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.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.

The Evolution of SlyA/RovA Transcription Factors from Repressors to Countersilencers in Enterobacteriaceae.

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.IMPORTANCE Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.

Identifying G protein-coupled receptor dimers from crystal packings.

Dimers of G protein-coupled receptors (GPCRs) are believed to be important for signaling with their associated G proteins. Low-resolution electron microscopy has shown rhodopsin dimers in native retinal membranes, and CXCR4 dimers have been found in several different crystal structures. Evidence for dimers of other GPCRs is more indirect. An alternative to computational modeling studies is to search for parallel dimers in the packing environments of the reported crystal structures of GPCRs. Two major structural types of GPCR dimers exist (as predicted by others), but there is considerable structural variation within each cluster. The different structural variants described here might reflect different functional properties and should provide a range of model structures for computational and experimental examination.

A Streptavidin Binding Site Mutation Yields an Unexpected Result: An Ionized Asp128 Residue Is Not Essential for Strong Biotin Binding.

We report a detailed study of a point mutation of the crucial binding site residue, D128, in the biotin-streptavidin complex. The conservative substitution, D128N, preserves the detailed structure observed for the wild-type complex but has an only minimal impact on biotin binding, even though previous experimental and computational studies suggested that a charged D128 residue was crucial for high-affinity binding. These results show clearly that the fundamental basis for streptavidin's extremely strong biotin binding affinity is more complex than assumed and illustrate some of the challenges that may arise when analyzing extremely strong ligand-protein binding interactions.

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides.

BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.

Comparison of epsilon- and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster.

Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta- and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5 Šresolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon- and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.

Crystal and molecular structure of the analgesic tetrapeptide, L-Phe-L-Leu-L-Pro-L-Ser.

The tetrapeptide, L-Phe-L-Leu-L-Pro-L-Ser (FLPS), alleviates pain in a rat model of post-surgery pain. The crystal structure of the tetrapeptide is solved at high resolution (0.54 Å). The asymmetric unit contains two FLPS molecules, one Zn ion, and four molecules of water with a formula of [Zn(C23H33N4O6)2(H2O)4]. Each Zn ion is octahedrally coordinated with Phe and Ser residues from four peptide molecules [2N+4O]. The linking of Phe and Ser residues of one FLPS molecule to three other FLPS molecules by Zn ion forms a complex consisting of chains of metal ions and FLPS molecules oriented along the b axis. Analysis of molecular packing reveals the coexistence of two FLPS conformers in the same crystal. The crystallographic parameters for [Zn(C23H33N4O6)2(H2O)4] are as follows: space group P21 21 21 , a = 9.8698(2) Å, α = 90°, b = 20.1844(4) Å, ß = 90°, c = 25.9302(6) Å, γ = 90°. Volume = 5165.71(19) Å(3), Z = 4, density (calc) = 1.364 Mg/cm(3), and agreement factor R1 = 4.13%.

The structure of rice weevil pectin methylesterase.

Rice weevils (Sitophilus oryzae) use a pectin methylesterase (EC 3.1.1.11), along with other enzymes, to digest cell walls in cereal grains. The enzyme is a right-handed ß-helix protein, but is circularly permuted relative to plant and bacterial pectin methylesterases, as shown by the crystal structure determination reported here. This is the first structure of an animal pectin methylesterase. Diffraction data were collected to 1.8 Šresolution some time ago for this crystal form, but structure solution required the use of molecular-replacement techniques that have been developed and similar structures that have been deposited in the last 15 years. Comparison of the structure of the rice weevil pectin methylesterase with that from Dickeya dandantii (formerly Erwinia chrysanthemi) indicates that the reaction mechanisms are the same for the insect, plant and bacterial pectin methylesterases. The similarity of the structure of the rice weevil enzyme to the Escherichia coli lipoprotein YbhC suggests that the evolutionary origin of the rice weevil enzyme was a bacterial lipoprotein, the gene for which was transferred to a primitive ancestor of modern weevils and other Curculionidae. Structural comparison of the rice weevil pectin methylesterase with plant and bacterial enzymes demonstrates that the rice weevil protein is circularly permuted relative to the plant and bacterial molecules.

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin.

Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing the biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide-biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the ß-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin-biotin complex.

Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction.

To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.

Structural basis for type VI secretion effector recognition by a cognate immunity protein.

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1-3 (Tse1-3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2-Tsi2 effector-immunity pair has features distinguishing it from previously characterized toxin-immunity and toxin-antitoxin systems.

Second-contact shell mutation diminishes streptavidin-biotin binding affinity through transmitted effects on equilibrium dynamics.

We report a point mutation in the second contact shell of the high-affinity streptavidin-biotin complex that appears to reduce binding affinity through transmitted effects on equilibrium dynamics. The Y54F streptavidin mutation causes a 75-fold loss of binding affinity with 73-fold faster dissociation, a large loss of binding enthalpy (ΔΔH = 3.4 kcal/mol at 37 °C), and a small gain in binding entropy (TΔΔS = 0.7 kcal/mol). The removed Y54 hydroxyl is replaced by a water molecule in the bound structure, but there are no observable changes in structure in the first contact shell and no additional changes surrounding the mutation. Molecular dynamics simulations reveal a large increase in the atomic fluctuation amplitudes for W79, a key biotin contact residue, compared to the fluctuation amplitudes in the wild-type. The increased W79 atomic fluctuation amplitudes are caused by loss of water-mediated hydrogen bonds between the Y54 hydroxyl group and peptide backbone atoms in and near W79. We propose that the increased atomic fluctuation amplitudes diminish the integrity of the W79-biotin interaction and represents a loosening of the "tryptophan collar" that is critical to the slow dissociation and high affinity of streptavidin-biotin binding. These results illustrate how changes in protein dynamics distal to the ligand binding pocket can have a profound impact on ligand binding, even when equilibrium structure is unperturbed.

Streptavidin and its biotin complex at atomic resolution.

Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 Å, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the ß-sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45-52). In three of the unliganded subunits the loop is in an `open' conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop `closes' over the biotin-binding site. The `closed' loop contributes to the protein's high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.

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.

Donor strand exchange and conformational changes during E. coli fimbrial formation.

Fimbriae and pili are macromolecular structures on the surface of Gram negative bacteria that are important for cellular adhesion. A 2.7Å resolution crystal structure of a complex of Escherichia coli fimbrial proteins containing FimH, FimG, FimF, and FimC provides the most complete model to date for the arrangement of subunits assembled in the native structure. The first three proteins form the tip of the fimbriae while FimC is the chaperone protein involved in the usher/chaperone assembly process. The subunits interact through donor strand complementation where a ß-strand from a subunit completes the ß-sandwich structure of the neighboring subunit or domain closer to the tip of the fimbria. The function of FimC is to provide a surrogate donor strand before delivery of each subunit to the FimD usher and the growing fimbria. Comparison of the subunits in this structure and their chaperone-bound complexes show that the two FimH domains change their relative orientation and position in forming the tip structure. Also, the non-chaperone subunits undergo a conformational change in their first ß-strand when the chaperone is replaced by the native donor strand. Some residues move as much as 14Å in the process. This structural shift has not been noted in structural studies of other bacterial adhesion sub-structures assembled via donor strand complementation. The domains undergo a significant structural change in the donor strand binding groove during fimbrial assembly, and this likely plays a role in determining the specificity of subunit-subunit interactions among the fimbrial proteins.

Structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta 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.