A general chemical principle for creating closure-stabilizing integrin inhibitors.

Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbß3 and α4ß1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbß3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4ß1.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Force interacts with macromolecular structure in activation of TGF-ß.

Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.

ß-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbß3 Headpiece.

The platelet integrin αIIbß3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and ß-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the ß3-subunit. Ligand binding induces headpiece opening, with conformational change in the ß-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the ßI-domain ß1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with ß3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbß3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbß3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the ß3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

Structural basis for quinine-dependent antibody binding to platelet integrin αIIbß3.

Drug-induced immune thrombocytopenia (DITP) is caused by antibodies that react with specific platelet-membrane glycoproteins when the provoking drug is present. More than 100 drugs have been implicated as triggers for this condition, quinine being one of the most common. The cause of DITP in most cases appears to be a drug-induced antibody that binds to a platelet membrane glycoprotein only when the drug is present. How a soluble drug promotes binding of an otherwise nonreactive immunoglobulin to its target, leading to platelet destruction, is uncertain, in part because of the difficulties of working with polyclonal human antibodies usually available only in small quantities. Recently, quinine-dependent murine monoclonal antibodies were developed that recognize a defined epitope on the ß-propeller domain of the platelet integrin αIIb subunit (GPIIb) only when the drug is present and closely mimic the behavior of antibodies found in human patients with quinine-induced thrombocytopenia in vitro and in vivo. Here, we demonstrate specific, high-affinity binding of quinine to the complementarity-determining regions (CDRs) of these antibodies and define in crystal structures the changes induced in the CDR by this interaction. Because no detectable binding of quinine to the target integrin could be demonstrated in previous studies, the findings indicate that a hybrid paratope consisting of quinine and reconfigured antibody CDR plays a critical role in recognition of its target epitope by an antibody and suggest that, in this type of drug-induced immunologic injury, the primary reaction involves binding of the drug to antibody CDRs, causing it to acquire specificity for a site on a platelet integrin.

Latent TGF-ß structure and activation.

Transforming growth factor (TGF)-ß is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-ß1 requires the binding of α(v) integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-ß binding proteins. Crystals of dimeric porcine proTGF-ß1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between α(v)ß(6) integrin and the prodomain is insufficient for TGF-ß1 release. Force-dependent activation requires unfastening of a 'straitjacket' that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-ß family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis.

Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces.

The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

Domain 1 of mucosal addressin cell adhesion molecule has an I1-set fold and a flexible integrin-binding loop.

Mucosal addressin cell adhesion molecule (MAdCAM) binds integrin α4ß7. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α4ß7 is unusual in its ability to mediate either rolling adhesion or firm adhesion of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution 1.7- and 1.4-Å structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the integrin-binding loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF domain topology from the I2- to I1-set, with a switch of integrin-binding loop from CC' to CD. The I1-set fold and CD loop appear biologically relevant. The different conformations seen in crystal structures suggest that the integrin-binding loop of MAdCAM is inherently flexible. This contrasts with rigidity of the corresponding loops in vascular cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm adhesion by binding to different α4ß7 integrin conformations.

Structure of an integrin with an alphaI domain, complement receptor type 4.

We report the structure of an integrin with an alphaI domain, alpha(X)beta(2), the complement receptor type 4. It was earlier expected that a fixed orientation between the alphaI domain and the beta-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the alphaI domain is highly flexible, enabling two betaI domain conformational states to couple to three alphaI domain states, and greater accessibility for ligand recognition. Although alpha(X)beta(2) is bent similarly to integrins that lack alphaI domains, the terminal domains of the alpha- and beta-legs, calf-2 and beta-tail, are oriented differently than in alphaI-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the beta(2)-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

α(V)ß(3) integrin crystal structures and their functional implications.

Many questions about the significance of structural features of integrin α(V)ß(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)ß(3) ectodomain linked to C-terminal coiled coils (α(V)ß(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)ß(3)-1TM), respectively. The α(V) and ß(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the ßI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of the α(V) linker. α(V)ß(3)-AB and α(V)ß(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and ß(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)ß(3)-1TM, and differed markedly between α(V)ß(3)-1TM and α(V)ß(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)ß(3)-AB and α(V)ß(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.

Closed headpiece of integrin αIIbß3 and its complex with an αIIbß3-specific antagonist that does not induce opening.

The platelet integrin α(IIb)ß(3) is essential for hemostasis and thrombosis through its binding of adhesive plasma proteins. We have determined crystal structures of the α(IIb)ß(3) headpiece in the absence of ligand and after soaking in RUC-1, a novel small molecule antagonist. In the absence of ligand, the α(IIb)ß(3) headpiece is in a closed conformation, distinct from the open conformation visualized in presence of Arg-Gly-Asp (RGD) antagonists. In contrast to RGD antagonists, RUC-1 binds only to the α(IIb) subunit. Molecular dynamics revealed nearly identical binding. Two species-specific residues, α(IIb) Y190 and α(IIb) D232, in the RUC-1 binding site were confirmed as important by mutagenesis. In sharp contrast to RGD-based antagonists, RUC-1 did not induce α(IIb)ß(3) to adopt an open conformation, as determined by gel filtration and dynamic light scattering. These studies provide insights into the factors that regulate integrin headpiece opening, and demonstrate the molecular basis for a novel mechanism of integrin antagonism.

Camel nanobodies broadly neutralize SARS-CoV-2 variants.

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: Dromedary camel ( Camelus dromedarius ) V H H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

The upcoming subatomic resolution revolution.

Subatomic resolution macromolecular crystallography has been revealing the most fascinating details of macromolecular structures for many years. This most extreme form of macromolecular crystallography is going through rapid changes. A new generation of superbrilliant X-ray sources and detectors is facilitating the rapid acquisition of high-quality datasets. Equally important, a new breed of methods and highly integrated advanced computational tools for structure refinement and analysis is poised to change the way we use subatomic resolution data and reposition high-resolution macromolecular crystallography in medicinal chemistry studies. Subatomic resolution macromolecular crystallography may soon be a routine source of detailed molecular information besides precise geometries, including binding energies and other chemical descriptors, opening new possibilities of application.

Synthesis and biological activity of some novel derivatives of 4-amino-3-(D-galactopentitol-1-yl)-5-mercapto-1,2,4-triazole.

To discover new 1,2,4-triazole derivatives which may possess significant biological activities, we synthesized a series of novel 6-aryl-3-(D-galactopentitol-1-yl)-7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines and 4-(arylmethylidene)amino-5-(D-galactopentitol-1-yl)-3-mercapto-4H-1,2,4-triazoles from 4-amino-3-(D-galactopentitol-1-yl)-5-mercapto-1,2,4-triazole. All the title compounds were characterized by elemental analysis, IR, 1H- and 13C-NMR. Plant growth-regulating activity tests showed that these compounds have remarkable effects on the growth of radish and wheat.

Complete integrin headpiece opening in eight steps.

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbß3 integrin headpiece. Starting from the closed ßI domain conformation, we saw six intermediate ßI conformations and finally the fully open ßI with the hybrid domain swung out in the crystal lattice. The ß1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca(2+), α1 helix, α1' helix, ß6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the ßI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.

GARP regulates the bioavailability and activation of TGFß.

Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-ß (proTGFß) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFß activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFß1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFß1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFß-binding protein for binding to proTGFß1. Association between GARP and proTGFß1 prevents the secretion of TGFß1. Integrin α(V)ß(6) and to a lesser extent α(V)ß(8) are able to activate TGFß from the GARP-proTGFß1 complex. Activation requires the RGD motif of latent TGFß, disulfide linkage between GARP and latent TGFß, and membrane association of GARP. Our results show that GARP is a latent TGFß-binding protein that functions in regulating the bioavailability and activation of TGFß.

Structural specializations of α(4)ß(7), an integrin that mediates rolling adhesion.

The lymphocyte homing receptor integrin α(4)ß(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)ß(1) and α(4)ß(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)ß(7). A long binding groove at the α(4)-ß(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)ß(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)ß(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.

Structure-guided design of a high-affinity platelet integrin αIIbß3 receptor antagonist that disrupts Mg²âº binding to the MIDAS.

An integrin found on platelets, α(IIb)ß(3) mediates platelet aggregation, and α(IIb)ß(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the ß subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the ß(3) subunits. They induce conformational changes in the ß(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)ß(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in ß(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)ß(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the ß(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)ß(3) antagonists and may offer advantages as a therapeutic agent.

Structural basis for distinctive recognition of fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3.

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin alpha(IIb)beta(3) on platelets, resulting in platelet aggregation. alpha(v)beta(3) binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's alpha subunit. alpha(IIb)beta(3) also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the gamma subunit (gammaC peptide). These distinct modes of fibrinogen binding enable alpha(IIb)beta(3) and alpha(v)beta(3) to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin alpha(IIb)beta(3)-gammaC peptide interface, and, for comparison, integrin alpha(IIb)beta(3) bound to a lamprey gammaC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in gammaC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg(2+) ion binds the gammaC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca(2+) ion binds the gammaC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered gammaC peptide enhances our understanding of the involvement of gammaC peptide and integrin alpha(IIb)beta(3) in hemostasis and thrombosis.

A new arrangement of (beta/alpha)8 barrels in the synthase subunit of PLP synthase.

Pyridoxal 5'-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP. In the 2.2-A crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (beta/alpha)8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.