Biology subjects

Butera, D.

Publications and source records attributed to Butera, D..

3 recordsLinked to original sources

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↗

bioRxiv2019

Protein function achieved through multiple covalent states

The structure of proteins is defined by two main types of covalent bonds; the peptide bonds that link the amino acid residues and disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds are introduced during protein folding and their formation is assumed to be complete in the mature, functional protein. We tested this assumption by quantifying the redox state of disulfide bonds in human blood proteins in their native environment. Using a differential cysteine alkylation and mass spectrometry method, we measured the redox state of disulfide bonds in circulating fibrinogen and von Willebrand factor. There is an extraordinary disulfide lability in the proteins, with 27 bonds in the two proteins ranging from 3 to 50% reduced in healthy human donors. Modelling of the data indicates that the proteins exist in hundreds of different disulfide-bonded states in the circulation. Different covalent states of fibrinogen are associated with different binding activities and their distribution is changed by fluid shear forces and altered in patients with cardiovascular disease, indicating that the different states have different functions and are dynamic. These findings have implications for protein function generally and how proteins are targeted in experimental settings and for therapeutic purposes.

biochemistry↗

bioRxiv2019

Evaluation of a potential redox switch in blood coagulation tenase

Blood coagulation factor IXa (FIXa) activates factor X that leads to thrombin and fibrin formation and a stable thrombus. FIXa catalytic efficiency is markedly enhanced when bound to the co-factor, factor VIII (FVIII), and a negatively charged phospholipid surface in the tenase complex. Small redox active peptides and protein oxidoreductases have been shown previously to have some FIXa co-factor activity and thiol modifying agents have been reported to influence FVIII activity. These observations suggested that FIXa might contain an allosteric disulfide that is regulated by FVIII. This idea was tested by measuring the influence of FVIII on the redox state of FIXa disulfide bonds and the effect of plasma oxidoreductases on FIXa activity. The redox state of nine of the 11 disulfide bonds in FIXa was measured using differential cysteine labelling and mass spectrometry and all were oxidized in the protein, and this did not change upon binding of the enzyme to FVIII. All eight disulfide bonds in FVIII were also predominantly oxidized and this did not appreciably change upon FIXa binding. In addition, relevant protein reductants in the circulation inhibited rather than activated FIXa activity. In conclusion, we found no evidence that the co-factor function of FVIII involves a change in the redox state of one or more FIXa disulfide bonds.

biochemistry↗