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Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

ABSTRACT: Glycosaminoglycan (GAG)-protein interactions modulate many important biological processes. Structure-function studies on GAGs may reveal probes and drugs, but their structural complexity and highly acidic nature confound such work. Productivity will increase if we are able to identify tight-binding oligosaccharides in silico. An extension of the CHARMM force field is presented to enable modeling of polysaccharides containing sulfamate functionality, and is used to develop a reliable alchemical free-energy perturbation protocol that estimates changes in affinity for the prototypical heparin-antithrombin system to within 2.3 kcal/mol using modest simulation times. Inclusion of water is crucial during simulation as solvation energy was equal in magnitude to the sum of all other thermodynamic factors. In summary, we have identified and optimized a reliable method for estimation of GAG-protein binding affinity, and shown that solvation is a crucial component in GAG-protein interactions.

SUBMITTER: Sarkar A

PROVIDER: S-EPMC5072149 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

Sarkar Aurijit A Yu Wenbo W Desai Umesh R UR MacKerell Alexander D AD Mosier Philip D PD

Glycobiology 20160718 10

Glycosaminoglycan (GAG)-protein interactions modulate many important biological processes. Structure-function studies on GAGs may reveal probes and drugs, but their structural complexity and highly acidic nature confound such work. Productivity will increase if we are able to identify tight-binding oligosaccharides in silico. An extension of the CHARMM force field is presented to enable modeling of polysaccharides containing sulfamate functionality, and is used to develop a reliable alchemical f ...[more]

PMID: 27496757

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Project description:Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

Dataset Information

Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

Publications

Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

Similar Datasets

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