Talin as a critical focal adhesion mechanosensor exhibits force-dependent folding dynamics and concurrent interactions. Being a cytoplasmic protein, talin also might interact with several cytosolic chaperones; however, the roles of chaperones in talin mechanics remain elusive. To address this question, we investigated the force response of a mechanically stable talin domain with a set of well-known unfoldase (DnaJ, DnaK) and foldase (DnaKJE, DsbA) chaperones, using single-molecule magnetic tweezers. Our findings demonstrate that chaperones could affect adhesion proteins’ stability by changing their folding mechanics; while unfoldases reduce their unfolding force from ~11 pN to ~6 pN, foldase shifts it upto ~15 pN. Since talin is mechanically synced within 2 pN force ranges, these changes are significant in cellular conditions. Furthermore, we determined that chaperones directly reshape the energy landscape of talin: unfoldases decrease the unfolding barrier height from 26.8 to 21.7 kBT, while foldases increase it to 33.5 kBT. We reconciled our observations with eukaryotic Hsp70 and Hsp40 and observed their similar function of decreasing the talin unfolding barrier. Quantitative mapping of this chaperone-induced talin folding landscape directly illustrates that chaperones perturb the adhesion protein stability under physiological force, thereby, influencing their force-dependent interactions and adhesion dynamics.

Talin as an adhesion protein, exhibits a strong force-dependent structure-function dynamics. Being a mechanosensitive focal adhesion (FA) protein, talin might interact to several FA targeting drugs; however, the molecular mechanism of talin-drug interactions remains elusive. Here we combined magnetic tweezers and molecular dynamics (MD) simulation to explore mechanical stability of talin with three drugs based on their talin specificity. Interestingly, our study revealed that talin displays a bimodal force distribution with a low and high unfolding force population. We observed that talin nonspecific drugs (tamoxifen and letrozole) display biphasic effect: increase talin mechanical stability upto optimum concentration, followed by a decrease in stability with further concentration increase. By contrast, talin-specific cyanidin 3-O-glucoside promotes a steady increase to talin mechanical stability with its concentration. We reconciled our observation from the simulation study: tamoxifen enters into talin hydrophobic core, eventually destabilizing the protein; whereas cyanidin 3-O-glucoside stabilizes the protein core by maintaining the inter-helix distance. Finally, we observed a strong correlation among hydrophobicity and cavity analysis, illustrating a detailed mechanistic analysis of drug effect on the mechanosensitive protein. Overall this study presents a novel perspective for drug designing against mechanosensitive proteins and studying off-target effects of already known drugs.

Recent single-molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (below 5 pN), folded talin binds RIAM for integrin activation; whereas at high force (above 5 pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic protein, talin might interact with several cytosolic chaperones: however, the role of chaperones in talin mechanics is unknown.To address this question, we investigated the force response of a mechanically stable talin domain with a set of well-known holdase (DnaJ, DnaK, Hsp70, and Hsp40) and foldase (DnaKJE, DsbA) chaperones, using single-molecule magnetic tweezers. Our findings demonstrate that chaperone could affect adhesion proteins stability by changing their folding mechanics; while holdase chaperones reduce their unfolding force to ~6 pN, foldase chaperones shift it up to ~15 pN. Since talin is mechanically synced within 2 pN force ranges, these changes are significant in cellular condition. Furthermore, we determined the fundamental mechanism of this altered mechanical stability, where chaperones directly reshape their energy landscape: unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.7 kBT, while foldase chaperone (DsbA) increases it to 33.5 kBT. We reconciled our observations with eukaryotic Hsp70 and Hsp40 chaperones and observed their similar function of decreasing the talin unfolding barrier to 23.1 kBT. The quantitative mapping of this chaperone-induced talin folding landscape directly illustrates that chaperones perturb the adhesion protein stability under physiological force, thereby influencing their force-dependent interactions and adhesion dynamics.

Methotrexate is a well-known antineoplastic drug used to prevent cancer aggravation. Despite being a targeted therapeutic approach, its administration comes with the risk of cancer recurrence, plausibly through its proven off-target effect on focal adhesions. Since FA dynamics is dependent on force transmission through its constituent proteins, including talin, methotrexate might affect the mechanical activity of these proteins. Here we have combined single-molecule studies, computational dynamics, cell-based assays, and genomic analysis to unveil the focal adhesion-regulating role of methotrexate central to its effect on talin dynamics and downstream pathways. Interestingly, our single-molecule force spectroscopic study shows that methotrexate modulates the bimodal force distribution of talin in a concentration-dependent manner. Steered molecular dynamics reveal that methotrexate-talin interactions alter talin mechanical stability exposing their vinculin binding sites. Finally, we found that methotrexate-regulated talin-dynamics remodel cancer cell mechanical phenotypes like cell polarity, adhesion, and migration by regulating talin-vinculin association-mediated YAP signaling. These results further correlate with genomic analysis of methotrexate-treated patients, demonstrating its clinical importance. Taken together, these findings disseminate the effects of methotrexate-modulated mechanosensitivity of adhesion proteins on cellular events.