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BackgroundRecognition proteins are critical in many biotechnology applications and would be even more useful if their binding could be regulated. The current gold standard for recognition molecules, antibodies, lacks convenient regulation. Alternative scaffolds can be used to build recognition proteins with new functionalities, including regulated recognition molecules. Here we test the use of the bacterial adhesin FimH as a scaffold for regulated molecular recognition. FimH binds to its native small molecule target mannose in a conformation-dependent manner that can be regulated by two types of noncompetitive regulation: allosteric and parasteric.ResultsWe demonstrate that conformational regulation of FimH can be maintained even after reengineering the binding site to recognize the non-mannosylated targets nickel or Penta-His antibody, resulting in an up to 7-fold difference in KDbetween the two conformations. Moreover, both the allosteric and parasteric regulatory mechanisms native to FimH can be used to regulate binding to its new target. In one mutant, addition of the native ligand mannose parasterically improves the mutant’s affinity for Penta-His 4-fold, even as their epitopes overlap. In another mutant, the allosteric antibody mab21 reduces the mutant’s affinity for Penta-His 7-fold. The advantage of noncompetitive regulation is further illustrated by the ability of this allosteric regulator to induce 98% detachment of Penta-His, even with modest differences in affinity.ConclusionsThis illustrates the potential of FimH, with its deeply studied conformation-dependent binding, as a scaffold for conformationally regulated binding via multiple mechanisms.

The binding of bacteria to platelets is thought to be a central event in the pathogenesis of infective endocarditis. The serine-rich repeat (SRR) glycoproteins of viridans group streptococci have been shown to mediate platelet binding in vitro and to contribute to virulence in animal models. However, it is not known whether SRR adhesins can mediate streptococcal binding under the high fluidic shear stress conditions present on the endocardial surface. We found that three streptococcal SRR adhesins (GspB, Hsa, and SrpA) with differing structures and sialoglycan binding specificities nevertheless exhibited similar biomechanical properties. All three adhesins mediated shear-enhanced streptococcal binding to immobilized platelets through the platelet receptor GPIbα. Shear-enhanced adhesion was manifested in three ways. First, the number of circulating streptococci binding via SRR adhesins to immobilized platelet receptors peaked at 1 dyn/cm 2 . Second, bound streptococci switched from weak rolling to strong stationary adhesion as shear stress increased to 10 dyn/cm 2 . Third, while a few streptococci detached each time the flow was increased, the majority of streptococci bound to platelets remained firmly attached through 20 to 80 dyn/cm 2 (shear levels typical of arteries and the endocardium). Thus, all three adhesins mediated shear-enhanced streptococcal binding to platelets under the flow conditions found in heart valves. The ability of the SRR adhesins to mediate shear-enhanced binding strongly suggests that they form catch bonds that are activated by tensile force and provides a mechanism for the selective targeting of bacteria to platelet receptors immobilized on the endocardial surface.

Many receptors display conformational flexibility, in which the binding pocket has an open inactive conformation in the absence of ligand and a tight active conformation when bound to ligand. Here we study the bacterial adhesin FimH to address the role of the inactive conformation of the pocket for initiating binding by comparing two variants: a wild-type FimH variant that is in the inactive state when not bound to its target mannose, and an engineered activated variant that is always in the active state. Not surprisingly, activated FimH has a longer lifetime and higher affinity, and bacteria expressing activated FimH bound better in static conditions. However, bacteria expressing wild-type FimH bound better in flow. Wild-type and activated FimH demonstrated similar mechanical strength, likely because mechanical force induces the active state in wild-type FimH. However, wild-type FimH displayed a faster bond association rate than activated FimH. Moreover, the ability of different FimH variants to mediate adhesion in flow reflected the fraction of FimH in the inactive state. These results demonstrate a new model for ligand-associated conformational changes that we call the kinetic-selection model, in which ligand-binding selects the faster-binding inactive state and then induces the active state. This model predicts that in physiological conditions for cell adhesion, mechanical force will drive a nonequilibrium cycle that uses the fast binding rate of the inactive state and slow unbinding rate of the active state, for a higher effective affinity than is possible at equilibrium.

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., Author Summary A common approach in the development of selective inhibitors for ligand-receptor interactions is targeting the receptor binding site with the expectation that inhibitors will sterically interfere with ligand binding and thus block receptor function via a competitive (orthosteric) mechanism. However, using monoclonal antibodies specific for the mannose-binding Escherichia coli adhesin, FimH, we demonstrate that the binding site epitopes allow for non-competitive inhibition that is more effective than orthosteric blocking. FimH, similar to other binding proteins, exhibits conformational flexibility of the ligand-binding pocket shifting between open (inactive) and tight (active) conformations, with relatively low- and high- affinity towards mannose. We show that an antibody that binds just one of the mannose-binding pocket loops prevents the shift from the inactive to the active conformation and hence blocks formation of high-affinity ligand-receptor complexes. This antibody type was more effective in inhibition of bacterial adhesion than anti-FimH antibodies competitively blocking mannose binding, and unlike the latter or a soluble ligand, showed the ability to detach an established bacterial biofilm from a ligand-coated surface. As the newly described antibody can bind the FimH pocket simultaneously with ligand, we refer to it as a parasteric (next-to-ligand) inhibitor that exhibits non-competitive inhibition from within the binding-pocket of the receptor.

Many bacteria and eukaryotic cells express adhesive proteins at the end of tethers that elongate reversibly at constant or near constant force, which we refer to as yielding elasticity. Here we address the function of yielding elastic adhesive tethers with Escherichia coli bacteria as a model for cell adhesion, using a combination of experiments and simulations. The adhesive bond kinetics and tether elasticity was modeled in the simulations with realistic biophysical models that were fit to new and previously published single molecule force spectroscopy data. The simulations were validated by comparison to experiments measuring the adhesive behavior of E. coli in flowing fluid. Analysis of the simulations demonstrated that yielding elasticity is required for the bacteria to remain bound in high and variable flow conditions, because it allows the force to be distributed evenly between multiple bonds. In contrast, strain-hardening and linear elastic tethers concentrate force on the most vulnerable bonds, which leads to failure of the entire adhesive contact. Load distribution is especially important to noncovalent receptor-ligand bonds, because they become exponentially shorter lived at higher force above a critical force, even if they form catch bonds. The advantage of yielding is likely to extend to any blood cells or pathogens adhering in flow, or to any situation where bonds are stretched unequally due to surface roughness, unequal native bond lengths, or conditions that act to unzip the bonds., Author Summary Cells adhere to surfaces and each other in the presence of forces that would easily overpower the individual noncovalent receptor-ligand bonds that mediate this adhesion, raising the question as to how these bonds cooperate to withstand such high forces. Here we show that cooperation and robust adhesion depends on the elastic properties of the bonds. A type of nonlinear elasticity referred to as elastic yielding ensures that the total force is distributed equally across the individual bonds regardless of geometry. In contrast, with linear or strain-hardening elasticity, the bonds that are stretched the most are exposed to higher forces, which cause them to fail sequentially. This work explains why elastic yielding is found in structurally and evolutionarily diverse adhesive complexes.

In allosteric regulation, an effector molecule binding a protein at one site induces conformational changes, which alter structure and function at a distant active site. Two key challenges in the computational modeling of allostery are the prediction of the structure of one allosteric state starting from the structure of the other, and elucidating the mechanisms underlying the conformational coupling of the effector and active sites. Here we approach these two challenges using the Rosetta high-resolution structure prediction methodology. We find that the method can recapitulate the relaxation of effector-bound forms of single domain allosteric proteins into the corresponding ligand-free states, particularly when sampling is focused on regions known to change conformation most significantly. Analysis of the coupling between contacting pairs of residues in large ensembles of conformations spread throughout the landscape between and around the two allosteric states suggests that the transitions are built up from blocks of tightly coupled interacting sets of residues that are more loosely coupled to one another., Author Summary A common means of biological regulation is allostery, in which an effector molecule binds to one site on a protein and induces a conformational change which changes activity at a distant active site. Frequently high resolution structures are determined for one state of an allosteric protein but not the other. To probe the allosteric conformational changes in such cases, we describe a computational method for predicting the structure of one allosteric state of a protein starting with knowledge of another. Our method also provides a detailed map of the free energy landscape traversed in an allosteric transition and reveals the coupling between interacting residue pairs that underlies the transition.

Signal peptides (SPs) are critical for protein transport across cellular membranes, have a highly conserved structure, and are cleaved from the mature protein upon translocation. Here, we report that naturally occurring mutations in the SP of the adhesive, tip-associated subunit of type 1 fimbriae (FimH) are positively selected in uropathogenic Escherichia coli . On the one hand, these mutations have a detrimental effect, with reduced FimH transport across the inner membrane, fewer FimH and fimbriae expressed on the bacterial surface, and decreased bacterial adhesion under flow conditions. On the other hand, the fimbriae expressed by the mutants are significantly longer on average, with many fimbriae able to stretch to >20 μm in length. More surprisingly, the SP mutant bacteria display an increased ability to resist detachment from the surface upon a switch from high to low flow. This functional effect of longer fimbriae highlights the importance of the nonadhesive fimbrial rod for adhesive function. Also, whereas bacterial adhesion to bladder epithelial cells was preserved in most mutants, binding to and killing by human neutrophils was decreased, providing an additional reason the SP mutations are relatively common among uropathogenic strains. Thus, this study demonstrates how mutations in an SP, while decreasing transport function and not affecting the final structure of the translocated protein, can lead to functional gains of the extracellular organelles that incorporate the protein and overall adaptive changes in the organism's fitness.

Carcinoembryonic antigen (CEA) related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N-terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgV-like domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis, and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.

We determined whether the molecular structures through which force is applied to receptor–ligand pairs are tuned to optimize cell adhesion under flow. The adhesive tethers of our model system, Escherichia coli, are type I fimbriae, which are anchored to the outer membrane of most E. coli strains. They consist of a fimbrial rod (0.3–1.5 μm in length) built from a helically coiled structural subunit, FimA, and an adhesive subunit, FimH, incorporated at the fimbrial tip. Previously reported data suggest that FimH binds to mannosylated ligands on the surfaces of host cells via catch bonds that are enhanced by the shear-originated tensile force. To understand whether the mechanical properties of the fimbrial rod regulate the stability of the FimH–mannose bond, we pulled the fimbriae via a mannosylated tip of an atomic force microscope. Individual fimbriae rapidly elongate for up to 10 μm at forces above 60 pN and rapidly contract again at forces below 25 pN. At intermediate forces, fimbriae change length more slowly, and discrete 5.0 ± 0.3–nm changes in length can be observed, consistent with uncoiling and coiling of the helical quaternary structure of one FimA subunit at a time. The force range at which fimbriae are relatively stable in length is the same as the optimal force range at which FimH–mannose bonds are longest lived. Higher or lower forces, which cause shorter bond lifetimes, cause rapid length changes in the fimbria that help maintain force at the optimal range for sustaining the FimH–mannose interaction. The modulation of force and the rate at which it is transmitted from the bacterial cell to the adhesive catch bond present a novel physiological role for the fimbrial rod in bacterial host cell adhesion. This suggests that the mechanical properties of the fimbrial shaft have codeveloped to optimize the stability of the terminal adhesive under flow., PLoS Biology, 4 (9), ISSN:1544-9173, ISSN:1545-7885

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli . Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.

Triiodothyronine in vitro at concentrations between 10 −13 and 1O −11 M very rapidly activates oxidative phosphorylation in hypothyroid rat liver mitochondria. Comparing the concentrations of hormone with estimates of the amounts of respiratory chain components present suggests that this activation may involve an amplification mechanism. Here we present evidence that while no changes in phosphorylation were detected following hormone administration, nicotinamide, an inhibitor of mono ADP-ribosylation reported to occur rapidly and reversibly in mitochondria, prevented activation by hormone. Moreover incubation with nicotinamide of euthyroid mitochondria and derived intact inner membrane vesicles revealed lowered ADP/O ratios under the same conditions as shown by hypothyroid preparations. While this lesion could be reversed simply by washing the intact mitochondria, the membrane vesicles required triiodothyronine addition.

Some recently studied biological noncovalent bonds have shown increased lifetime when stretched by mechanical force. In each case these counterintuitive “catch-bonds” have transitioned into ordinary “slip-bonds” that become increasingly shorter lived as the tensile force on the bond is further increased. We describe analytically how these results are supported by a physical model whereby the ligand escapes the receptor binding site via two alternative routes, a catch-pathway that is opposed by the applied force and a slip-pathway that is promoted by force. The model predicts under what conditions and at what critical force the catch-to-slip transition would be observed, as well as the degree to which the bond lifetime is enhanced at the critical force. The model is applied to four experimentally studied systems taken from the literature, involving the binding of P- and L-selectins to sialyl LewisX oligosaccharide-containing ligands. Good quantitative fit to the experimental data is obtained, both for experiments with a constant force and for experiments where the force increases linearly with time.

Regulation of the bond between platelet Glycoprotein Ib (GPIb) and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and/or tensile mechanical force, but the structural basis of this mechanical regulation remained unknown. To address this question, we expressed a number of A1 constructs that differed in one or more residues or domains and anchored them to the surface at known concentrations and orientations. We then measured rolling velocities of platelets or GPIb-coated microspheres to determine the importance of different structural regions and the manner in which force is applied. We show that an intrinsically disordered peptide region N-terminal to the A1 domain has strong inhibitory activity. In addition, the A1-GPIb bond is much stronger when the A1 domain is anchored via the C-terminus rather than the N-terminus. This suggests that shear stress activates A1-GPIb binding in two ways. First, it applies tensile force across multimeric VWF, activating the A1 domain by dislodging this inhibitory region. Second, it applies tensile force across the A1-GPIb bond, inducing a conformational change in the C-terminal region of VWF that allosterically activates the A1 domain, perhaps in a way analogous to that seen in integrins.

Bacteria can adhere to mannose present on the surface of epithelial cells via the FimH-mannose bond. Surprisingly, they have been shown to enhance their adhesion to the cells when fluid flow is increased. These conditions would normally weaken traditional ‘slip’ bonds. Catch bonds have been defined as non-covalent bonds whose lifetime increases with tensile force on the bond, instead of decreasing as expected for slip bonds.Here we present results confirming catch bonds in FimH-mannose complexes at the single molecule level using the Atomic Force Microscope (AFM). This is the first AFM measurement of catch bond lifetimes using a constant force mode. In constant force mode (or force clamp), a feedback loop maintains the force at a predefined level, correcting for changes in conformation of the molecule. We observe multiple lifetimes and that force enhances the proportion of bonds with a long lifetime. This is the first measurement that resolves multiple lifetimes in catch bonds while verifying that only a single molecule is tethered. We discuss the implications of these findings in the context of the mechanism giving rise to bacterial catch bonds.

The initial arrest of platelets at a wound site requires the binding of glycoprotein Ib-IX-V complex (GPIb-IX-V) to the extracellular matrix protein von Willebrand factor (VWF). Increasing forces on these bonds increases the bond lifetime, known as a catch bond. Recently, we have shown that platelets are able to transmit cytoskeletal forces through the GPIbα subunit of GPIb-IX-V, which binds to the A1 domain of VWF. This provides an internal force on the bond to maintain adhesion in the absence of external forces. Integrin engagement and force transmission are known to require receptor clustering. We therefore investigate whether the clustering of GPIb-IX-V receptor is needed for the transmission of cytoskeletal forces to VWF. We first examined whether GPIb-IX-V clusters formed in spreading platelets. GPIbα-positive punctate structures were visualized by confocal microscopy and correlated with dark regions imaged using interference reflection microscopy (IRM), indicating that GPIb-IX-V forms adhesive contacts that are similar in size to integrin-related focal adhesions in platelets. To investigate the effect of ligand density on platelet spreading, we prepared molecularly-defined adhesive ligand spots, which were separated 28 or 108 nm apart by nonadhesive regions using self-assembling gold nanoparticles. These nanoparticles were coated with A1 domain of VWF. When seeded on nanoparticles with 28 nm spacing, platelets appeared well-spread and formed punctate adhesions. However, when seeded on 108 nm spacing, platelet adhesion was significantly reduced and those platelets that did adhere spread poorly. These findings demonstrate that an upper limit for ligand density exists where platelet adhesion and spreading are impeded. Investigating the effects of ligand spacing will facilitate an understanding of GPIbα clustering in platelet adhesion and spreading, thus providing insight into thrombotic diseases and congenital bleeding disorders.

Many blood cells and pathogens bind to other cells or tissues in the presence of flowing fluid. These diverse cells have evolved mechanisms to withstand and even utilize the associated drag forces to strengthen adhesion, so that many of them display a shear enhanced adhesion in which they detach at low shear but roll along the tissue surface or even stick firmly at higher shear. In this talk we use Escherichia coli as a model system to determine the role of various molecules in this counterintuitive behavior. We use force spectroscopy to characterize the mechanical properties of simple molecular complexes, including the adhesive molecules of E. coli, which form catch bonds that are longer lived under increased tensile force, and the tethers anchoring these bonds, which elongate long distances at a constant force. We then incorporate these behaviors into simulations to understand the how each property contributes to the adhesive behavior of cells or artificial adhesives. Other pathogens and blood cells display a remarkable convergent evolution, because their adhesive bonds and tethers catch and elongate with similar mechanical properties as those of E. coli, although they have no genetic or structural similarities. This suggests that the principles established from studying this model system can be extended to a large number of cells adhering in flow.