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, Dynamics, and Branch Migration of a DNA Holliday Junction: A Single-Molecule Fluorescence and Modeling Study

AbstractThe 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

Mapping out molecular locations in biological liposomes by fluorescence nanotomography

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structural information on nanomolecular systems revealed by FRET

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D-A distance distribution function ρDA(r).We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers

An allosteric redox switch in domain V of \u3b22-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition

\u3b22-glycoprotein I (\u3b22GPI) is an abundant multi-domain 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 \u3b22GPI 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 \u3b22GPI 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 \u3b22GPI'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 \u3b22GPI 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 \u3b22GPI'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