Gianluca Interlandi

Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like β Sheet Twisting

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow.

The catch bond mechanism between von Willebrand factor and platelet surface receptors investigated by molecular dynamics simulations

The multi‐domain protein von Willebrand factor is crucial in the blood coagulation process at high shear. The A1 domain binds to the platelet surface receptor glycoprotein Ibα (GpIbα) and this interaction is known to be strengthened by tensile force. The molecular mechanism behind this observation was investigated here by molecular dynamics simulations. The results suggest that the proteins unbind through two distinct pathways depending whether a high‐tensile force is applied or whether unbinding happens through thermal fluctuations. In the high‐force unbinding pathway the A1 domain was observed to rotate away from the C‐terminus of GpIbα. In contrast, during thermal unbinding the A1 domain rotated in the opposite direction as in the high‐force pathway and the distance between the terminii of A1 and the GpIbα C‐terminus shortened. This shortening was reduced and the lifetime of the bond extended if a moderate tensile force was applied across the complex. This suggests that the thermal unbinding pathway is inhibited by a moderate tensile force which is in agreement with the catch bond property shown previously in single molecule experiments. A designed mutant of GpIbα is suggested here in order to test in vitro the thermal unbinding pathway observed in silico. Proteins 2010.

Allosteric Coupling in the Bacterial Adhesive Protein FimH

Background: The bacterial adhesin FimH is allosterically regulated. Results: Mutations designed to control the allosteric state of the protein created low or high affinity variants as predicted. Conclusion: Three regulatory regions are strongly coupled together, whereas the active site is more weakly coupled to those regions. Significance: Allosteric regulation can be used to develop antiadhesive therapies for bacterial infections. 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.

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.

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations

The ankyrin repeat is one of the most common protein motifs and is involved in protein–protein interactions. It consists of 33 residues that assume a β‐hairpin helix‐loop‐helix fold. Mutagenesis and kinetic experiments (Φ‐value analysis of the folding transition state) have shown that the tumor suppressor p16INK4a, a four‐repeat protein, unfolds sequentially starting from the two N‐terminal repeats. Here, the flexibility of p16INK4a at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 μs. The transition state ensemble (TSE) of p16INK4a was identified by monitoring both the deviation from the experimental Φ values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid‐body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp15, Pro75, and Ala76 side‐chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on‐pathway intermediates with partial α‐helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect. Proteins 2006.

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 6 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 6 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 αIIbβ3.

INTRODUCTION The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. METHODS We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance. RESULTS AND CONCLUSIONS Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, 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 αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

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.

Tight Conformational Coupling between the Domains of the Enterotoxigenic Escherichia coli Fimbrial Adhesin CfaE Regulates Binding State Transition

Background: The fimbrial tip adhesin CfaE comprises stacked adhesin and pilin domains. Results: The CfaE adhesin domain mutation G168D loosens the interdomain interface and perturbs only the adjoining pilin domain structure, yet alters CfaE adhesive properties. Conclusion: The structure-function relationship of CfaE reveals an intimately coupled, bipartite molecular assembly. Significance: These findings underscore the dynamic nature of host-pathogen interactions. 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 β-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.