of the two complexes. Our simulations reveal that the transition state structure of SUMO1 gets perturbed on binding with S12. In contrast, the binding of CUE-2 has minimal structural impact of ubiquitin. Thus our study demonstrates that a direct interaction with the b-clamp does not necessarily modulate the mechanical stability of proteins, an observation in contrast with previous studies. Instead ligand binding far from the b-clamp can reinforce the mechanical stability of the proteins. Our study highlights the importance of ligand binding site in modulating mechanical stability of ubiquitin family proteins.