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Aponte-Santamaria, C.

Publications and source records attributed to Aponte-Santamaria, C..

8 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

Mechanical forces control the valency of the malaria adhesin VAR2CSA by exposing cryptic glycan binding sites

Plasmodium falciparum (Pf) is responsible for the most lethal form of malaria. VAR2CSA is an adhesin protein expressed by this parasite at the membrane of infected erythrocytes for attachment on the placenta, leading to pregnancy-associated malaria. VAR2CSA is a large 355 kDa multidomain protein composed of nine extracellular domains, a transmembrane helix, and an intracellular domain. VAR2CSA binds to Chondroitin Sulphate A (CSA) of the proteoglycan matrix of the placenta. Shear flow, as the one occurring in blood, has been shown to enhance the (VAR2CSA-mediated) adhesion of Pf -infected erythrocytes on the CSA-matrix. However, the underlying molecular mechanism governing this enhancement has remained elusive. Here, we address this question by using equilibrium, force-probe, and docking-based molecular dynamics simulations. We subjected the VAR2CSA protein-CSA sugar complex to a force mimicking the elongational tension exerted on this system due to the shear of the flowing blood. We show that upon this force exertion, VAR2CSA undergoes a large opening conformational transition before the CSA sugar chain dissociates from its main binding site. This preferential order of events is caused by the orientation of the molecule during elongation as well as the strong electrostatic attraction of the sugar to the main protein binding site. Upon opening, two additional cryptic CSA binding sites get exposed and a functional dodecameric CSA molecule can be stably accommodated at these force-exposed positions. Thus, our results suggest that mechanical forces, increase the avidity of VAR2CSA, by turning it from a monovalent to a multivalent state. We propose this to be the molecular cause of the observed shear-enhanced adherence. Mechanical control of the valency of VAR2CSA is an intriguing hypothesis that can be tested experimentally and which is of relevance for the understanding of the malaria infection and for the development of anti placental-malaria vaccines targeting VAR2CSA.

biophysics↗
bioRxiv2023

Energetics and kinetics of membrane permeation of photoresists for bioprinting

Three-dimensional (3D) bioprinting is a promising technology which typically uses bioinks to pattern cells and their scaffolds. The selection of cytocompatible inks is critical for the printing success. In laserbased 3D bioprinting, photoresist molecules are used as bioinks. We propose that cytotoxicity can be a consequence of the interaction of photoresists with lipid membranes and their permeation into the cell. Here, molecular dynamics simulations and in vitro assays address this issue, retrieving partition coefficients, free energies, and permeabilities for eight commonly-used photoresists in model lipid bilayers. Crossing the hydrophobic center of the membrane constitutes the rate limiting step during permeation. In addition, three photoresists feature a preferential localization site at the acyl chain headgroup interface. Photoresist permeabilities range over eight orders of magnitude, with some molecules being membrane-permeable on bioprinting timescales. Moreover, permeation correlates well with the oil-water partition coefficients and is severely hampered by the lipid ordering imposed by the lipid saturation. Overall, the mechanism of interaction of photoresists with model lipid bilayers is provided here, helping to classify them according to their residence in the membrane and permeation through it. This is useful information to guide the selection of cytocompatible photoresists for 3D bioprinting.

biophysics↗
bioRxiv2022

The interplay between adsorption and aggregation of von Willebrand factor chains in shear flows

AO_SCPLOWBSTRACTC_SCPLOWVon Willebrand factor (VWF) is a giant extracellular glycoprotein that carries out a key adhesive function during primary hemostasis. Upon vascular injury and triggered by the shear of flowing blood, VWF establishes specific interactions with several molecular partners in order to anchor platelets to collagen on the exposed sub-endothelial surface. VWF also interacts with itself to form aggregates that, adsorbed on the surface, provide more anchor sites for the platelets. However, the interplay between elongation and subsequent exposure of cryptic binding sites, self-association, and adsorption on the surface, remained unclear for VWF. In particular, the role of shear flow in these three processes is not well understood. In this study, we address these questions by using Brownian dynamics simulations at a coarse-grained level of resolution. We considered a system consisting of multiple VWF-like self-interacting chains that also interact with a surface under a shear flow. By a systematic analysis, we reveal that chain-chain and chain-surface interactions coexist non-trivially to modulate the spontaneous adsorption of VWF and the posterior immobilization of secondary tethered chains. Accordingly, these interactions tune VWFs extension and its propensity to form shear-assisted functional adsorbed aggregates. Our data highlights the collective behavior VWF self-interacting chains have when bound to the surface, distinct from that of isolated or flowing chains. Furthermore, we show that the extension and the exposure to solvent have a similar dependence on shear flow, at a VWF-monomer level of resolution. Overall, our results highlight the complex interplay that exists between adsorption, cohesion, and shear forces and its relevance for the adhesive hemostatic function of VWF.

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↗
bioRxiv2022

How talin allosterically activates vinculin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single molecule magnetic tweezer experiments, Molecular Dynamics simulations, actin bundling assays, and adhesion assembly experiments in live cells, we here discover a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at 7-10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The allosteric vinculin mutant is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

biophysics↗
bioRxiv2022

Thermodynamic stabilization of the von Willebrand Factor A1 domain due to loss-of-function disease-related mutations.

The von Willebrand disease (vWD) is the most common hereditary bleeding disorder, caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets at sites of vascular lesion. vWF carries out this essential homeostatic task, via the specific protein-protein interaction between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIB). Upon the vWF activation triggered by the shear of the flowing blood. The two naturally occurring mutations G1324A and G1324S at the A1 domain, near the GPIB binding site, result in a dramatic decrease of platelets adhesion, a bleeding disorder classified as type 2M vWD. However, it remained unclear how these two supposedly minor modifications lead to this drastic phenotypic response. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and non-equilibrium MD-based free energy simulations. Our data confirm that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. These mutations locally diminished the flexibility of the binding site to GPIB and induced a conformational change that affected the nearby secondary structure elements. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously-reported mutation-augmented melting temperatures. Overall, our results support the idea of thermodynamic conformational restriction of A1-- before the binding to GPIB--as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.

biophysics↗