Biology subjects

Graeter, F.

Publications and source records attributed to Graeter, F..

6 recordsLinked to original sources

bioRxiv2023

Insights into human outer kinetochore assembly and force transmission from a structure-function analysis of the KMN network

The biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cells chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. The kinetochore outer layer consists of a 10-subunit apparatus comprising Knl1C, Mis12C, and Ndc80C subcomplexes (KMN network). The KMN network is highly elongated and docks on kinetochores and microtubules using interfaces at its opposite extremes. Here, we combine cryo-EM reconstructions and AlphaFold2 predictions to generate a model of the KMN network that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments and molecular dynamics simulations we demonstrate this mutual organization stabilizes the KMN network. Our work reports the first comprehensive structural and functional analysis of the microtubule binding machinery of kinetochores and elucidates a path of microtubule-generated force transmission

molecular biology↗

bioRxiv2023

DOPA residues endow collagen with radical scavenging capacity

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.

biophysics↗

bioRxiv2022

Myristoyl's dual role in allosterically regulating and localizing Abl kinase

c-Abl kinase, a key signalling hub in many biological processes ranging from cell development to proliferation, is tightly regulated by two inhibitory Src homology domains. An N-terminal myristoyl-modification can bind to a hydrophobic pocket in the kinase C-lobe, which stabilizes the auto-inhibitory assembly. Activation is triggered by myristoyl release. We used molecular dynamics simulations to show how both myristoyl and the Src homology domains are required to impose the full inhibitory effect on the kinase domain, and reveal the allosteric transmission pathway at residue-level resolution. Importantly, we find myristoyl insertion into a membrane to thermodynamically compete with binding to c-Abl. Myristoyl thus not only localizes the protein to the cellular membrane, but membrane attachment at the same time enhances activation of c-Abl by stabilizing its pre-activated state. Our data put forward a model in which lipidation tightly couples kinase localization and regulation, a scheme that currently appears to be unique for this non-receptor tyrosine kinase.

biophysics↗

bioRxiv2022

The role of hydrodynamic flow in the self-assembly of dragline spider silk proteins

Hydrodynamic flow in the spider duct induces conformational changes in dragline spider silk proteins (spidroins) and drives their assembly, but the underlying physical mechanisms are still elusive. Here we address this challenging multiscale problem with a complementary strategy of atomistic and coarse-grained Molecular Dynamics (MD) simulations with uniform flow. The conformational changes at the molecular level were analyzed for single tethered spider silk peptides. Uniform flow leads to coiled-to-stretch transitions and pushes alanine residues into {beta}-sheet and Poly-Proline II (PPII) conformations. Coarse-grained simulations of the assembly process of multiple semi-flexible block copolymers using multi-particle collision dynamics reveal that the spidroins aggregate faster but into low-order assemblies when they are less extended. At medium-to-large peptide extensions (50%-80%), assembly slows down and becomes reversible with frequent association and dissociation events, while spidroin alignment increases and alanine repeats form ordered regions. Our work highlights the role of flow in guiding silk self-assembly into tough fibers by enhancing alignment and kinetic reversibility, a mechanism likely relevant for other proteins whose function depends on hydrodynamic flow.

biophysics↗

bioRxiv2022

Dynamic Disulfide Bond Topologies in von-Willebrand-Factor's C4-Domain Undermine Platelet Binding

BackgroundThe von Willebrand Factor (vWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large multi-domain mechano-sensitive protein stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the vWF-C4 domain which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. ObjectiveTo quantitatively determine C4s disulfide topologies and their implication in vWFs platelet-binding function via integrin. MethodsWe employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. ResultsWe quantitatively show that two disulfide bonds in the vWF-C4 domain, namely the two major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the RGD-integrin binding motif, and thereby impair integrin-mediated platelet binding. Our combined approach also reveals that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all six vWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. ConclusionOverall, our data put forward a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of vWF with integrin and potentially other partners, thereby critically influencing its hemostatic function. EssentialsO_LIPlatelet integrins interact with the disulfide-bonded C4 domain of von Willebrand Factor C_LIO_LIThe redox state of vWF-C4s disulfide bonds is studied by molecular simulations and experiments C_LIO_LITwo bonds are reduced causing C4 unfolding and disulfide swapping C_LIO_LIOpening of disulfide bonds impairs integrin-mediated platelet binding C_LI

biophysics↗

bioRxiv2021

ATP allosterically stabilizes Integrin-linked kinase for efficient force generation

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:-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.

biophysics↗