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Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.

Transformations of extracellular matrix (ECM) accompany pathological tissue changes, yet how cell-ECM crosstalk drives these processes remains unknown as adequate tools to probe forces or mechanical strains in tissues are lacking. Here, we introduce a new nanoprobe to assess the mechanical strain of fibronectin (Fn) fibers in tissue, based on the bacterial Fn-binding peptide FnBPA5. FnBPA5 exhibits nM binding affinity to relaxed, but not stretched Fn fibers and is shown to exhibit strain-sensitive ECM binding in cell culture in a comparison with an established Fn-FRET probe. Staining of tumor tissue cryosections shows large regions of relaxed Fn fibers and injection of radiolabeled 111 In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111 In-FnBPA5 in tumor with prolonged retention compared to other organs. The herein presented approach enables to investigate how Fn fiber strain at the tissue level impacts cell signaling and pathological progression in different diseases.

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Although soluble inhibitors are frequently used to block cell binding to the extracellular matrix (ECM), mechanical stretching of a protein fibre alone can physically destroy a cell-binding site. Here, we show using binding assays and steered molecular dynamics that mechanical tension along fibronectin (Fn) fibres causes a structural mismatch between Fn-binding proteins from Streptococcus dysgalactiae and Staphylococcus aureus. Both adhesins target a multimodular site on Fn that is switched to low affinity by stretching the intermodular distances on Fn. Heparin reduces binding but does not eliminate mechanosensitivity. These adhesins might thus preferentially bind to sites at which ECM fibres are cleaved, such as wounds or inflamed tissues. The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow. Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.