Stretching fibronectin fibres disrupts binding of bacterial adhesins by physically destroying an epitope

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

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

Multidomain Assembler (MDA) Generates Models of Large Multidomain Proteins

Homology modeling predicts protein structures using known structures of related proteins as templates. We developed MULTIDOMAIN ASSEMBLER (MDA) to address the special problems that arise when modeling proteins with large numbers of domains, such as fibronectin with 30 domains, as well as cases with hundreds of templates. These problems include how to spatially arrange nonoverlapping template structures, and how to get the best template coverage when some sequence regions have hundreds of available structures while other regions have a few distant homologs. MDA automates the tasks of template searching, visualization, and selection followed by multidomain model generation, and is part of the widely used molecular graphics package UCSF CHIMERA (University of California, San Francisco). We demonstrate applications and discuss MDA's benefits and limitations

Eyelid Closure Behavior of Patients with Idiopathic and Nonorganic Hypersomnia, Narcolepsy-Cataplexy, and Healthy Controls in the Maintenance of Wakefulness Test.

PURPOSE Differential diagnosis of central disorders of hypersomnolence remains challenging, particularly between idiopathic (IH) and nonorganic hypersomnia (NOH). We hypothesized that eyelid closure behavior in the maintenance of wakefulness test (MWT) could be a valuable biomarker. PATIENTS AND METHODS MWT recordings of patients with IH, NOH, narcolepsy-cataplexy (NC), and healthy sleep-deprived controls (H) were retrospectively analyzed (15 individuals per group). For each MWT trial, visual scoring of face videography for partial (50-80%) and full eyelid closure (≥80%) was performed from "lights off" to the first microsleep episode (≥3 s). RESULTS In all groups, the frequency and cumulative duration of periods with partial and full eyelid closure gradually increased toward the first microsleep episode. On the group level, significant differences occurred for the latency to the first microsleep episode (IH 21 min (18-33), NOH 23 min (17-35), NC 11 min (7-19), H 10 min (6-25); p = 0.009), the ratio between partial and full eyelid closure duration (IH 2.2 (0.9-3.1), NOH 0.5 (0-1.2), NC 2.8 (1.1-5), H 0.7 (0.4-3.3); p = 0.004), and the difference between full and partial eyelid closure duration in the five minutes prior to the first microsleep episode (∆full - partial eyelid closure duration: IH -16 s (-35 to 28); NOH 46 s (9-82); NC -6 s (-26 to 5); H 10 s (-4 to 18); p = 0.007). IH and NOH significantly differed comparing the ratio between partial and full eyelid closure (p = 0.005) and the difference between ∆full - partial eyelid closure duration in the five minutes prior to the first microsleep episode (p = 0.006). CONCLUSION In the MWT, eyelid closure behavior (∆full - partial) in the period prior to the first microsleep episode could be of value for discriminating NOH from other etiologies of excessive daytime sleepiness, particularly IH

Simulation schematics for case studies.

<p>Key perturbations to the experimental structure are indicated in red text. (A) Binding and cooperativity of a negative allosteric modulator (NAM) in the M2 muscarinic receptor (M2R). The binding pose of the NAM and its cooperativity with the orthosteric ligand are probed by performing unguided binding simulations with an unliganded receptor and a receptor with bound orthosteric ligand. Negative cooperativity leads to stronger binding of the NAM to the unliganded M2R. Simulations indicate that cooperativity in this system is due both to direct electrostatic repulsion between cationic ligands and coupled conformational changes of the two binding sites. (B) Activation mechanism of the β₂-adrenergic receptor (β₂AR). Removal of the bound G protein from the active-state, agonist-bound crystal structure of β₂AR leads to a spontaneous transition to the inactive state in simulation, capturing the activation process in reverse. (C) Mechanism of GPCR-catalyzed nucleotide release from a heterotrimeric G protein. Simulations of a G protein with and without bound guanosine diphosphate (GDP) suggested that nucleotide release from a G protein—which leads to G protein activation—takes place via a previously unexpected mechanism. (D) Force-induced uncoupling of a bacterial adhesin from a fibronectin fragment. MD simulations of a fibronectin–adhesin complex led to the discovery that application of stretching forces to fibronectin reduces its affinity to adhesin.</p

Bidirectional symmetry in allosteric systems.

<p>Suppose that a protein can bind two different ligands, A and B, each at their respective sites. The difference in free energy Δ<i>G</i>_0→AB between a state with both ligands bound and one with neither ligand bound is independent of the order in which the ligands bind, so we can write it both as Δ<i>G</i>_0→AB = Δ<i>G</i>_0→B + Δ<i>G</i>_B→AB and as Δ<i>G</i>_0→AB = Δ<i>G</i>_0→A + Δ<i>G</i>_A→AB, implying that Δ<i>G</i>_0→B + Δ<i>G</i>_B→AB = Δ<i>G</i>_0→A + Δ<i>G</i>_A→AB. Rearranging this equation yields Δ<i>G</i>_A→AB − Δ<i>G</i>_0→B = Δ<i>G</i>_B→AB − Δ<i>G</i>_0→A. That is, the difference in the binding energy of ligand A with or without ligand B bound is the same as the difference in the binding energy of ligand B with or without ligand A bound. If one side of this equation is negative, the other must also be negative, and if one side is positive, the other must also be positive. Thus, if binding of ligand B is more favorable in the presence of A, then binding of ligand A is more favorable in the presence of ligand B (positive cooperativity). Likewise, if the presence of ligand A disfavors the binding of ligand B, then the presence of ligand B disfavors the binding of ligand A (negative cooperativity).</p

Novel peptide probes to assess the tensional state of fibronectin fibers in cancer

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 111In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111In-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.ISSN:2041-172