Comparison of human and mouse E-selectin binding to Sialyl-Lewisx
© The Author(s). 2016
Received: 29 March 2016
Accepted: 21 June 2016
Published: 2 July 2016
During inflammation, leukocytes are captured by the selectin family of adhesion receptors lining blood vessels to facilitate exit from the bloodstream. E-selectin is upregulated on stimulated endothelial cells and binds to several ligands on the surface of leukocytes. Selectin:ligand interactions are mediated in part by the interaction between the lectin domain and Sialyl-Lewis x (sLex), a tetrasaccharide common to selectin ligands. There is a high degree of homology between selectins of various species: about 72 and 60 % in the lectin and EGF domains, respectively. In this study, molecular dynamics, docking, and steered molecular dynamics simulations were used to compare the binding and dissociation mechanisms of sLex with mouse and human E-selectin. First, a mouse E-selectin homology model was generated using the human E-selectin crystal structure as a template.
Mouse E-selectin was found to have a greater interdomain angle, which has been previously shown to correlate with stronger binding among selectins. sLex was docked onto human and mouse E-selectin, and the mouse complex was found to have a higher free energy of binding and a lower dissociation constant, suggesting stronger binding. The mouse complex had higher flexibility in a few key residues. Finally, steered molecular dynamics was used to dissociate the complexes at force loading rates of 2000–5000 pm/ps2. The mouse complex took longer to dissociate at every force loading rate and the difference was statistically significant at 3000 pm/ps2. When sLex-coated microspheres were perfused through microtubes coated with human or mouse E-selectin, the particles rolled more slowly on mouse E-selectin.
Both molecular dynamics simulations and microsphere adhesion experiments show that mouse E-selectin protein binds more strongly to sialyl Lewis x ligand than human E-selectin. This difference was explained by a greater interdomain angle for mouse E-selectin, and greater flexibility in key residues. Future work could introduce similar amino acid substitutions into the human E-selectin sequence to further modulate adhesion behavior.
KeywordsE-selectin Receptor Cell adhesion Molecular dynamics Docking Steered molecular dynamics
Selectins are a family of transmembrane adhesion molecules that mediate the inflammatory response and the cancer metastasis cascade. There are three members of the selectin family: P(latelet)-selectin, E(ndothelial)-selectin, and L(eukocyte)-selectin. All three contain an N-terminal lectin domain, epidermal-growth-factor-like (EGF) domain, a varying number of consensus repeat units, a transmembrane portion, and a cytoplasmic tail [1–3]. During inflammation, fast binding and dissociation of bonds between cells and endothelium contributes to rolling. Selectin:ligand interactions are mediated partially by the interaction between the lectin domain and Sialyl Lewis x (sLex), a tetra saccharide on cell surface proteins common to selectin ligands. E-selectin binds particularly well to PSGL-1, CD44, and ESL-1 [1, 4].
Molecule conformational changes are essential to physiological processes . Selectin interdomain hinge flexibility greatly affects the on-rate of selectin:ligand binding. All the selectins have shown “open” and “closed” states that correspond to whether or not they are in complex; for instance, there is a 52° increase in the interdomain angle from unliganded P-selectin to P-selectin in complex . Hydrodynamic forces in the bloodstream favor the open conformation as it can strengthen selectin:ligand bonds . A flexible hinge encourages the oscillation between the two states, which facilitates greater range of motion for the lectin domain and thus provides more opportunity for binding [8, 9]. Lou et al. used molecular dynamics (MD) and site mutagenesis at the interdomain hinge of L-selectin to learn that increasing hinge flexibility via mutation caused an increase in binding on- and off-rates of selectin:ligand interactions . Of particular interest are the binding site and interdomain angle, since prior dissociation studies of P-selectin:sLex suggest these to be important modulators of dissociation time and final conformation .
MD simulations are a useful tool to study the movement of a protein chain over time, given specified starting parameters . The goal of this study was to determine how the structural differences between human and mouse E-selectin affect their corresponding binding and thus cell rolling behavior. MD, docking, and steered molecular dynamics (SMD) were used in conjugation with microtube rolling experiments to address this link between molecular properties and cellular scale adhesion phenomena under flow.
MD to prepare receptor (E-selectin or mutants) for docking
The lectin and EGF crystal structure of human E-selectin (1ESL) was obtained from the Protein Data Bank to provide starting atomic coordinates. The lectin and EGF domains are the effective binding unit of E-selectin. The E-selectin:sLex complex crystal structure (1G1T) was not used as a starting structure as the bound complex does not allow for full flexibility of E-selectin when amino acid substitutions are made. MD, docking, and SMD simulations were performed using the YASARA (YASARA Biosciences GmbH, Vienna, Austria) package of MD programs with the YAMBER3 self-parameterizing force field. For all simulations, the temperature and pressure were held constant at 298 K and 1 atm, respectively. Other parameters used include periodic boundary conditions, the particle mesh Ewald method for electrostatic interactions, and the recommended 7.86 Å force cutoff for long-range interactions . A predicted model of mouse E-selectin was created using human E-selectin as a template and substituting 29 residues.
For equilibrium simulations, human and mouse E-selectin were each solvated in a water box and neutralized by adding Na+ and Cl- ions to a concentration of ~50 mM. To allow for free protein rotation, the water box was defined as a cube with sides 80 Å, at least 10 Å from the structure. The conformational stresses were removed using short steepest-descent minimizations followed by simulated annealing until sufficient convergences were reached. Free dynamics simulations were run for 10 ns. Similar equilibration simulations were run for sLex (taken from the 1G1T PDB structure) with a water box of size 30 × 30 × 30 Å. The average structure for each simulation run was used for further simulation steps.
Binding sLex to human and mouse E-selectin
Molecular docking predicts the conformation of a protein-ligand complex and enables calculation of the binding affinity . sLex was docked to the human and mouse E-selectin structures using the AutoDock program with YAMBER3 force field. sLex was allowed full flexibility and E-selectin had a fixed backbone with flexible sidechains. 250 docking runs were completed, and the AutoDock scoring function sorted the runs by binding energy. Complex conformations were assumed to be different if the ligand RMSD was greater than 5 Å. Of the final conformations with positive binding energy, those for which there was no contact (5 Å or less) between the fucose residue of sLex and the calcium ion were eliminated as they would not be physiologically realistic. The docked complexes were solvated using the same MD steps as before with a water box of size 100 × 100 × 100 Å. The distance from the ligand to the calcium ion was analyzed over the simulation, and if it remained relatively constant, the complex was considered stable. The average free dynamics complex structures were used for the subsequent dissociation steps.
SMD to simulate dissociation under applied force
SMD was used to simulate dissociation under applied force. Constant acceleration was applied to the ligand center of mass to move it away from the receptor center of mass. The simulations were run until all the hydrogen bonds between sLex and E-selectin broke and the two proteins dissociated.
Microrenathane tubes (300 μm i.d. and 50 cm long; Braintree Scientific, Braintree, MA) were sterilized with 75 % ethanol for 15 min. After three washes with PBS, the inner luminal surface was functionalized with recombinant human E-selectin (5 μg/mL) by incubating for 2 h, to allow for passive adsorption to the surface. Next, the microtubes were then incubated with dry milk powder (5 % w/v) in PBS for 1 h to prevent nonspecific adhesion. For control experiments, microtubes were prepared as indicated above except that E-selectin was replaced with BSA.
SuperAvidin-coated microspheres (9.94 μm diameter; CP01N, Bangs Laboratories, Fishers, ID) were washed with PBS buffer per manufacture instruction. Next, the microspheres were incubated with Sialyl-LewisX-biotin at specified concentrations for 1 h with gentle mixing every 15 min. Finally, the microspheres were washed twice and resuspended in flow buffer (PBS supplemented with 2 mM Ca2+). The surface density of sLex on the microspheres was not measured in this study, however our previous work with similar sLex-coated microspheres and selectin surface coatings show that these materials recreate the physiological rolling behavior of leukocytes in the vasculature, with comparable rolling velocities .
Functionalized microspheres (2x106/mL) suspended in flow buffer were perfused through the microtubes using a syringe pump at 8 dyne/cm2. Recorded videos of rolling microbeads were captured and analyzed using ImageJ similarly to prior publications [15, 16].
Mouse E-selectin homology model exhibits a greater interdomain angle than human E-selectin
Mouse E-selectin is predicted to bind more strongly to sLex than human E-selectin
Differences in dissociation time among complexes are caused more by interdomain flexibility rather than by contacts between receptor and ligand
Mouse E-selectin complex takes longer to dissociate than human E-selectin
Excessive leukocyte extravasation out of the bloodstream has been linked with chronic inflammation . Thus, potential therapies for controlling the inflammatory response could involve inhibiting or moderating the selectin adhesion that mediates leukocyte tethering and rolling to the blood vessel walls. Homology modeling and amino acid substitutions, particularly those that affect molecular flexibility, and have been shown to be highly effective in changing adhesion and inhibitive function [24–26]. In this study, a mouse homology model comprising 29 point substitutions to the human E-selectin crystal structure greatly affected dissociation of sLex from the resulting complex. The adhesive characteristics of the mouse E-selectin homology model qualitatively match results from experiments that showed slower rolling velocity of sLex-coated microspheres. These results provide new insight into the connection between structure and function of species-specific E-selectin. These results suggest that differences in dissociation time result more from interdomain flexibility than by contacts between receptor and ligand.
Docking a homology model structure does accumulate more errors than using a crystal structure , but in this case, a crystal structure for mouse E-selectin was not available. The docking algorithm accounts for two important details: protein flexibility is a key determinant in binding, and physiologically, complexes are solvated in a salt solution . The docking algorithm included flexibility in the E-selectin side chains and full flexibility in the sLex. The docked structures were solvated after docking using 10-ns MD simulations to allow for more physiological conditions. Intramolecular distortion of the lectin and EGF domains was not evident for most simulations, particularly at higher force-induced loading rates. It has been shown that shear flow can have a contribution to intramolecular distortion , but as with most selectin:ligand dissociation simulations , shear flow is not directly considered in these SMD simulations.
This study demonstrates the significance of combining simulations with experimental rolling studies to gain insights into the functional differences between proteins that share sequence similarity. The differences in amino acid structure can be exploited for applications such as selectin-based leukocyte and circulation tumor cell isolation . The combined methodology involving docking, SMD, and MD simulations of receptor:ligand interactions holds possibility as a means for rational drug design .
Molecular simulations were used to elucidate the binding of sLex to mouse and human E-selectin. Docking simulations predicted that mouse E-selectin would bind more strongly to sLex than human E-selectin, and SMD simulations predicted that the mouse E-selectin:sLex complex would exhibit a longer dissociation time. Mouse E-selectin alone and bound to sLex exhibited a greater interdomain angle than human E-selectin, and there were fewer receptor:ligand contacts. When tested experimentally, sLex-coated microspheres rolled more slowly in tubes coated with mouse E-selectin rather than human E-selectin.
BSA, bovine serum albumin; EGF, epidermal growth factor; MD, molecular dynamics; PBS, phosphate-buffered saline; PDB, protein data bank; RMSD, root mean square deviation; RMSF, root mean square fluctuation; sLex, sialyl Lewis x; SMD, steered molecular dynamics
This work was funded by the U.S. National Institutes of Health, Grant no. HL018208 to M.R.K.
Availability of data and materials
The raw simulation output data will not be published, but will be made available upon request.
ADR performed the computational study and wrote the manuscript. TC performed the experiments. TT provided computational methods. MRK conceived of the study and edited the manuscript. All authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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