Exploring ligands that target von Willebrand factor selectively under oxidizing conditions through docking and molecular dynamics simulations.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets, tethering them to the site of vascular injury and initiating blood coagulation. This process is critical for the normal hemostatic response, but especially under inflammatory conditions, it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation, as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding, in particular intracranial hemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIbα, and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such an auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations, and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface, with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in the presence of inflammatory conditions.

Exploring ligands that target von Willebrand factor selectively under oxidizing conditions through docking and molecular dynamics simulations.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets tethering them to the site of vascular injury initiating blood coagulation. This process is critical for the normal haemostatic response, but especially under inflammatory conditions it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding in particular intracranial haemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIbα and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in inflammatory conditions.

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called FimH FocH , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and FimH FocH are almost identical, experimental evidence shows that FimH FocH is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in FimH FocH . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in FimH FocH than FimH. In conclusion, the mutations in FimH FocH shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin.

Allosteric proteins transition between 'inactive' and 'active' states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH - the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.

Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear.

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an 'inactive' conformation with fast binding to mannose to an 'active' conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor.

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details of how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ibα (GpIbα), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIbα-binding site in the A1 domain.

Oxidation-induced destabilization of the fibrinogen αC-domain dimer investigated by molecular dynamics simulations.

Upon activation, fibrinogen is converted to insoluble fibrin, which assembles into long strings called protofibrils. These aggregate laterally to form a fibrin matrix that stabilizes a blood clot. Lateral aggregation of protofibrils is mediated by the αC domain, a partially structured fragment located in a disordered region of fibrinogen. Polymerization of αC domains links multiple fibrin molecules with each other enabling the formation of thick fibrin fibers and a fibrin matrix that is stable but can also be digested by enzymes. However, oxidizing agents produced during the inflammatory response have been shown to cause thinner fibrin fibers resulting in denser clots, which are harder to proteolyze and pose the risk of deep vein thrombosis and lung embolism. Oxidation of Met476 located within the αC domain is thought to hinder its ability to polymerize disrupting the lateral aggregation of protofibrils and leading to the observed thinner fibers. How αC domains assemble into polymers is still unclear and yet this knowledge would shed light on the mechanism through which oxidation weakens the lateral aggregation of protofibrils. This study used temperature replica exchange molecular dynamics simulations to investigate the αC-domain dimer and how this is affected by oxidation of Met476 . Analysis of the trajectories revealed that multiple stable binding modes were sampled between two αC domains while oxidation decreased the likelihood of dimer formation. Furthermore, the side chain of Met476 was observed to act as a docking spot for the binding and this function was impaired by its conversion to methionine sulfoxide.

Destabilization of the von Willebrand factor A2 domain under oxidizing conditions investigated by molecular dynamics simulations.

The protein von Willebrand factor (VWF) is key for the adhesion of blood platelets to sites of vascular injury. Recent studies have shown that the release of oxidative agents during inflammation increases the platelet-tethering activity of VWF contributing to a pro-thrombotic state. This has been linked to the oxidation of methionine residues in the A1, A2 and A3 domains of VWF. The A1 domain binds to platelet surface receptors glycoprotein Ib α (GpIbα). This interaction has been shown to be inhibited under static conditions by the neighboring A2 domain. Tensile force exerted by blood flow unfolds the A2 domain normally leading to its cleavage by the metalloprotease ADAMTS13 preventing pathological thrombus formation. However, oxidizing conditions inhibit proteolysis through ADAMTS13. Here, molecular dynamics simulations tested the hypothesis whether methionine oxidation induced by inflammatory conditions favors unfolding of the A2 domain contributing to the experimentally observed activation of VWF. The results indicate that oxidation of methionine residues located near the C-terminal helix of the A2 domain reduce the force necessary to initiate unfolding. Furthermore, oxidation of methionine residues shifts the thermodynamic equilibrium of the A2 domain fold towards the denatured state. This work suggests a mechanism whereby oxidation reduces the kinetic and thermodynamic stability of the A2 domain removing its inhibitory function on the binding of the A1 domain to GpIbα.

Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis.

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets.

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.

Predicting the orientation of protein G B1 on hydrophobic surfaces using Monte Carlo simulations.

A Monte Carlo algorithm was developed to predict the most likely orientations of protein G B1, an immunoglobulin G (IgG) antibody-binding domain of protein G, adsorbed onto a hydrophobic surface. At each Monte Carlo step, the protein was rotated and translated as a rigid body. The assumption about rigidity was supported by quartz crystal microbalance with dissipation monitoring experiments, which indicated that protein G B1 adsorbed on a polystyrene surface with its native structure conserved and showed that its IgG antibody-binding activity was retained. The Monte Carlo simulations predicted that protein G B1 is likely adsorbed onto a hydrophobic surface in two different orientations, characterized as two mutually exclusive sets of amino acids contacting the surface. This was consistent with sum frequency generation (SFG) vibrational spectroscopy results. In fact, theoretical SFG spectra calculated from an equal combination of the two predicted orientations exhibited reasonable agreement with measured spectra of protein G B1 on polystyrene surfaces. Also, in explicit solvent molecular dynamics simulations, protein G B1 maintained its predicted orientation in three out of four runs. This work shows that using a Monte Carlo approach can provide an accurate estimate of a protein orientation on a hydrophobic surface, which complements experimental surface analysis techniques and provides an initial system to study the interaction between a protein and a surface in molecular dynamics simulations.

Mechanism of allosteric propagation across a ß-sheet structure investigated by molecular dynamics simulations.

The bacterial adhesin FimH consists of an allosterically regulated mannose-binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter-domain region, the mannose binding site and a large ß sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter-domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central ß sheet demonstrated a soft spring-like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called "population shift" model whereby binding of the lectin domain to either the pilin domain or mannose locks the ß sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990-1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli.

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.

Variable content of von Willebrand factor mutant monomer drives the phenotypic variability in a family with von Willebrand disease.

Von Willebrand disease (VWD) is an inherited bleeding disorder characterized by incomplete penetrance and variable expressivity. We evaluated a 24-member pedigree with VWD type 2 caused by a T>G mutation at position 3911 that predicts a methionine to arginine (M1304R) change in the platelet-binding A1 domain of von Willebrand factor (VWF). This mutation manifests as an autosomal-dominant trait, with clinical and biochemical phenotypic variability among affected individuals, including differences in bleeding tendency and VWF quantity, activity, and multimer pattern. Sequencing of all VWF coding regions in 3 affected individuals did not identify additional mutations. When expressed in heterologous cells, M1304R was secreted in lower quantities, failed to drive formation of storage granules, and was defective in multimerization and platelet binding. When cotransfected in equal quantities with the wild-type complementary DNA, the mutant complementary DNA depressed VWF secretion, although multimerization was only mildly affected. A llama nanobody (AU/VWFa-11) that detects the mutant A1 domain demonstrated highly variable binding to VWF from different affected members, indicating that the VWF contained different percentages of mutant monomers in different individuals. Thus, the observed variability in VWD phenotypes could in part be determined by the extent of mutant monomer incorporation in the final multimer structure of plasma VWF.

Allosteric coupling in the bacterial adhesive protein FimH.

The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH.

Tight conformational coupling between the domains of the enterotoxigenic Escherichia coli fimbrial adhesin CfaE regulates binding state transition.

CfaE, the tip adhesin of enterotoxigenic Escherichia coli colonization factor antigen I fimbriae, initiates binding of this enteropathogen to the small intestine. It comprises stacked ß-sandwich adhesin (AD) and pilin (PD) domains, with the putative receptor-binding pocket at one pole and an equatorial interdomain interface. CfaE binding to erythrocytes is enhanced by application of moderate shear stress. A G168D replacement along the AD facing the CfaE interdomain region was previously shown to decrease the dependence on shear by increasing binding at lower shear forces. To elucidate the structural basis for this functional change, we studied the properties of CfaE G168D (with a self-complemented donor strand) and solved its crystal structure at 2.6 Å resolution. Compared with native CfaE, CfaE G168D showed a downward shift in peak erythrocyte binding under shear stress and greater binding under static conditions. The thermal melting transition of CfaE G168D occurred 10 °C below that of CfaE. Compared with CfaE, the atomic structure of CfaE G168D revealed a 36% reduction in the buried surface area at the interdomain interface. Despite the location of this single modification in the AD, CfaE G168D exhibited structural derangements only in the adjoining PD compared with CfaE. In molecular dynamics simulations, the G168D mutation was associated with weakened interdomain interactions under tensile force. Taken together, these findings indicate that the AD and PD of CfaE are conformationally tightly coupled and support the hypothesis that opening of the interface plays a critical modulatory role in the allosteric activation of CfaE.

Structural basis of type 2A von Willebrand disease investigated by molecular dynamics simulations and experiments.

The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered in silico the tensile force necessary to undock the C-terminal helix α6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix α6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.

Heparin modulates the conformation and signaling of platelet integrin αIIbß3.

INTRODUCTION: The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbß3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbß3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. METHODS: We assessed conformational changes in αIIbß3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbß3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the ß3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbß3, and of recombinant fragments of αIIb and ß3, by surface plasmon resonance. RESULTS AND CONCLUSIONS: Heparin binding results in conformational changes in αIIbß3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin ß3 cytoplasmic domain, but the presence of heparin increased both ß3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant ß3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbß3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbß3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

The bacterial fimbrial tip acts as a mechanical force sensor.

There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.