Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles.

Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a') and two inactive (b, b'), organized to form a flexible abb'a' U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI's structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2, and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2 and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Bioorthogonal Chemistry Enables Single-Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase.

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs' structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

The J-elongated conformation of ß2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome.

ß2-Glycoprotein I (ß2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound ß2GPI to that in solution. ß2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, ß2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant ß2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound ß2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of ß2GPI provides a strategy to block pathogenic aPLs in APS.

Discovery and characterization of 2 novel subpopulations of aPS/PT antibodies in patients at high risk of thrombosis.

Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen-autoantibody recognition in APS.

Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants.

Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, "closed" (~80%) and "open" (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the "closed"/"open" equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.

Structure of prothrombin in the closed form reveals new details on the mechanism of activation.

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.

Solubility and diffusion of oxygen in phospholipid membranes.

The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl- and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state.

Structural studies of interactions between cardiac troponin I and actin in regulated thin filament using Förster resonance energy transfer.

The Ca(2+)-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we have investigated changes of this interaction in response to strong cross-bridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Förster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188, and 210 of cTnI and the nonfluorescent acceptor 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca(2+) induced large increases in the distances from actin to the cTnI sites, indicating a Ca(2+)-triggered separation of cTnI from actin. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca(2+). The two ligand-induced increases were independent of each other. These two-step changes in distances provide a direct link of structural changes at the interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca(2+)-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca(2+). These results suggest bound Ca(2+) or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca(2+) and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg(2+) state, the regulatory region appears more flexible than the inhibitory region, and in the Ca(2+) state the inhibitory region becomes more flexible. PKA phosphorylation of cTnI at Ser23 and Ser24 distance from actin to cTnI residue 131 by 2.2-5.2 A in different biochemical states and narrowed the distributions of the distances from actin to the inhibitory and regulatory regions of cTnI. The observed phosphorylation effects are likely due to an intramolecular interaction of the phosphorylated N-terminal segment and the inhibitory region of cTnI.

Structure, dynamics, and branch migration of a DNA Holliday junction: a single-molecule fluorescence and modeling study.

The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.