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77 Publications visible to you, out of a total of 77
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

Mechano-redox control of Mac-1 de-adhesion from ICAM-1 by protein disulfide isomerase promotes directional movement of neutrophils under flow

Abstract (Expand)

Macrophage-1 antigen or Mac-1 (CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion of neutrophils, lymphocytes and monocytes against flow when recruited to the endothelium. To migrate … to the site of inflammation, leukocytes require coordinated adhesion and de-adhesion for directional movement. The vascular thiol isomerase, protein disulfide isomerase (PDI), was found by fluorescence microscopy to colocalize with high affinity Mac-1 at the trailing edge of stimulated neutrophils when adhered to ICAM-1 under fluid shear. From differential cysteine alkylation and mass spectrometry studies, PDI cleaves two allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and in mutagenesis and cell transfection studies, cleavage of the C224-C264 disulfide bond was shown to selectively control Mac-1 dis-engagement from ICAM-1 under fluid shear. Molecular dynamics simulations and binding of conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain that allosterically alters exposure of an αI domain epitope and shifts Mac-1 to a lower affinity state. From studies of neutrophil adherence to ICAM-1 under fluid shear, these molecular events promote neutrophil motility in the direction of flow at high shear stress. In summary, shear-dependent PDI cleavage of neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Authors: Alexander Dupuy, Camilo Aponte-Santamaría, Adva Yeheskel, Frauke Gräter, Philip J. Hogg, Freda H. Passam, Joyce Chiu

Date Published: 30th 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

Phosphorylation tunes elongation propensity and cohesiveness of INCENP's intrinsically disordered region.

Abstract (Expand)

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access … its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP's IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP's IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP's function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined "dog-leash" model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.

Authors: Isabel M Martin, Camilo Aponte-Santamaría, Lisa Schmidt, Marius Hedtfeld, Adel Iusupov, Andrea Musacchio, Frauke Gräter

Date Published: 15th Jan 2022

Publication Type: Journal

Longitudinal strand ordering leads to shear thinning in Nafion.

Abstract

hts into the mechanism governing the shear thinning effects observed in Nafion solutions, through the use of non-equilibrium, coarse-grained, molecular dynamic simulations.

Authors: Nicholas Michelarakis, Florian Franz, Konstantinos Gkagkas, Frauke Gräter

Date Published: 24th Nov 2021

Publication Type: Journal

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