Bacterial Adhesin FimH Research Articles

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 , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and are almost identical, experimental evidence shows that 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 . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in than FimH. In conclusion, the mutations in shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

Binding site plasticity regulation of the FimH catch-bond mechanism

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional “local” catch-bond effect could delay unbinding between the bacteria and the host cell before the “global” allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state.

Serial crystallography and kinetics reveal how the FimH bacterial lectin tweaks between mono‐ and multivalent binding of high‐mannose N‐glycans

BackgroundThe lectin domain of the fimbrial adhesin FimH from Escherichia coli recognizes with the highest affinity and in seemingly monovalent fashion oligomannosides‐3 and ‐5 N‐glycans. This happens with a high affinity (Kd=20 nM) because the alpha1,3‐linked arm remains free from any further alpha1,2‐mannose substitution. The latter causes an at least 10‐fold affinity loss in creating the oligomannose‐6 N‐glycan (1).MethodsThe binding of FimH to pauci‐ and oligomannoside‐containing N‐glycans and similarly glycosylated proteins, as well as its dimannoside endings Manalpha1,2, Manalpha1,3 and Manalpha1,6, have been profiled using the FimH Lectprofile kit (GlycoDiag, Applied Glycomics, France) and a newly designed paucimannose‐containing glycan array. The kinetics of binding, ka and kd, were analysed using surface plasmon resonance and compared with earlier incomplete data (2).ResultsMono‐ and multivalent binding were found in co‐crystal structures of the FimH lectin domain with alpha1,6‐fucosylated oligomannose‐3 and with oligoannose‐6, respectively. The kinetics of binding indicate that once alpha1,2‐linked mannose comes into play, the complexes are not so stable, as previously also measured using calorimetry (3), and accompanied with a high dissociation constant. Molecular dynamics simulations further support that it is not the total interaction energy that determines the final retained complex (metastable complexes), but the kinetics of interaction with the three possible dimannoside endings.ConclusionsKinetics and stability of the complexes drive the most favoured arrangement, via either a unique or multivalent interaction, between the N‐glycan and the FimH protein in the crystal.References :(1) Bouckaert, J., Mackenzie, J., de Paz, J. L., Chipwaza, B., Choudhury, D., Zavialov, A., Mannerstedt, K., Anderson, J., Pierard, D., Wyns, L., Seeberger, P. H., Oscarson, S., De Greve, H., Knight, S. D. (2006) The affinity of the FimH fimbrial adhesin is receptor‐driven and quasi‐independent of Escherichia colipathotypes. Molecular Microbiology 61, 1556‐1568(2) Sauer, M. M., Jakob, R. P., Luber, T., Canonica, F., Navarra, G., Ernst, B., Maier, T., Glockshuber, R. (2019) Binding of the bacterial adhesin FimH to its natural, multivalent high‐mannose type glycan targets. J Am Chem Soc 141, 936‐944(3) Dumych, T., Bridot, C., Gouin, S. G., Paryzhak, S., Szunerits, S., Bilyy, R., Bouckaert, J., (2018) A novel integrated way for deciphering the glycan code for the FimH lectin. Molecules 23, 2794

Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH

Glycoprotein 2 (GP2) and uromodulin (UMOD) filaments protect against gastrointestinal and urinary tract infections by acting as decoys for bacterial fimbrial lectin FimH. By combining AlphaFold2 predictions with X-ray crystallography and cryo-EM, we show that these proteins contain a bipartite decoy module whose new fold presents the high-mannose glycan recognized by FimH. The structure rationalizes UMOD mutations associated with kidney diseases and visualizes a key epitope implicated in cast nephropathy.

Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

FimH as a scaffold for regulated molecular recognition

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 KD between 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.

Inflammatory Bowel Disease-associated GP2 Autoantibodies Inhibit Mucosal Immune Response to Adherent-invasive Bacteria.

Adherent-invasive Escherichia coli have been suggested to play a pivotal role within the pathophysiology of inflammatory bowel disease (IBD). Autoantibodies against distinct splicing variants of glycoprotein 2 (GP2), an intestinal receptor of the bacterial adhesin FimH, frequently occur in IBD patients. Hence, we aimed to functionally characterize GP2-directed autoantibodies as a putative part of IBD's pathophysiology. Ex vivo, GP2-splicing variant 4 (GP2#4) but not variant 2 was expressed on intestinal M or L cells with elevated expression patterns in IBD patients. The GP2#4 expression was induced in vitro by tumor necrosis factor (TNF)-α. The IBD-associated GP2 autoantibodies inhibited FimH binding to GP2#4 and were decreased in anti-TNFα-treated Crohn's disease patients with ileocolonic disease manifestation. In vivo, mice immunized against GP2 before infection with adherent-invasive bacteria displayed exacerbated intestinal inflammation. In summary, autoimmunity against intestinal expressed GP2#4 results in enhanced attachment of flagellated bacteria to the intestinal epithelium and thereby may drive IBD's pathophysiology.

D-Mannose Treatment neither Affects Uropathogenic Escherichia coli Properties nor Induces Stable FimH Modifications.

Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts “antibiotic-like” activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH’s capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs.

Comparison of affinity ranking by target-directed dynamic combinatorial chemistry and surface plasmon resonance

Target-directed dynamic combinatorial chemistry (tdDCC) is a powerful method to screen ligands for pharmacologically relevant targets. Generating a dynamic library from reversibly reacting building blocks in the presence of a target protein leads to the amplification of the most potent library constituents. In previous studies on tdDCC, these compounds were identified in a qualitative “hit/no-hit”-manner. However, the precise relationship between the degree of amplification and the affinity of the library constituent has not yet been evaluated. To study the amplification–affinity relationship, we compared tdDCC experiments, employing reversible acylhydrazone formation and the bacterial adhesin FimH as a target, with affinities of the library constituents as determined by surface plasmon resonance.

Binding of the Bacterial Adhesin FimH to Its Natural, Multivalent High-Mannose Type Glycan Targets.

Multivalent carbohydrate-lectin interactions at host-pathogen interfaces play a crucial role in the establishment of infections. Although competitive antagonists that prevent pathogen adhesion are promising antimicrobial drugs, the molecular mechanisms underlying these complex adhesion processes are still poorly understood. Here, we characterize the interactions between the fimbrial adhesin FimH from uropathogenic Escherichia coli strains and its natural high-mannose type N-glycan binding epitopes on uroepithelial glycoproteins. Crystal structures and a detailed kinetic characterization of ligand-binding and dissociation revealed that the binding pocket of FimH evolved such that it recognizes the terminal α(1-2)-, α(1-3)-, and α(1-6)-linked mannosides of natural high-mannose type N-glycans with similar affinity. We demonstrate that the 2000-fold higher affinity of the domain-separated state of FimH compared to its domain-associated state is ligand-independent and consistent with a thermodynamic cycle in which ligand-binding shifts the association equilibrium between the FimH lectin and the FimH pilin domain. Moreover, we show that a single N-glycan can bind up to three molecules of FimH, albeit with negative cooperativity, so that a molar excess of accessible N-glycans over FimH on the cell surface favors monovalent FimH binding. Our data provide pivotal insights into the adhesion properties of uropathogenic Escherichia coli strains to their target receptors and a solid basis for the development of effective FimH antagonists.

KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists.

Affinity data, such as dissociation constants (KD ) or inhibitory concentrations (IC50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant koff ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant kon ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon .

Targeting Dynamical Binding Processes in the Design of Non-Antibiotic Anti-Adhesives by Molecular Simulation-The Example of FimH.

Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.

Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system

For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. One example is the bacterial adhesin FimH, where the C-terminal pilin domain exerts negative allosteric control over binding of the N-terminal lectin domain to mannosylated ligands on host cells. When the lectin and pilin domains are separated under shear stress, the FimH-ligand interaction switches in a so-called catch-bond mechanism from the low- to high-affinity state. So far, it has been assumed that the pilin domain is essential for the allosteric propagation within the lectin domain that would otherwise be conformationally rigid. To test this hypothesis, we generated mutants of the isolated FimH lectin domain and characterized their thermodynamic, kinetic, and structural properties using isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, and X-ray techniques. Intriguingly, some of the mutants mimicked the conformational and kinetic behaviors of the full-length protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. Thus, these mutants represent a minimalistic allosteric system of FimH, useful for further mechanistic studies and antagonist design.

Urinary Tract Infection: Which Conformation of the Bacterial Lectin FimH Is Therapeutically Relevant?

Frequent antibiotic treatment of urinary tract infections has resulted in the emergence of antimicrobial resistance, necessitating alternative treatment options. One such approach centers around FimH antagonists that block the bacterial adhesin FimH, which would otherwise mediate binding of uropathogenic Escherichia coli to the host urothelium to trigger the infection. Although the FimH lectin can adopt three distinct conformations, the evaluation of FimH antagonists has mainly been performed with a truncated construct of FimH locked in one particular conformation. For a successful therapeutic application, however, FimH antagonists should be efficacious against all physiologically relevant conformations. Therefore, FimH constructs with the capacity to adopt various conformations were applied. By examining the binding properties of a series of FimH antagonists in terms of binding affinity and thermodynamics, we demonstrate that depending on the FimH construct, affinities may be overestimated by a constant factor of 2 orders of magnitude. In addition, we report several antagonists with excellent affinities for all FimH conformations.

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

ABSTRACTThe 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 β 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 β 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 β 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.

Catch-bond mechanism of the bacterial adhesin FimH.

Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.

Second generation of thiazolylmannosides, FimH antagonists for E. coli-induced Crohn's disease.

The anti-adhesive strategy, consisting of disrupting bacterial attachment to the host cells, is widely explored as an alternative to antibiotic therapies. Recently, thiazolylmannosides (TazMans) have been identified as strong anti-adhesives of E. coli strains implied in the gut inflammation of patients with Crohn's disease. In this work, we developed a second generation of TazMans with improved chemical stability. The anomeric nitrogen was substituted by short linkers and the compounds were assessed against the bacterial adhesin FimH and the clinically isolated LF82 E. coli strain in four in vitro assays. The results obtained on the FimH adhesin alone and the whole bacteria enabled the identification of a candidate for further in vivo evaluations.

Inactive conformation enhances binding function in physiological conditions

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.

Inhibition of the Bacterial Adhesin FimH May Potentially Treat Inflammatory Bowel Diseases (IBD).

Inhibition of the Bacterial Adhesin FimH May Potentially Treat Inflammatory Bowel Diseases (IBD).

Catch bonds

Catch bonds