| 1. |
- Asquith, Nathan L., et al.
(author)
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Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds
- 2022
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In: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 6:13, s. 4015-4027
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Journal article (peer-reviewed)abstract
- Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, yD297N, yE323Q, and yK356Q, and a triple variant yDEK (yD297N/yE323Q/yK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for yD297N and enhanced for the yK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that yDEK and yE323Q produced denser clots, whereas yD297N and yK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only yDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.
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| 2. |
- Jansen, Karin A., et al.
(author)
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Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling
- 2020
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In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-683X .- 1744-6848. ; 16:35, s. 8272-8283
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Journal article (peer-reviewed)abstract
- Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots, yet the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing of fibrin fibers. Here the packing structure of fibrin fibers is quantitatively assessed by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin reconstituted under a wide range of conditions with computational molecular modeling of fibrin protofibrils. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.
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| 3. |
- Kliuchnikov, Evgenii, et al.
(author)
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CellDynaMo-stochastic reaction-diffusion-dynamics model : Application to search-and-capture process of mitotic spindle assembly
- 2022
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In: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 18:6
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Journal article (peer-reviewed)abstract
- We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly. Author summary The CellDynaMo package models, in 3D, any cellular subsystem where sufficient detail of the macromolecular players and the kinetics of relevant reactions are available. The package is based on the Stochastic Reaction-Diffusion-Dynamics model that combines the stochastic description of chemical kinetics, Brownian diffusion-based description of molecular transport, and Langevin dynamics-based representation of mechanical processes most pertinent to the system. We apply the model to test the Search-and-Capture mechanism of mitotic spindle assembly. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of chromosome connections, that chromosome arms improve the attachment accuracy by slowing down chromosome movements, that Aurora A kinase and kinetochore deformations have small positive effects on the accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy.
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| 4. |
- Kliuchnikov, Evgenii, et al.
(author)
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Microtubule assembly and disassembly dynamics model : Exploring dynamic instability and identifying features of Microtubules' Growth, Catastrophe, Shortening, and Rescue
- 2022
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In: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 953-974
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Journal article (peer-reviewed)abstract
- Microtubules (MTs), a cellular structure element, exhibit dynamic instability and can switch stochastically from growth to shortening; but the factors that trigger these processes at the molecular level are not understood. We developed a 3D Microtubule Assembly and Disassembly DYnamics (MADDY) model, based upon a bead-per-monomer representation of the alpha beta-tubulin dimers forming an MT lattice, stabilized by the lateral and longitudinal interactions between tubulin subunits. The model was parameterized against the experimental rates of MT growth and shortening, and pushing forces on the Dam1 protein complex due to protofilaments splaying out. Using the MADDY model, we carried out GPU-accelerated Langevin simulations to access dynamic instability behavior. By applying Machine Learning techniques, we identified the MT characteristics that distinguish simultaneously all four kinetic states: growth, catastrophe, shortening, and rescue. At the cellular 25 mu M tubulin concentration, the most important quantities are the MT length L, average longitudinal curvature kappa(long), MT tip width w, total energy of longitudinal interactions in MT lattice U-long, and the energies of longitudinal and lateral interactions required to complete MT to full cylinder U-long(add) and U-lat(add) . At high 250 mu M tubulin concentration, the most important characteristics are L, kappa(long), number of hydrolyzed alpha beta-tubulin dimers n(hyd) and number of lateral interactions per helical pitch n(lat) in MT lattice, energy of lateral interactions in MT lattice U-lat, and energy of longitudinal interactions in MT tip u(long). These results allow greater insights into what brings about kinetic state stability and the transitions between states involved in MT dynamic instability behavior.
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