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23 Publications visible to you, out of a total of 23
Disulfide Bond Reduction and Exchange in von-Willebrand-Factor’s C4-Domain Undermines Platelet Binding

Abstract

Not specified

Authors: Fabian Kutzki, Diego Butera, Angelina J. Lay, Denis Maag, Joyce Chiu, Heng-Giap Woon, Tomáš Kubař, Marcus Elstner, Camilo Aponte-Santamaría, Philip J. Hogg, Frauke Gräter

Date Published: 12th Apr 2023

Publication Type: Journal

Structure and dynamics of the von Willebrand Factor C6 domain

Abstract (Expand)

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain … plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF’s hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.

Authors: Po-Chia Chen, Fabian Kutzki, Angelika Mojzisch, Bernd Simon, Emma-Ruoqi Xu, Camilo Aponte-Santamaría, Kai Horny, Cy Jeffries, Reinhard Schneppenheim, Matthias Wilmanns, Maria A. Brehm, Frauke Gräter, Janosch Hennig

Date Published: 18th Nov 2022

Publication Type: Journal

A Conformational Transition of the D’D3 Domain primes von Willebrand Factor for Multimerization

Abstract (Expand)

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the (trans-)Golgi is … still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D’D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose two buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we are able to quantify the kinetics of the transition and the stability of the interface. We find a pronounced destabilization of the interface upon lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D’D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII (FVIII), providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D’D3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Authors: Sophia Gruber, Achim Löf, Adina Hausch, Fabian Kutzki, Res Jöhr, Tobias Obser, Gesa König, Reinhard Schneppenheim, Camilo Aponte-Santamaría, Frauke Gräter, Maria A. Brehm, Martin Benoit, Jan Lipfert

Date Published: 2nd Jun 2022

Publication Type: Journal

Martini 3 coarse-grained force field for poly(para-phenylene ethynylene)s.

Abstract (Expand)

Poly(<i>para</i>-phenylene ethynylene)s, or short PPEs, are a class of conjugated and semi-flexible polymers with a strongly delocalized π electron system and increased chain stiffness. Due … to this, PPEs have a wide range of technological applications. Although the material properties of single-chains or mixtures of few PPE chains have been studied in detail, the properties of large assemblies remain to be fully explored. Here, we developed a coarse-grained model for PPEs with the Martini 3 force field to enable computational studies of PPEs in large-scale assembly. We used an optimization geometrical approach to take the shape of the π conjugated backbone into account and also applied an additional angular potential to tune the mechanical bending stiffness of the polymer. Our Martini 3 model reproduces key structural and thermodynamic observables of single PPE chains and mixtures, such as persistence length, density, packing and stacking. We show that chain entanglement increases with the expense of nematic ordering with growing PPE chain length. With the Martini 3 PPE model at hand, we are now able to cover large spatio-temporal scales and thereby to uncover key aspects for the structural organization of PPE bulk systems. The model is also predicted to be of high applicability to investigate out-of-equilibrium behavior of PPEs under mechanical force.

Authors: Matthias Brosz, Nicholas Michelarakis, Uwe H F Bunz, Camilo Aponte-Santamaría, Frauke Gräter

Date Published: 4th May 2022

Publication Type: Journal

ATP allosterically stabilizes integrin-linked kinase for efficient force generation

Abstract (Expand)

Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic … and intrinsic forces, but how the essential adhesion component intergrin-linked kinase (ILK) dynamically responds to mechanical force and what role ATP bound to this pseudokinase plays remains elusive. Here, we apply force-probe molecular dynamics simulations of human ILK:α-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key saltbridge-forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a new role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.

Authors: Isabel Martin, Michele Nava, Sara Wickström, Frauke Gräter

Date Published: 8th Mar 2022

Publication Type: Journal

Accurate Free Energies for Complex Condensed-Phase Reactions Using an Artificial Neural Network Corrected DFTB/MM Methodology.

Abstract (Expand)

Semiempirical methods like density functional tight-binding (DFTB) allow extensive phase space sampling, making it possible to generate free energy surfaces of complex reactions in condensed-phase … environments. Such a high efficiency often comes at the cost of reduced accuracy, which may be improved by developing a specific reaction parametrization (SRP) for the particular molecular system. Thiol-disulfide exchange is a nucleophilic substitution reaction that occurs in a large class of proteins. Its proper description requires a high-level ab initio method, while DFT-GAA and hybrid functionals were shown to be inadequate, and so is DFTB due to its DFT-GGA descent. We develop an SRP for thiol-disulfide exchange based on an artificial neural network (ANN) implementation in the DFTB+ software and compare its performance to that of a standard SRP approach applied to DFTB. As an application, we use both new DFTB-SRP as components of a QM/MM scheme to investigate thiol-disulfide exchange in two molecular complexes: a solvated model system and a blood protein. Demonstrating the strengths of the methodology, highly accurate free energy surfaces are generated at a low cost, as the augmentation of DFTB with an ANN only adds a small computational overhead.

Authors: Claudia L Gómez-Flores, Denis Maag, Mayukh Kansari, Van-Quan Vuong, Stephan Irle, Frauke Gräter, Tomáš Kubař, Marcus Elstner

Date Published: 8th Feb 2022

Publication Type: Journal

Mechanical force can enhance c-Src kinase activity by impairing autoinhibition.

Abstract (Expand)

Cellular mechanosensing is pivotal for virtually all biological processes, and many molecular mechano-sensors and their way of function are being uncovered. In this work, we suggest that c-Src kinase … acts as a direct mechano-sensor. c-Src is responsible for, among others, cell proliferation, and shows increased activity in stretched cells. In its native state, c-Src has little basal activity, because its kinase domain binds to an SH2 and SH3 domain. However, it is known that c-Src can bind to p130Cas, through which force can be transmitted to the membrane. Using molecular dynamics simulations, we show that force acting between the membrane-bound N-terminus of the SH3 domain and p130Cas induces partial SH3 unfolding, thereby impeding rebinding of the kinase domain onto SH2/SH3 and effectively enhancing kinase activity. Forces involved in this process are slightly lower or similar to the forces required to pull out c-Src from the membrane through the myristoyl linker, and key interactions involved in this anchoring are salt bridges between negative lipids and nearby basic residues in c-Src. Thus, c-Src appears to be a candidate for an intriguing mechanosensing mechanism of impaired kinase inhibition, which can be potentially tuned by membrane composition and other environmental factors.

Authors: Csaba Daday, Svenja de Buhr, Davide Mercadante, Frauke Gräter

Date Published: 2nd Feb 2022

Publication Type: Journal

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