School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK
Received on August 9, 1999; revised on October 5, 1999; accepted on October 5, 1999.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: glycosaminoglycan/x-ray diffraction/dermatan sulfate/molecular dynamics/water
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unlike hyaluronan, chondroitin and dermatan are not found free in the extracellular matrix, but are covalently linked to protein in complexes such as proteoglycans. Chondroitin sulfate proteoglycans have been localized to cartilage, bone, cornea, and intervertebral disc, whereas dermatan sulfate proteoglycans are found in skin and aorta. Dermatan and chondroitin sulfate have been implicated in interacting with collagen to stabilize the extracellular matrix, binding to growth factors with a high degree of specificity to regulate growth factor activity, and have also been shown to have anticoagulant, antilipemic, antiangiogenic, and antitumor activities (Hardingham and Bayliss, 1990; Hardingham and Fosang, 1992
).
The interaction of carbohydrates with molecular water is central to their biological functions, but is not well understood presently. Molecular dynamics (MD), performed with explicit inclusion of solvent, is an excellent technique for studying these interactions. MD has previously been used to provide detailed information about the water structure around monosaccharides (Brady, 1989; Liu and Brady, 1997
) and the presence of intramolecular hydrogen-bonds between linked sugar units (Engelsen and Pérez, 1996
; Ueda and Brady, 1996
). However, carbohydrate force-fields and water models require extensive testing by comparison with experimental data, such as that provided by x-ray crystallography or NMR (Homans, 1990
). Our previous work concentrated on studying the interaction of water with hyaluronan using aqueous molecular dynamics simulations. We noticed in our MD studies of hyaluronan tetrasaccharides that the predicted energy surface around their central linkages contained a single minima. However the ends of these molecules were predicted to be complex multi-minima surfaces, due to end effects (Almond et al., 1998a
). Subsequently, we have demonstrated that simulations of hyaluronan tetrasaccharides in solution predict structures similar to those found in x-ray fiber diffraction. We also compared conformations predicted by these simulations with data from NMR (Almond et al., 1997
) and hydrodynamics (Almond et al., 1998b
). Thus, our hypothesis is that if aqueous MD simulations of carbohydrates can be compared effectively with structures proposed by x-ray crystallography, and other experimental techniques, then they could have a predictive capacity. We have noticed that most glycosaminoglycan structures refined by x-ray fiber diffraction are left-handed helices, the only exceptions being the 8-fold helices proposed for chondroitin and dermatan sulfate which are right-handed. Our aim in this paper is to understand the interaction of water with these structures. In particular the stability of intramolecular hydrogen-bonds in solution, and thus throw light onto the apparently anomalous right-handed helices.
In the present study simulations were performed on chondroitin and dermatan tetrasaccharides. However, only data for the central linkages, which do not have end-effects, are being presented. Detailed analysis of the simulations was performed to determine whether long-lived hydrogen-bonds are present in the related structures of chondroitin and dermatan, which we characterized in hyaluronan. Particular emphasis was placed on understanding the dynamics of intramolecular hydrogen-bonds and water interaction. Simulations of dermatan were performed with 4C1, 2SO, and 1C4 iduronate starting ring geometries, internal ring flexibility was allowed thereafter. We calculated the helical symmetries and axial rises for each of these structures as a function of conformation, allowing the ensemble of calculated MD structures to be compared with raw x-ray fiber diffraction data. Finally, we present our preferred solution conformations and discuss how they relate to x-ray diffraction refinements.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We identified the presence of intramolecular hydrogen-bonds (see Materials and methods) in chondroitin tetrasaccharide simulations, Figure 1 (bottom) and compared them to our previous hyaluronan simulations, Figure 1 (top). The same hydrogen-bonds were observed across the ß(14) linkages of chondroitin and hyaluronan, namely: GlcA OH3...GalNAc O5, labeled (A) in Figure 1, and GalNAc NH...GlcA COO, labeled (B). Consequently, the ß(1
4) linkage of chondroitin was found to maintain a similar average conformation to hyaluronan, centered upon (
,
) = (50°,0°). Our definitions of
and
are presented in Materials and methods. In contrast, the average ß(1
3) conformation for chondroitin, during a 500 ps molecular dynamics simulation in water, is (
,
) = (50°,-30°) whereas for hyaluronan it is (
,
) = (50°,0°).
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparing hyaluronan with chondroitin, the overall predicted effect of hexosamine OH4 epimerization is to change the average conformation from approximately left-handed 4-fold to 3-fold helices. This would appear to be reflected in previous x-ray fiber diffraction refinements. Hyaluronan prefers 43 helices (Mitra et al., 1983a) when crystallized with weakly perturbing monovalent sodium or potassium ions, and under neutral pH conditions. Chondroitin sulfate, on the other hand, has not been observed to crystallize into any 4-fold structures. However, both chondroitin-4-sulfate and chondroitin-6-sulfate have been refined into a 32 packing arrangement in the presence of sodium ions (Millane et al., 1983
). A 2-fold helix has been observed in chondroitin-4-sulfate (Cael et al., 1978
), but this was in the presence of calcium, which can be seen to be coordinated between carboxyl and sulfate moieties in the refinement, and is therefore dominated by the effect of the highly charged calcium ion. Similarly, a 2-fold helix was observed with chondroitin-6-sulfate at low pH, a situation that cannot be compared to the conditions under which we performed our simulations. As far as we are aware, the 8-fold helix observed in chondroitin-6-sulfate has never been accurately refined (Arnott et al., 1973
), although Arnott et al. originally represented the structure as an 85 helix. Subsequent reviews have come to represent the structure as an 83 helix on the basis of later refinements performed on dermatan sulfate (Ernst et al., 1995
).
Based on our data, and the x-ray fiber diffraction literature, it would appear that at neutral pH and in the presence of sodium it is the satisfaction of intramolecular hydrogen-bonds that dominates the conformation of chondroitin sulfate, rather than the distribution of sulfation. In fact, none of the possible targets for sulfation in chondroitin have been observed to be involved in intramolecular hydrogen-bonds, supporting our claim. Conversely, in the hyaluronan we predict that these groupings do play important roles, involving themselves in intramolecular hydrogen-bonds, as shown in Figure 1. Hyaluronan, in contrast to chondroitin, has not been found to be sulfated.
The presence of strong intra-molecular hydrogen-bonds in chondroitin sulfate has experimental backing, in the form of periodate oxidation kinetic measurements (Scott and Tigwell, 1978). In Figure 8 possible 2-, 3-, and 8-fold helices have been constructed which are closest to the chondroitin MD energy minima. The dihedral angles applied to the linkages are detailed in Table I. In each diagram the structural intra-molecular hydrogen-bonds, GalNAc NH...GlcA COO and GlcA O2...GalNAc O7, have been labeled by (A) and (B) respectively. Both hydrogen-bonds, (A) and (B), can be observed in the 21, 85, and 32 structures. However, in the right-handed 83 structure hydrogen-bond (B) cannot form, and this is labeled by (X).
|
Our simulations suggest that dermatan structures containing IdoA residues with a 4C1 ring geometry can maintain strong interactions between the carboxyl and adjacent acetamido moieties. This interaction is predicted to occur at the expense of the hydrogen-bond between OH3 on IdoA and the ring oxygen on hexosamine, resulting in the observed shift of its ß(14) linkage with respect to chondroitin. However, the conformation of the acetamido is such that interaction with IdoA OH2 perturbs the
(1
3) linkage and allows the hydrogen-bond GalNAc OH4...IdoA O5 (ring oxygen) to exist simultaneously. The preferred helices sampled at the predicted MD energy minima (Figure 4b), with IdoA in a 4C1 conformation, are all left handed (2.5- to 4.5-fold) in contrast to the 8-fold dermatan fiber diffraction refinement, which is right-handed. However, these models have been rejected by x-ray refinement on the basis of packing considerations. Accepting this, our molecular dynamics studies would suggest a left-handed 85 helical conformation containing 2SO IdoA sugars. In this conformation the chains can maintain intramolecular hydrogen-bonds and at the same time produce the required axial-rise. This model would agree with solid state and current solution NMR data (Rao et al., 1995
). Taken together these observations suggest that the 8-fold dermatan x-ray diffraction data should be re-refined with inclusion of left-handed 2SO IdoA models. Based on our MD simulations, we can depict minimum energy models for helices containing the three IdoA forms (Figure 9). The model containing IdoA residues with 1C4 ring geometry has a sinuous contracted structure. Such structures could be present in solution, but the process of drying and fiber stretching may discriminate against them. We have compared a skew boat model (2SO) with 85 symmetry, lying close to the minimum predicted by MD, with the literature model of the 83 helix. This highlights a difference in intramolecular hydrogen-bonds. Based on our simulations, we also conclude that chains containing IdoA with either 4C1 or 2SO ring geometry can be used as starting models for the observed 2-fold and 3-fold diffraction patterns of dermatan fibers (Nieduszynski, 1985
, and references therein). Similarly, we suggest that in chondroitin fibers an 85 helix is more energetically favorable than an 83 helix, and in the absence of other data the former structure should be assumed. If these conclusions are true, then all known x-ray diffraction structures of glycosaminoglycans would be extended 2-fold or left-handed conformations which have optimized intramolecular hydrogen-bonds.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular dynamics integration was carried out using the leap-frog formulation (Hockney, 1970) of the Verlet algorithm (Verlet, 1967
), and hydrogen covalent bond length were kept constant using the SHAKE procedure (van Gunsteren and Berendsen, 1977
). An integration step size of 1 fs was used to provide precise trajectories. No explicit hydrogen-bonding function was used in the simulations, as it is assumed that they are well represented by the partial atom charges and van der Waals parameters (Brady and Schmidt, 1993
).
Solvated simulations used 32 Å water boxes, filled with 1000 TIP3P previously equilibrated water molecules (Jorgensen et al., 1983). The initial configuration was achieved by minimization using adopted basis Newton-Raphson approach, heating for 3 ps at a rate of 100K ps1, followed by 50 ps of diabatic equilibration at 300K. Subsequently 500 ps of adiabatic dynamics was performed. Coordinates were written every 0.05 ps, and the nonbonded lists were updated using the grid search cubing algorithm at a heuristic frequency. Edge effects were overcome by implementing cubic periodic boundary conditions, and the electrostatics were reduced to zero over the range 812 Å using the shifting function.
Calculation of dihedral angles and hydrogen-bonds
The linkage conformation was represented by dihedral angles (H1-C1-Ox-Cx) and
(C1-Ox-Cx-Hx) defined by the hydrogen atoms. Hydrogen-bonds were calculated as in our previous work (Almond et al., 1997
, 1998a). Our definition of a hydrogen-bond is identical to that used by other authors who have analyzed carbohydrate simulations (Brady and Schmidt, 1993
), the distance of D (hydrogen donor) to A (hydrogen acceptor) is less than 3.5 Å, and the angle D-A...A is less than 60°. Hydrogen-bonds were then calculated between all relevant groups during each simulation. The data is grouped into 4 ps intervals, and a persistent hydrogen-bond is counted if it is present for 50% of the time over this period. Any groups which have no persistence of hydrogen-bonding during any of these time periods are ignored.
Calculation of n and h parameters
Calculation of the helical fold (n) as a function of polysaccharide linkage conformation was based on a general method described in the literature (Miyazawa, 1961; Sugeta and Miyazawa, 1967
). In this method the polymer is built up from a series of virtual vectors which describe the smallest repeating unit (a disaccharide in this case). Therefore, short oligomeric sections with specific (
1
3,
1
3) and (
1
4,
1
4) angles at the two linkages were constructed. The positions of the glycosidic oxygen atoms were then extracted and used to calculate n and h for each conformation. Further details are provided in our previous paper (Almond et al., 1998b
). Contour plots were calculated by varying a single linkage while constraining the other to a particular conformation (Figures 2, 4b, 6b, 7b). Dihedral angles relating to specific x-ray diffraction data were calculated by varying all linkages and selecting those conformations which satisfied the helical parameters. The dihedrals which were closest to our calculated solution MD energy for that particular structure were then selected, and tabulated. Three-dimensional structures shown in Figures 8 and 9 were rendered with the package Raster3D (Merritt and Bacon, 1997
).
Calculation of energy contours
Pairs of dihedral angles (,
) were extracted as a function of time for each of the linkages. The conformational space was then split into a grid at 5° intervals, and the probability of pairs of dihedral angles occurring at different grid points calculated. If it is assumed that the space has been efficiently sampled, and that the energy states are nondegenerate, then the energy at each point is proportional to the logarithm of the probability. Thus, the energy was calculated at each point and negated, and then these were rescaled such that the lowest energy corresponded to the zero energy. This data was used to construct contour plots in Figure 3. An identical approach was used to calculate the Cremer-Pople contour surface of Figure 5.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almond,A., Brass,A. and Sheehan,J.K. (1998a) Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data. Glycobiology, 8, 973980.
Almond,A., Brass,A. and Sheehan,J.K. (1998b) Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: solution simulations of hyaluronan tetrasaccharides compared with hydrodynamic and x-ray fiber diffraction data from the polymer. J. Mol. Biol., 284, 14251437.[ISI][Medline]
Arnott,S., Guss,J.M., Hukins,D.W.L. and Mathews,M.B. (1973) Mucopolysaccharides: comparison of chondroitin sulfate conformations with those of related polyanions. Science, 180, 743745.[ISI][Medline]
Brady,J.W. (1989) Molecular dynamics simulations of -D-glucose in aqueous solution. J. Am. Chem. Soc., 111, 51555165.[ISI]
Brady,J.W. and Schmidt,R.K. (1993) The role of hydrogen bonding in carbohydrates: molecular dynamics simulations of maltose in aqueous solution. J. Phys. Chem., 97, 958966.[ISI]
Brooks,B.R., Bruccoleri,R.E., Olafson,B.D., States,D.J., Swaminathan,S. and Karplus,M. (1983) CHARMm: a program for macromolecular energy minimization and dynamics calculations. J. Comp. Chem., 4, 187217.[ISI]
Cael,J.J., Winter,W.T. and Arnott,S. (1978) Calcium chondroitin sulfate: molecular conformation and organization of polysaccharide chains in a proteoglycan. J. Mol. Biol., 125, 2142.[ISI][Medline]
Casu,B., Choay,J., Ferro,D.R., Gatti,G., Jacquinet,J.C., Petitou,M., Provasoli,A., Regazzi,M., Sinay,P. and Torri,G. (1986) Controversial glycosaminoglycan conformation. Nature, 322, 215216.[Medline]
Cremer,D. and Pople,J.A. (1975) General definition of ring puckering parameters. J. Am. Chem. Soc., 97, 13541358.[ISI]
Dowd,M.K., French,A.D. and Reilly,P.J. (1994) Modeling of aldopyranosyl ring puckering with MM3 (92). Carbohydr. Res., 264, 119.[ISI]
Engelsen,S.B. and Pérez,S. (1996) The hydration of sucrose. Carbohydr. Res., 292, 2138.[ISI]
Ernst,S., Langer,R., Cooney,C.L. and Sasisekharan,R. (1995) Enzymatic degradation of glycosaminoglycans. Crit. Rev. Biochem. Mol. Biol., 30, 387444.[Abstract]
Faham,S., Hileman,R.E., Fromm,J.R., Lindhardt,R.J. and Rees,D.C. (1996) Heparin structure and interactions with basic fibroblast growth factor. Science, 271, 11161120.[Abstract]
Frisch,M.J., Trucks,G.W., Schlegel,H.B., Scuseria,G.E., Robb,M.A., Cheeseman,J.R., Zakrzewski,V.G., Montgomery,J.A., Stratmann,R.E., Burant,J.C., Dapprich,S., Millam,J.M., Daniels,A.D., Kudin,K.N., Strain,M.C., Farkas,O., Tomasi,J., Barone,V., Cossi,M., Cammi,R., Mennucci,B., Pomelli,C., Adamo,C., Clifford,S., Ochterski,J., Petersson,G.A., Ayala,P.Y., Cui,Q., Morokuma,K., Malick,D.K., Rabuck,A.D., Raghavachari,K., Foresman,J.B., Cioslowski,J., Ortiz,J.V., Stefanov,B.B., Liu,G., Liashenko,A., Piskorz,P., Komaromi,I., Gomperts,R., Martin,R.L., Fox,D.J., Keith,T., Al-Laham,M.A., Peng,C.Y., Nanayakkara,A., Gonzalez,C., Challacombe,M., Gill,P.M.W., Johnson,B.G., Chen,W., Wong,M.W., Andres,J.L., Head-Gordon,M., Replogle,E.S. and Pople,J.A. (1998) Gaussian 98 (revision A.2), Gaussian, Inc., Pittsburgh, PA.
Hardingham,T.E. and Bayliss,M.T. (1990) Proteoglycans of articular cartilage changes in ageing and in joint disease. Semin. Arth. Rheum. Suppl., 1, 1233.
Hardingham,T.E. and Fosang,A.J. (1992) Proteoglycans: many forms and many functions. FASEB J., 6, 861870.
Hockney,R.W. (1970) The potential calculation and some applications. Methods Comp. Phys., 9, 135211.
Homans,S.W. (1990) A molecular force field for the conformational analysis of oligosaccharides: comparison of theoretical and crystal structures of Man13Manß14GlcNAc. Biochemistry, 29, 91109118.[ISI][Medline]
Jorgensen,W.L., Chandrasekhar,J., Madura,J.D., Impey,R.W. and Klein,M.L. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys., 79, 926.[ISI]
Liu,Q. and Brady (1997) Anisotropic solvent structuring in aqueous sugar solutions. J. Am. Chem. Soc., 118, 1227612286.[ISI]
Lindahl,U., Kushe-Gullberg,M. and Kjellén,L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem., 273, 2497924982.
Merritt,E.A. and Bacon,D.J. (1997) Raster3D: photorealistic molecular graphics. Methods Enzymol., 277, 505524.[ISI]
Millane,R.P., Mitra,A.K. and Arnott,S. (1983) Chondroitin-4-sulfate: comparison of the structures of the potassium and sodium salts. J. Mol. Biol., 169, 903920.[ISI][Medline]
Mitra,A.K., Raghunathan,S., Sheehan,J.K. and Arnott,S. (1983a) Hyaluronic acid: molecular conformations and interactions in the orthorhombic and tetragonal forms containing sinuous chains. J. Mol. Biol., 169, 829859.[ISI][Medline]
Mitra,A.K., Arnott,S., Atkins E.D.T. and Isaac,D.H. (1983b) Dermatan sulfate: molecular conformations and interactions in the condensed state. J. Mol. Biol., 169, 873901.[ISI][Medline]
Miyazawa,T. (1961) Molecular vibrations and structure of high polymers. II. Helical parameters of infinite polymer chains as functions of bond lengths, bond angles and internal rotation angles. J. Polymer Sci., 55, 215231.[ISI]
Mulloy,B., Forster,M.J., Jones,C., Drake,A.F., Johnson,E.A. and Davies,D.B. (1994) The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modeling study. Carbohydr. Res., 255, 126.[ISI][Medline]
Nieduszynski,I.A. (1985) Connective tissue polysaccharides. In Atkins,E.D.T. (ed.), Polysaccharides. Topics in Structure and Morphology. Macmillan, Southampton, UK, pp. 107139.
Ornitz,D.M., Herr,A.B., Nilsson,M., Westman,J., Svahn,C. and Waksman,G. (1995) FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides. Science, 268, 432436.[ISI][Medline]
Ragazzi,M., Ferro,D.R. and Provasoli,A. (1986) A force-field study of the conformational characteristics of the iduronate ring. J. Comp. Chem., 7, 105112.[ISI]
Ragazzi,M., Provasoli,A. and Ferro,D.R. (1990) Molecular mechanics and the structure of iduronate constaining carbohydrates. In French,A.D. and Brady,J.W. (eds.), Computer Modeling of Carbohydrate Molecules. Vol. 430. American Chemical Society, Washington, DC, pp. 332344.
Rao,V.S.R., Balaji,P.V. and Qasba,P.K. (1995) Controversial iduronate ring conformation in dermatan sulfate. Glycobiology, 5, 273279.[ISI][Medline]
Scott,J.E. and Tigwell,M.J. (1978) Periodate oxidation and the shapes of glycosaminoglycuronans in solution. Biochem. J., 173, 103114.[ISI][Medline]
Sugeta,H. and Miyazawa,T. (1967) General method for calculating helical parameters of polymer chains from bond lengths, bond angles and internal-rotation angles. Biopolymers, 5, 673679.[ISI]
Ueda,K. and Brady,J.W. (1996) The effect of hydration upon the conformation and dynamics of neocarrabiose, a repeat unit of ß-carrageenan. Biopolymers, 38, 461469.[ISI][Medline]
van Gunsteren,W.F. and Berendsen,H.J.C. (1977) Algorithms for macromolecular dynamics and constraint dynamics. Mol. Phys., 34, 13111327.[ISI]
Venkataraman,G., Sasisekharan,V., Cooney,C.L., Langer,R. and Sasisekharan,R. (1994) A stereochemical approach to pyranose ring flexibility: its implications for the conformation of dermatan sulfate. Proc. Natl. Acad. Sci. USA, 91, 61716175.[Abstract]
Verlet,L. (1967) Computer experiments on classical fluids. I. Thermodynamical properties of lennard-jones molecules. Phys. Rev., 159, 98103.[ISI]
Winter,W.T., Taylor,M.G., Stevens,E.S., Morris,E.R. and Rees,D.A. (1986) Solid-state 13C NMR and x-ray fiber diffraction of dermatan sulfate. Biochem. Biophys. Res. Commun., 137, 8793.[ISI][Medline]