Antithrombin (AT), a member of the serpin protease inhibitors family, is conformationally activated when bound to heparin, so becoming capable to inhibit some of the coagulation cascade serine proteinases, like thrombin and fXa. This serpin occurs in the blood as a glycoprotein that co-exists in two isoforms, a- and b-AT, which differ in the amount of glycosylation attached to the protein.  Moreover, b-AT has the higher affinity for heparin, functioning as the major inhibitory form in vivo, although it is the less abundant one. Considering the lack of structural information regarding the effect of glycosylation in heparin-AT recognition, this work intends to analyze and properly describe the conformational profile of the a- and b-AT glycosylation structures using molecular modeling techniques.

                Using a protocol previously described by the group (Verli and Guimarães, Carbohidr. Res. 2004, 339, 281), applying both GROMACS force field and simulation suite, energy contour plots were constructed for each of AT glycan glycosidic linkages at the disaccharide level. The global minimum of the obtained contour plots, further refined in a 5.0ns molecular dynamics (MD) simulation, were used as reference geometry for each glycosidic linkage of AT glycosylation. The average geometries so obtained were used to build the whole glycan structure, which was conformationally sampled in a 10.0ns MD simulation.

            The geometry associated to the global minimum of each contour plot was well related to the average geometry sampled in MD simulations. Moreover, the obtained a- and b-AT glycosylation structures were compared to X-ray crystallographic structures of AT with different levels of glycosylation, suggesting the occurrence of packing effects over this flexible carbohydrate moiety.

The data so obtained opens the perspective to build the complete structure of AT, both in its a- and b- isoforms, as well as to evaluate, at the atomic level, the molecular recognition responsible for the distinct activation of each AT isoform by heparin. Since it allows a description of the dynamics of AT closest to its biological properties, it can be expected that these data contribute in the understanding of biological processes dependent on glycoproteins, as well as in the design of new synthetic derivatives of heparin.

Supported by: CNPq and CAPES