The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK).

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Computational studies on glycosaminoglycan recognition of sialyl transferases.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65 nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

Glycan Modulation of Insulin-like Growth Factor-1 Receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.

Combinatorial Virtual Library Screening Study of Transforming Growth Factor-ß2-Chondroitin Sulfate System.

Transforming growth factor-beta (TGF-ß), a member of the TGF-ß cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-ß2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-ß2-CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-ß2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-ß2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-ß2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG-protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-ß2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-ß2-CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein-CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-ß2 and may out-compete common natural CS biopolymers.

Enzyme immobilization offers a robust tool to scale up the production of longer, diverse, natural glycosaminoglycan oligosaccharides.

Although structurally diverse, longer glycosaminoglycan (GAG) oligosaccharides are critical to understand human biology, few are available. The major bottleneck has been the predominant production of oligosaccharides, primarily disaccharides, upon enzymatic depolymerization of GAGs. In this work, we employ enzyme immobilization to prepare hexasaccharide and longer sequences of chondroitin sulfate in good yields with reasonable homogeneity. Immobilized chondroitinase ABC displayed good efficiency, robust operational pH range, broad thermal stability, high recycle ability and excellent distribution of products in comparison to the free enzyme. Diverse sequences could be chromatographically resolved into well-defined peaks and characterized using LC-MS. Enzyme immobilization technology could enable easier access to diverse longer GAG sequences.

Rigorous analysis of free solution glycosaminoglycan dynamics using simple, new tools.

Heparin/heparan sulfates (H/HS) are ubiquitous biopolymers that interact with many proteins to induce a range of biological functions. Unfortunately, how these biopolymers recognize their preferred protein targets remain poorly understood. It is suggested that computational simulations offer attractive avenues but a number of challenges, e.g., difficulty of selecting a comprehensive force field, few simple tools to interpret data, among others, remain. This work addresses several such challenges so as to help ease the implementation and analysis of computational experiments. First, this work presents a rigorous comparison of two different recent force fields, CHARMM36 and GLYCAM06, for H/HS studies. Second, it introduces two new straightforward parameters, i.e., end-to-end distance and minimum volume enclosing ellipsoid, to understand the myriad conformational forms of oligosaccharides that evolve over time in water. Third, it presents an application to elucidate the number and nature of inter and intramolecular, nondirect bridging water molecules, which help stabilize unique forms of H/HS. The results show that nonspecialists can use either CHARMM36 or GLYCAM06 force fields because both gave comparable results, albeit with small differences. The comparative study shows that the HS hexasaccharide samples a range of conformations with nearly equivalent energies, which could be the reason for its recognition by different proteins. Finally, analysis of the nondirect water bridges across the dynamics trajectory shows their importance in stabilization of certain conformational forms, which may become important for protein recognition. Overall, the work aids nonspecialists employ computational studies for understanding the solution behavior of H/HS.

Studies on fragment-based design of allosteric inhibitors of human factor XIa.

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa's active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.

Inhibition of Human Cytomegalovirus Entry into Host Cells Through a Pleiotropic Small Molecule.

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.

Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin.

Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, ß3-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation.

Molecular principles for heparin oligosaccharide-based inhibition of neutrophil elastase in cystic fibrosis.

Cystic fibrosis (CF) is a multifactorial disease in which dysfunction of protease-antiprotease balance plays a key role. The current CF therapy relies on dornase α, hypertonic saline, and antibiotics and does not address the high neutrophil elastase (NE) activity observed in the lung and sputum of CF patients. Our hypothesis is that variants of heparin, which potently inhibit NE but are not anticoagulant, would help restore the protease-antiprotease balance in CF. To realize this concept, we studied molecular principles governing the effectiveness of different heparins, especially 2-O,3-O-desulfated heparin (ODSH), in the presence of sputum components and therapeutic agents. Using sputa from CF patients and an NE activity assay, we found that heparins are ineffective if used in the absence of dornase. This is true even when mucolytics, such as DTT or N-acetylcysteine, were used. Computational modeling suggested that ODSH and DNA compete for binding to an overlapping allosteric site on NE, which reduces the anti-NE potential of ODSH. NE inhibition of both DNA and ODSH is chain length-dependent, but ODSH chains exhibit higher potency per unit residue length. Likewise, ODSH chains exhibit higher NE inhibition potential compared with DNA chains in the presence of saline. These studies suggest fundamental differences in DNA and ODSH recognition and inhibition of NE despite engaging overlapping sites and offer unique insights into molecular principles that could be used in developing antiprotease agents in the presence of current treatments, such as dornase and hypertonic saline.

anexVis: visual analytics framework for analysis of RNA expression.

Summary: Although RNA expression data are accumulating at a remarkable speed, gaining insights from them still requires laborious analyses, which hinder many biological and biomedical researchers. This report introduces a visual analytics framework that applies several well-known visualization techniques to leverage understanding of an RNA expression dataset. Our analyses on glycosaminoglycan-related genes have demonstrated the broad application of this tool, anexVis (analysis of RNA expression), to advance the understanding of tissue-specific glycosaminoglycan regulation and functions, and potentially other biological pathways. Availability and implementation: The application is accessible at https://anexvis.chpc.utah.edu/, source codes deposited on GitHub. Supplementary information: Supplementary data are available at Bioinformatics online.

A small group of sulfated benzofurans induces steady-state submaximal inhibition of thrombin.

Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric inhibition of thrombin against fibrinogen (ΔY = 55-75%), the first time that a small molecule (MW  < 800) has been found to thwart macromolecular cleavage by a monomeric protease in a controlled manner. The work leads to the promising concept that it should be possible to develop allosteric inhibitors that reduce clotting, but do not completely eliminate it, thereby avoiding major bleeding complications that beset anticoagulants today.

2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity.

High mobility group box 1 (HMGB1) is an alarmin released from macrophages after infection or inflammation and is a biomarker of lung disease progression in patients with cystic fibrosis. We reported that 2-O, 3-O desulfated heparin (ODSH) inhibits the release of HMGB1 from murine macrophages triggered by neutrophil elastase both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this study, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase that has been linked to HMGB1 acetylation and release. ODSH inhibited both neutrophil elastase and LPS-triggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration-dependent manner. Fluorescein-labeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus, suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro, resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognizes a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (dissociation constant ∼1 nM) in a highly cooperative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.

Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions.

Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, ß3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

Immobilization alters heparin cleaving properties of heparinase I.

We report here a novel observation that immobilization of heparinase I on CNBr-activated Sepharose results in heparin degradation properties that are different from heparinase I in the free solution form. Studies over a range of pHs (5-8) and temperatures (5-50°C) as well as under batch and flow conditions show that immobilized heparinase 1 displays altered pH and temperature optima, and a higher propensity for generation of longer chains (hexa- and octa-) with variable sulfation as compared to that in the free form, which is known to yield disaccharides. The immobilized enzyme retained good eliminase activity over at least five cycles of reuse. In combination, results suggest that heparinase I immobilization may offer a more productive route to longer, variably sulfated sequences.

Sulfated dehydropolymer of caffeic acid: In vitro anti-lung cell death activity and in vivo intervention in emphysema induced by VEGF receptor blockade.

Induced lung cell death and impaired hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) signaling are proposed as a pathobiologic mechanism for alveolar structural destruction and loss in emphysema. We hypothesized that our sulfated dehydropolymer of caffeic acid, CDSO3, exerts anti-cell death activities and therapeutic interventions in emphysema by virtue of Fe2+ chelation-based HIF-1α/VEGF stabilization and elevation. The Fe2+ chelating activity was determined in the chromogenic ferrozine-Fe2+ chelation inhibitory assay. The in vitro anti-cell death activities and their Fe2+ and HIF-1α dependence were assessed against a range of emphysematous insults in the lung endothelial (HMVEC-L) and epithelial (A549) cells. CDSO3 was spray-dosed to the lung for three weeks (day 1-21) in an in vivo rat model of apoptotic emphysema induced with a VEGF receptor antagonist SU5416. Post-treatment treadmill exercise endurance, airspace enlargement, and several lung biomarkers/proteins were measured. CDSO3 was a potent Fe2+ chelating molecule. At 10 µM, CDSO3 inhibited HMVEC-L and A549 cell death induced by histone deacetylase inhibition with trichostatin A, VEGF receptor blockade with SU5416, and cigarette smoke extract by 65-99%, which were all significantly opposed by addition of excess Fe2+ or HIF-1α inhibitors. As a potent elastase inhibitor and antioxidant, CDSO3 also inhibited elastase- and H2O2-induced cell death by 92 and 95%, respectively. In the rat model of SU5416-induced apoptotic emphysema, CDSO3 treatment at 60 µg/kg 1) produced 61-77% interventions against exercise endurance impairment, airspace enlargement [mean linear intercept] and oxidative lung damage [malondialdehyde activity]; 2) normalized the apoptotic marker [cleaved caspase-3]; 3) stimulated the VEGF signaling [VEGF receptor 2 phosphorylation] by 1.4-fold; and 4) elevated the HIF-1α and VEGF expression by 1.8- and 1.5-fold, respectively. All of these were consistent with CDSO3's Fe2+ chelation-based HIF-1α/VEGF stabilization and elevation against their pathobiologic deficiency, inhibiting lung cell death and development of apoptotic emphysema.

Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry.

Structural characterization of the microheterogeneity of heparin, heparan sulfate, and other glycosaminoglycans is a major analytical challenge. We present the use of a stable isotope-labeled hydrazide tag (INLIGHT™) with high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS, which was recently introduced for detailed study of N-glycan heterogeneity, to characterize heparinase-digested heparin (digHep) products without the use of semi-volatile ion pairing reagents. Using both full scan LC-MS and data-dependent LC-MS/MS, we identified 116 unique digHep species, a feat possible because of INLIGHT™ labeling. Of these, 83 digHep products were structurally identified, including the 12 standard disaccharides as well as 34 tetra- (DP4), 26 hexa- (DP6), 21 octa- (DP8), and 2 decasaccharides (DP10). Each of the 116 digHep species co-eluted with both light and heavy INLIGHT™ tags (L/Havg = 1.039 ± 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT™-based comparative analyses of different forms of heparin, heparan sulfate, and other GAGs with high quantitative precision using mainstay reverse-phase HRAM LC-MS/MS. Graphical Abstract Reducing end labeling strategy for mapping depolymerized heparin/heparan sulfate products by reverse-phase LC-MS/MS.

A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

BACKGROUND: The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. METHODS: In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. RESULTS: Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. CONCLUSIONS: An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. GENERAL SIGNIFICANCE: These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions.

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

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.