Effects of glycosylation on heparin binding and antithrombin activation by heparin

Abstract

Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β-AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135-linked oligosaccharide caused a bending in AT-bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α-AT. The obtained data supported the proposal of an atomic-level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β-sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135-linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic-level explanation for the Asn135-linked glycan influence over heparin binding and AT activation.

The Calcium Goes Meow: Effects of Ions and Glycosylation on Fel d 1, the Major Cat Allergen

“…This construction was made via Glycosciences.de modelling tools [ 37 , 38 ]. The glycosidic linkages composing such glycans had their geometries adjusted to each of their relative abundance as isolated disaccharides in water, thus determined as their main conformational states [ 39 , 40 ]. Such procedure was successfully applied in previous glycoprotein studies [ 41 , 42 ].…”

Dynamics on human Toll‐like receptor 4 complexation to MD‐2: The coreceptor stabilizing function

“…GROMACS simulations suite and GROMOS53a6 force field were used, employing a molecular dynamics protocol based on previous studies . Briefly, all systems were solvated with Simple Point Charge water model in rectangular boxes, except for the system (hTLR4–MD‐2) 2 , that was solvated in an octahedron box—due to its shape and size, by a layer of at least 10 Å from the solute atoms.…”

The mechanism of high affinity pentasaccharide binding to antithrombin, insights from Gaussian accelerated molecular dynamics simulations

“…diffraction structures (Huntington, 2011;Olson et al, 2010) and MD-based studies (Arantes et al, 2018;Pol-Fachin et al, 2011;T oth et al, 2015) have been used to investigate the details of the allosteric processes. In our GAMD simulations, we were able to capture a significant number of different binding modes for the pentasaccharide, including conformations with relatively large RMSD compared to the X-ray structures.…”

“…Notable examples for such studies include modelling the transition into latent conformation in plasminogen activator inhibitor 1 at atomistic level (Cazzolli et al, 2014), the elucidation of the pathway of RCL insertion in a1-antitrypsin (Andersen et al, 2017), and protein folding studies aimed at explaining the misfolding of mutated serpins (Wang et al, 2018). Specifically, in case of AT, the role of Asn135 glycosylation (Pol-Fachin, Franco Becker, Almeida Guimarães, & Verli, 2011) as well as the RCL dynamics and the conformation of the critical Arg393 side chain was investigated (T oth, Fekete, Balogh, Bereczky, & Kom aromi, 2015). In a recent study, the binding of D-myo-inositol-3,4,5,6-tetrakisphosphate, a ligand with a scaffold different from heparinoids was studied and signs of allosteric activation such as higher flexibility of RCL were detected (Arantes, P erez-S anchez, & Verli, 2018).…”