Progress in Biophysics and Molecular Biology 114 (2014) 61e68

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Review

Solution NMR conformation of glycosaminoglycans Vitor H. Pomin* Program of Glycobiology, Institute of Medical Biochemistry Leopoldo de Meis, University Hospital Clementino Fraga Filho, Federal University of Rio de Janeiro, 255, HUCFF 4A01, Ilha do Fundão, Rio de Janeiro, RJ 21941-913, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 19 February 2014

Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structural, dynamical, conformational and intermolecular binding aspects of carbohydrates. Particularly in the field of conformation, NOE resonances, scalar couplings, residual dipolar couplings, and chemical shift anisotropy offsets have been the principal NMR parameters utilized. Molecular dynamics calculations restrained by NMR-data input are usually employed in conjunction to generate glycosidic bond dihedral angles. Glycosaminoglycans (GAGs) are a special class of sulfated polysaccharides extensively studied worldwide. Besides regulating innumerous physiological processes, these glycans are also widely explored in the global market as either clinical or nutraceutical agents. The conformational aspects of GAGs are key regulators to the quality of interactions with the functional proteins involved in biological events. This report discusses the solution conformation of each GAG type analyzed by one or more of the above-mentioned methods. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Chemical shift anisotropy Glycosaminoglycans Nuclear Overhauser effect Residual dipolar coupling Scalar coupling

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 Chondroitin sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Dermatan sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Heparin and heparan sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1. Introduction Glycosaminoglycans (GAGs) are sulfated polysaccharides composed of disaccharide repeating units whose structures vary according to families (Lindahl and Hook, 1978). The families are chondroitin sulfate (CS), dermatan sulfate (DS), heparin (Hp), heparan sulfate (HS), keratan sulfate (KS) and hyaluronic acid (HA). Structurally speaking, while CSs are composed of alternating

* Tel.: þ55 21 2562 2939; fax: þ55 21 2562 2090. E-mail addresses: [email protected], [email protected]. http://dx.doi.org/10.1016/j.pbiomolbio.2014.01.001 0079-6107/Ó 2014 Elsevier Ltd. All rights reserved.

3-linked N-acetyl b-D-galactosamine (GalNAc) and 4-linked b-D-glucuronic acid (GlcA) units, the closely related DS has 4-linked a-L-iduronic acid (IdoA) rather than b-D-GlcA (Fig. 1). This happens because of the C5 epimerization process during the biosynthesis of DS (Sugahara et al., 2003). Both CS and DS are highly sulfated. CS may bear 4-O- and/or 6-O-sulfonations at the GalNAc units, whereas DS can have 4-O- and 2-O-sulfonation respectively at GalNAc and IdoA units (Sugahara et al., 2003) (Fig. 1). Hp and HS share the same repeating disaccharide-composed backbone of [/4)-b-D-GlcA-(1 / 4)-a-D-GlcNAc-(1/] however, within different degrees of chain modifications (Rabenstein, 2002; Sasisekharan and Venkataraman, 2000) (Fig. 1). While HS is less

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V.H. Pomin / Progress in Biophysics and Molecular Biology 114 (2014) 61e68

Fig. 1. (A) Representative repeating disaccharide unit of chondroitin sulfate (CS) and dermatan sulfate (DS) structures. CSs are composed of alternating 4-linked b-D-glucuronic acid (GlcA) and 3-linked N-acetyl a-D-galactosamine (GalNAc) units. CS-A is mostly 4-sulfated at the GalNAc units while CS-C is predominantly 6-sulfated. CS-B, widely known as DS, has a-L-iduronic acid (IdoA) units rather than b-GlcA. The IdoA units in DS may contain 2-sulfation while the GalNAc units are mostly 4-sulfated. CS-D has sulfation at the GlcA and GalNAc units respectively at the 2- and 6-position. CS-E is majorly 2,4-di-sulfated at its GalNAc units. The OSCS is highly sulfated at all available hydroxyl sites such as those of the 2-, and 3-positions of the GlcA as well as of 4-, and 6-positions of the GalNAc units. (B) Representative repeating disaccharide unit of heparan sulfate (HS), and heparin (Hp) structures. Both are composed of alternating 4-linked uronic acid and 4-linked a-glucosamine (GlcNX) units. HS has b-D-glucuronic acid as its major uronic acid type, whereas Hp has a-Liduronic acid (IdoA). HS is frequently N-sulfated at the GlcNAc unit, with less extension of N-acetylation and just rare amounts of N-free (just the amino NH2 group), while Hp is predominantly composed of 2-sulfated IdoA together with N,6-di-sulfated GlcNAc units. Although the 3-O-sulfation at the GlcNAc unit occurs more often at Hp than HS, it still happens but rarely. (C) Representative repeating disaccharide unit of hyaluronic acid (HA) structure. It is composed of alternating 4-linked b-GlcA and 3-linked b-GlcNAc units. HA also known as hyaluronan is the only non-sulfated GAG. (D) Representative repeating disaccharide unit of keratan sulfate (KS) structure. It is composed of alternating 3-linked bgalactose (Gal) and 4-linked N-acetyl b-glucosamine units. KS can bear sulfation at the 6-position of each unit, although sulfation at GlcNAc occurs more often. In all panels the glycosidic bonds are indicated in ellipses, whereas monosaccharide types are indicated in rectangles.

processed by N-deacetylation/N-sulfation, which converts N-acetyl b-D-glucosamine (GlcNAc) into N-sulfo-b-D-glucosamine (GlcNS), and consequently less modified by both epimerization (GlcA into IdoA) and O-sulfonations (at the C2 position of IdoA, and C3 and C6 positions of GlcNS), Hp is extensively modified by these processes. This gives rise to a large number of structures for Hp (Sugahara and Kitagawa, 2002). On the other hand, HA and KS are the least processed GAGs. HA is structurally composed of alternating b-D-GlcNAc and b-D-GlcA units, with glycosidic linkages at positions C3 and C4, respectively (Fig. 1). HA is the only GAG which is not sulfated (Fig. 1) since no further chain modification after the polymerization process of HA backbone occur during its biosynthesis (Almond, 2007). KS is composed of 4-linked b-D-GlcA and 3-linked b-D-galactose (Gal) units (Fig. 1). The Osulfonations at the C6 positions of either monosaccharide are the only modification that occurs in KS chains. Usually, however, GlcA units in KS are more 6-sulfated than Gal units (Pomin et al., 2012) (Fig. 1). Biologically speaking, GAGs are highly relevant because of their ability of binding to a multitude of functional proteins (Hileman et al., 1998; Imberty et al., 2007; Spillmann and Lindahl, 1994). This leads to a great number of biological outcomes for GAGs including inflammation, coagulation, cell growth and tissue

development. The natural structural diversity, poly-anionic character and the typical location at the extracellular matrices are all important features that make GAGs extremely versatile in terms of functionality. GAG-protein interactions are key players of the most GAG-related biological functions (Gandhi and Mancera, 2008). The quality and nature of the GAG-protein complexes is not solely regulated by structural features of GAGs, like sulfation patterns or IdoA content, but ultimately driven by their conformations either free in solution or at bound-states (Hricovíni et al., 2001; Jin et al., 2009; Nieto et al., 2011, 2013). It is worth saying that the conformations of GAGs, and of their individual composing units, are intimately dependent on their intrinsic dynamic properties (Angulo et al., 2005). For instance, while GlcA, GalNAc and glucosamine (GlcNX) units are the least dynamic units because they adopt mostly the 4C1 chair conformation in solution, IdoA units are conversely way more flexible because they have the ability to undergo ring conformational changes in solution. Two chairs (4C1 and 1C4) and one skew-boat (2S0) conformers can be seen for IdoA ring, and for its 2-sulfated form (IdoA2S). In aqueous solution, the ratio of approximately 35:65% to 2S0:1C4 conformers, is observed for IdoA2S in HS and Hp chains (Jin et al., 2009; Nieto et al., 2011). The 4C1 conformation is detected just in very small amounts. The presence and content of IdoA2S units in Hp, HS, and

V.H. Pomin / Progress in Biophysics and Molecular Biology 114 (2014) 61e68

DS chains are very relevant to the biological activities of these GAGs. The conformational flexibility of IdoA2S is essential for the biological events of HS, Hp and DS as reported previously (Gandhi and Mancera, 2008; Hricovíni et al., 2001; Jin et al., 2009; Nieto et al., 2011; García-Mayoral et al., 2012; Nieto et al., 2013). In synthesis, understanding the conformational properties of GAGs and/or of their composing units in solution is an efficient path to also comprehend the underlying biological properties of these glycans. The 3D-structural properties of GAGs can be classically examined by three major analytical methods: (i) molecular dynamics (MD) which relies on computational calculations of molecules subjected to virtual simulations in which a series of parameters might be used as input for structural restraints; (ii) crystal structures obtained mainly from the complexes formed between GAGs and binding proteins; and (iii) liquid or media-aligned nuclear magnetic resonance (NMR) spectroscopy. Overall, NMR is the utmost reliable, advanced and information-rich technique for conformational studies. Lately, it has been giving a pivotal contribution in the conformational analyses of GAGs. This work aims to provide a compilation of the main findings generated so far regarding the solution NMR-assisted conformational aspects of GAGs and their derivatives. These conformational aspects are mostly analyzed by four NMR parameters: (i) nuclear Overhauser effect (NOE), (ii) scalar couplings (J), (iii) residual dipolar couplings (RDC), and (iv) chemical shift anisotropy (CSA). NOE signals are used to indicate through-space contacts of nuclei. NOE resonances are the base of biomolecular NMR for elucidation of the 3D structures of biomolecules, including carbohydrates (Pomin, 2012). Scalar coupling associated with the Karplus equation relationship infer the angles between coupled vicinal protons. This type of data is informative for analyses of sugar ring conformations (Pomin, 2012). RDC and CSA are obtained from anisotropic conditions, usually when the biomolecules are partially aligned. Both RDC and CSA offsets are supplemental data in conformational studies (Pomin, 2012). Besides these NMR methods, MD simulations usually restrained by NMR data input are frequently employed to provide glycosidic linkage dihedral angle values. These values are informative in terms of conformation. A review integrating the most up-to-date NMR-based conformation of each GAG type in solution is missing in the literature despite the growing importance of NMR spectroscopy and GAGs to the current glycomic era. This report aims to fill this lack in the field. 2. Chondroitin sulfate Usually, the GlcA units of CS molecules are conformationally found at their 4C1 form. Curiously, Maruyama and coworkers have detected additional conformations for this unit when it is fully Osulfonated (2,3-di-sulfated GlcA) (Maruyama et al., 1998). At 30 and 60  C, GlcA2,3S can also adopt the 1C4 and 2S0 conformations. This observation supports the conclusion that sulfation patterns of the CS-composing GlcA unit, at certain temperatures, are influential to the conformational population distribution of this residue within the polymer in solution (Maruyama et al., 1998). These conformations of GlcA might lead to differential biological responses. For instance, while the naturally occurring CS is a non-anticoagulant GAG, its fully O-sulfonated version is anticoagulant. Zsiska and Meyer (1993) studied the influence of positionally different sulfate groups on the conformation of differently sulfated and non-sulfated CS disaccharides. 1H- and 13C NMR data like 1He 1 H NOE, 1He1H rotating-frame nuclear Overhauser effect (ROE), 3 JH5-H6, and 13C-chemical shifts, were used to establish the correlation between conformational preferences and sulfation patterns.

63

The 3JH5-H6 coupling constants were obtained for all studied disaccharides and preferred rotamer populations were estimated based on parametrization of the Karplus equation (Karplus, 1959, 1963). The NOE- and ROE-based nuclear distances, especially those across the 1H1 with 1H4 or 1H3 of the adjacent units, were measured to predict geometries of the glycosidic linkages (Zsiska and Meyer, 1993). Taking scalar coupling and NOE/ROE datasets, the authors concluded that CS disaccharides bearing sulfation at 6position or at 4- and 6-positions together have a tendency to exhibit a small repulsive effect between 6-sulfation of GalNAc and the carboxylate group of the adjacent GlcA. On the other hand, the 4-sulfation alone does not affect the glycosidic linkage geometries. These results have led to the conclusion that 6-sulfation exerts more influence on the conformation of the CS disaccharides than 4-sulfation (Zsiska and Meyer, 1993). The small or lack of influence of the 4-sulfation on the overall conformation of CS, and derivatives, was also pointed in the work of Sattelle and co-authors (2010). In their work, the investigators have also used scalar coupling and NOE datasets to obtain 3D-structural information from some CS oligosaccharides. Fig. S1 illustrates the 25 lowest-energy 3D-models obtained for a non-sulfated CS hexasaccharide calculated based on a MD simulated annealing protocol using NOE-based distance as restraints (represented by dashed lines). From Fig. S1 averaged glycosidic linkage torsion angles were obtained and used comparatively with some published data about sulfated CS and non-sulfated HA oligosaccharides (Table 1). Based on Table 1 no big changes on the conformations of the GAG oligosaccharides can be seen. The results of NMR restrained MD simulation of 4-sulfated CS (Fig. 2A), non-sulfated CS (Fig. 2B) and HA (Fig. 2C) disaccharides indicated clearly minimal changes on the b(1 / 3) linkage geometries. In addition, the amino sugar types, GlcNAc in HA versus GalNAc in CS, also seem to present similar conformations in solution. Yu and coworkers used RDC and CSA offsets to determine the conformational preferences of a CS pentasaccharide of well-defined chemical structure. The structure studied is the following GalNAc6S(b1 / 4)-GlcA-(b1 / 3)-GalNAc4S-(b1 / 4)-GlcA-(b1 / 3)-GalNAc4S-ol. The acetyl groups were doubly labeled with carbon-13 through a series of chemical reactions to enhance sensitivity for the RDC- and CSA offset-based NMR experiments. Values of 13Ce13C RDCs and carbonyl carbon CSA offsets were obtained and they are shown at Table S1. Data of Table S1, although just used for the isotopically labeled acetyl groups, clearly gave structural information on the average orientation of this lateral group. To supplement this dataset, other sources of information such multiple 1He13C RDC values, inter-residue NOE-derived distances of some glycosidic bonds, and JH2-HN values were also generated (Table S2). Using the collection of data of Table S2 as structural restraints, especially the 1 He13C RDC values, the 3D-structural view of the CS pentasaccharide was successfully generated through a combination of the computational programs REDCAT (residual dipolar coupling analysis tool) (Valafar and Prestegard, 2004) and XPLOR-NIH. REDCAT was used to estimate alignment parameters. XPLOR-NIH was used to optimize and refine the structure through a simulated annealing protocol (Schwieters et al., 2003, 2006). The back-calculated RDC values and CSA offsets produced by REDCAT in the structure refinement stage used a three-structure-evaluation-process (Table S3) which has led to very refined values. The reducing end GalNAc unit was not used therein because of its high flexibility due to its open ring caused by a reduction reaction. The ten lowest energy structures obtained from XPLOR-NIH simulated annealing calculation are shown at Fig. S2. In the work of Yu and co-authors, there is also information about the glycosidic bond torsion angles for the final conformation of this CS pentasaccharide (Yu et al., 2007).

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V.H. Pomin / Progress in Biophysics and Molecular Biology 114 (2014) 61e68

Table 1 Comparison of glycosidic torsions of non-sulfated CS, sulfated CS and HA oligosaccharides (Sattelle et al., 2010). Torsion angleb

b(1 / 4)

b(1 / 4) ABS DCS6c

b(1 / 4)

b(1 / 4) ABS DCS6

4

73 117 67 124 98 174 69 180 79 111 70 120 80 110 71 116

e e 6 7 25 57 4 63 6 6 3 3 7 7 4 7

72 108 61 109 80 107 89 108 79 90 70 90 80 90 68 129

e e 11 1 8 1 17 0 7 18 2 18 8 18 8 20

Source (reference)

Structure/dpa

NMR/simulation (Sattelle et al., 2010)

Non-sulfated/6

NMR/simulation (Yu et al., 2007)

4-sulfated/5

4

X-ray fiber (Cael et al., 1978)

4-sulfated/4

4

X-ray chondroitinase B (Michel et al., 2004)

4-sulfated/2

d

j j j 4

j

Simulation (MM3) (Rodríguez-Carvajal et al., 2003)

4-sulfated/2

4

Simulation (CHARMM) (Almond and Sheehan, 2000)

Non-sulfated/4

4

NMR/simulation (Blanchard et al., 2007)

4-sulfated/8

4

NMR

HA/8

4

j j j j

a b c d

dp stands for degree of polymerization. Torsion angles are reported in units of degrees. ABS DCS6: absolute deviation from the non-sulfated CS hexasaccharide (Sattelle et al., 2010). The 4 and j values of non-sulfated CS hexasaccharide were average to facilitate comparison (Sattelle et al., 2010). 4 ¼ O5-C1-O1-CX and j ¼ C1-O1-CX-CXþ1.

3. Dermatan sulfate As stated earlier, DS has its backbone mostly composed of IdoA as the uronic acid type and GalNAc as hexosamine. Because IdoA units can be found in three different lowest-energy ring conformations in solution (1C4, 4C1, and 2S0) the conformational population distribution for this unit was investigated by Inuoe and coworkers (1990) for a series of DS oligosaccharides and their alditol derivatives. The work was carried out essentially on the basis of 3JH-H analyses at two temperatures, 20 and 80  C. The ring conformation distributions of both internal and terminal IdoA units in DS oligosaccharides were nearly equal regardless anomericity and temperature (Table 2). The non-reducing terminal IdoA units in the DS tetrasaccharides IdoA-GalNAc-IdoA-GalNAc and IdoAGalNAc-GlcA-GalNAc, and their alditols IdoA-GalNAc-IdoAGalNAc-ol and IdoA-GalNAc-GlcA-GalNAc-ol, have shown the equilibrium of the three conformers as expected (Table 2). Conversely the internal IdoA units have shown an equilibrium mixture of only 1C4 and 2S0 within different population percentages depending on structures (Table 2). Clearly, very little 4C1 proportions could be seen for internal IdoA units. The percentage of conformers changes again for internal IdoA units in a hexasaccharide (Table 2). Based on this work, the distribution conformation of internal IdoA units of DS hexasaccharides starts to be close to the distribution of the native DS polysaccharide, which is 60% 1C4, 4% 4C1, and 36% 2S0 (Inuoe et al., 1990). Besides showing that the conformation distribution of IdoA units are different according to their positions within the chain (internal versus terminal positions, Table 2), this work has also demonstrated that

2-sulfonation of DS influences just slightly the conformer population distribution of the IdoA unit in solution (Inuoe et al., 1990). The extensive work of Silipo et al. (2008) has employed a combination of NMR (scalar couplings, residual dipolar couplings, and ROE), with computational analyses (molecular modeling (MM), and MD) to investigate the solution conformation of a DS tetrasaccharide of well-defined chemical structure DHexA-(1 / 3)GalNAc4S-b(1 / 4)-IdoA-a-(1 / 3)-GalNAc4S-a/b. This DSderived tetrasaccharide has shown four major conformations in solution. Two of which are chemically different because of the anomeric influence of the reducing end GalNAc4S unit on the adjacent IdoA unit. The other two structures are consequences from the two major solution conformers (1C4 or 2S0) of the non-sulfated IdoA residue. The solution equilibrium of the interconvertible a4b anomers was measured to be 0.6:1 ratio. Ring conformer populations have been analyzed essentially by 3JH-H (Table S4), and inter-residual NOE or ROE contacts (Silipo et al., 2008). The GalNAc units were found exclusively at their 4C1 chair conformation, while the unsaturated uronic acid (DHexA) adopted predominantly the half-chair 1H2 conformation. The 1C4/2S0 ratio for the IdoA unit was measured as 4:1. The four structures generated from ROE-based distances combined with MD and MM data are presented in Fig. 3. From this picture, combined with RDC values, the resultant dihedral angles (4 and j) values were obtained (Table 3). Besides allowing improved refinement of the 3D-structures of the four DS conformations in solution, one-bond CeH RDC (1DCH) and threebond HeH (3DHH) RDC values were also used to validate the conformer population distributions for the IdoA unit (Silipo et al., 2008). As opposed to the highly dynamic behavior of DS, as one

Fig. 2. Comparison of the NMR-based b(1 / 3) linkage geometries for (A) chondroitin 4-sulfate, (B) non-sulfated CS, and (C) HA. Geometries from (A) Yu et al., 2007, (B) the lowest energy 3D-model of non-sulfated CS hexasaccharides from Sattelle et al. (2010), and (C) Almond et al., 2006. For clarity, all hydrogen atoms are hidden except for OH-4. Indicated distances ( A) between GalNAc-GlcNAc OH-4 and GlcA O5 atoms are represented by dotted lines. Modified with permission (Sattelle et al., 2010).

V.H. Pomin / Progress in Biophysics and Molecular Biology 114 (2014) 61e68 Table 2 IdoA conformer populations for DS disaccharides, tetrasaccharides, alditol derivatives, and hexasaccharides in solution, based on 3JH-H analysis (Inoue et al., 1990). Structure

Anomer Temp. Proportion of conformers (%) ( C) Internal IdoA Non-reducing IdoA 2

IdoA-GalNAc

IdoA-GalNAc-IdoA-GalNAc

GlcA-GalNAc-IdoA-GalNAc IdoA-GalNAc-GlcA-GalNAc IdoA-GalNAc-ol

a b a b a b a b a b a/b

IdoA-GalNAc-IdoA-GalNAc-ol GlcA-GalNAc-IdoA-GalNAc-ol IdoA-GalNAc-GlcA-GalNAc-ol IdoA-GalNAc-IdoA*-GalNAc- a/b IdoA-GalNAc

20 20 80 80 20 20 80 80 20 20 20 20 80 20 80 20 20 20

S0

42 42 26 28 44 44

1

C4

54 54 52 52 54 54

4

C1

4 4 22 20 2 2

66 50 66

28 34 28

6 16 6

38

50

12

Table 3 (a) Average values of dihedral angles (F and J) directly obtained from the MD simulation. (b) Best-fit average dihedral angle values from the RDC-based data, considering different sets of conformers. The set of conformers with the lowestenergy values of root-mean square deviation are given (Silipo et al., 2008). Chair and skew conformations refer to the IdoA unit. a/b configurations come from the reducing end unit. 4 ¼ O5-C1-O1-CX and j ¼ C1-O1-CX-CXþ1.

2

1

4

(a)

30 26 16 16 32 32 18 18

30 30 30 30 28 28 32 32

40 44 54 54 40 40 50 50

AeB

28 32 18 30 24

30 10 16 30 30

42 58 66 40 46

32 28

30 30

38 42

S0

C4

C1

65

F J

b-skew

a-skew

(b)

b-skew

a-skew

52.8 1

53.0 2.9

F J

50.3  7.6 6.2  11.3

51.9  7.3 3.5  11.4

47.9 13.8

47.6 24.4

F J

45.4  13.4 24.5  17.6

45.4  12.9 26.6  16.6

53.6 2.9 b-chair

53.6 1.2 a-chair

F J (b)

53.6  7.7 0.5  11.9 b-chair

52.7  8.2 6.1  12.0 a-chair

50.8 2.0

52.8 1.0

F J

48.2  9.1 0.0  9.6

47.8  8.5 4.13  9.4

43.8 17.7

43.5 20.1

F J

44.2  14.5 22.5  18.8

43.8  9.4 25.1  17.9

48.6 9.8

52.7 14.1

F J

48.3  7.8 13.0  8.7

50.9  6.7 16.3  7.5

BeC

F J CeD

F J (a) AeB

F J BeC

F J CeD

F J

The specific internal IdoA unit marked with the star (*) denotes the one analyzed for its conformer distribution.

should expect based on the great IdoA conformational mobility, the results from this work pointed more toward the conception of a moderate dynamic behavior for DS. This dynamic, although limited, is still driven by the IdoA unit. The glycosidic linkages of the central IdoA unit seemed to be the main contributor to the general motion of the DS tetrasaccharides as seen by MD analysis (Sillipo et al., 2008). 4. Heparin and heparan sulfate Hp is so far the mostly studied GAG type concerning its solution NMR 3D-structure (Mulloy et al., 1993, 1994; Mikhailov et al., 1996, 1997; Cros et al., 1997; Mulloy and Forster, 2000; Hricovíni and Bízik, 2007; Hricovíni et al., 2007; Rudd et al., 2007; Mobli and

Fig. 3. Representation of the four generated structures (combinations of a/b anomers plus the skew/chair conformers of the composing IdoA unit) of the studied DS tetrasaccharide. The possible inter- and intra-residue hydrogen bonds are also shown by dashed lines. (A) b-skew, (B) b-chair, (C) a-skew, and (D) a-chair conformations. The residues are labeled with the letters a-d for the following structure: DHexA-(1/3)GalNAc4S-b(1 / 4)-IdoA-a-(1 / 3)-GalNAc4S-a/b, starting from the non-reducing end. Modified with permission (Silipo et al., 2008).

Almond, 2007; Murphy et al., 2008; Jin et al., 2009; Hricovíni, 2011; Sattelle et al., 2013). In the classic references from Mulloy et al. (1993, 1994), the solution NMR conformation of Hp, a fully O- and N-desulfated re-N-acetylated derivative, and partially and fully O-desulfated derivatives were examined by scalar coupling constants, NOE-based protoneproton signal intensities, and NMRrestrained MM calculations to generate glycosidic dihedral angles of their lowest-energy conformers in solution (Table S5). Fig. S3 shows the final models of the unmodified (panels S3A and S3B), and the fully O- and N-desulfated re-N-acetylated Hp dodecasaccharides (panels S3C and S3D). These conformational models were generated assuming either 1C4 (panels S3A and S3C), or 2S0 (panels S3B and S3D) conformation of IdoA. Note the slight changes on the overall conformations of these structures regarding the two IdoA ring conformations. This indicates that no significant changes on the 3D-structural models of the Hp dodecassacharide can be noted in relation to the two different IdoA2S ring conformations (1C4 or 2S0). This lack of conformational influence of the IdoA2S conformers will be again seen by RDC-based analyses on a Hp tetrasaccharide, in the work of Jin as discussed further (Jin et al., 2009). Conversely, the presence or lack of sulfate groups has shown to make a greater influence on the conformation of the Hp models. Hence, chemical substitutions, like O-sulfation, seem to be influential to the overall Hp geometry more than the IdoA ring conformations (Mulloy et al., 1994). Nevertheless, N-substitution (sulfation or acetylation) exerts minimal influence on the conformation of the Hp derivatives (Table S5). This was also seen by experimental NOE-based inter-residue proton-pair distances measured for Hp and derivatives in the recent work of Zhang et al. (2008). As for DS, the IdoA ring conformation in Hp was shown to be quite dependent on its location within the backbone, in relation to its substitutions or adjacent residue’s substitutions (Ferro et al., 1990; Murphy et al., 2008). These works were accomplished by measuring J-coupling constant values of the IdoA units within different GAG types, including Hp oligosaccharides. Another work supporting the fact that conformational population distributions of IdoA units are sensitive to chemical substitutions, sulfation patterns and counterions, is the report from Rudd et al. (2007). In this

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work, the authors have investigated by NMR spectroscopy and synchrotron radiation circular dichroism, eight Hp disaccharides with distinct substitution patterns and with seven different cations as counterions. The conclusions were clear. Substitutions and cations are indeed able to induce significant conformational changes on the IdoA of Hp derivatives, although within different extensions depending on the chemical group, concentration and cation types (Rudd et al., 2007). This type of research was coincidently undertaken by Hricovíni (2011). In his work, Hricovíni has studied the effect of solvent and counterions upon the structure and thus protoneproton spinespin coupling constants of the Hp disaccharides. Hricovíni observed the same marked conformational influence of these components. Both works of Mikhailov et al. (1996, 1997) have used NOE, Jcoupling, iterative relaxation matrix approach, restrained MD simulations, and energy minimization calculations to distinguish the minor differences on the solution conformers of a Hp tetrasaccharide, and a Hp hexasaccharide, both of well-defined chemical structures. Final glycosidic bond dihedral angle values were obtained by calculations after using NMR-data as restraints. Both Hp hexasaccharide and tetrasaccharide had the same backbone constitution but, the dihedral angle values of the common residues were noted quite different. This indicates that Hp oligosaccharides of different lengths might have distinct averaged conformations in solution. Cros et al. used the combination of NMR (NOE and J-couplings) and MD analyses to investigate the conformation of a Hp trisaccharide. In their study, the IdoA2S moiety was proved to exhibit the three ring conformations in solution (1C4, 4C1, and 2S0). Curiously, dominance of the 2S0 conformation population was observed but, still in equilibrium with the 1C4 ring conformer (Cros et al., 1997). In contrast to the absence of changes in the Hp tetrasaccharide conformations as pointed out by Mulloy et al. (1993, 1994), the only reasonable explanation for the drastic change observed in the conformation of the Hp trisaccharide seen by Cros et al. (1997) would be the difference in length of the Hp fragments. As raised in the previous paragraph, the data discussed in this paragraph also go in favor of molecular-size effects on the overall conformations of the Hp fragments. Mulloy and Forster had deepened the conformational studies on Hp/HS in their classic publication of 2000 (Mulloy and Forster, 2000). Using solution-state NMR spectroscopy, fiber diffraction data, crystallographic data, and MM methods, the authors have shown that the solution 3D-structures of HS and Hp are indeed not affected by the IdoA ring conformer fluctuation. From this reference, they stated “heparin exhibits a well-defined overall shape within which iduronate ring forms can freely interconvert.” A more sophisticated representation (Fig. S3E and S3F) of the same Hp dodecasaccharide models previously studied by Mulloy and coworkers in 1993 (Fig. S3A and S3B) was generated in the newer reference (Mulloy and Forster, 2000). Note again the minimal influence of the two IdoA2S ring conformers on the overall conformations of the Hp oligosaccharides in solution. Taking the values of J-coupling constants from the references (Ferro et al., 1986; La Ferla et al., 1999), Table 4 can be built. The values displayed in this table concern the population distribution of the IdoA2S ring conformations as a function of structure and different oligosaccharide lengths, such as monosaccharides, oligosaccharides, and polysaccharides. Note that the population distribution of the different conformers 1C4, 2S0, and 4C1, although 4C1 at much lower percentage, changes accordingly to the sequences in which the IdoA2S units are found (Table 4). This table is not including the conformation percentages of the IdoA2S unit seen in intermolecular complexes with the mostly studied Hp-binding proteins such as antithrombin, fibroblast growth factors and

Table 4 Experimental (exp) and calculated (calc) three-bond protoneproton scalar coupling constant values (Hz) for the different composing units of Hp molecules (italic) and the percentage of the resultant conformers of the IdoA unit in solution. Unit/molecule

1

3

3

IdoA2SeOMe

H1eH2 H2eH3 H3eH4 H4eH5 H1eH2 H2eH3 H3eH4 H4eH5 H1eH2 H2eH3 H3eH4 H4eH5 H1eH2 H2eH3 H3eH4 H4eH5 H1eH2 H2eH3 H3eH4 H4eH5

1.8 3.3 3.4 2.2 2.3 4.6 3.4 2.6 3.6 11.4 9.5 10.4 4.0 7.5 3.6 3.1 2.6 5.9 3.4 3.1

1.8 3.2 3.3 2.4 2.4 4.4 3.1 3.2 3.4 11.3 9.5 8.9 3.5 7.4 3.9 3.3 2.7 5.6 3.6 2.9

GlcNS,6S-IdoA2SeOMe

GlcNS,6S

IdoA2S/pentasaccharide

IdoA2S/Hp

a b

Hn-1Hnþ1

JH-H (exp)a

JH-H (calc)b

1

S0

4

97

0

3

79

21

0

42

58

0

65

35

0

C4

2

C1

Ferro et al., 1986. La Ferla et al., 1999.

receptors. In complexes with these proteins, the conformation distribution of IdoA2S units can turn to approximately 100% 2S0 form, depending on the protein type (Nieto et al., 2011). This means that upon binding with certain proteins, selection or induction of the IdoA2S 2S0 conformation will occur (Nieto et al., 2011; Hricovíni and Torri, 1995; Jin et al., 1997; García-Mayoral et al., 2012; Nieto et al., 2013). The recent publication of Jin et al. (2009) has unequivocally proved the limited conformational flexibility of Hp in relationship to the convertible conformers of the IdoA unit. The authors from this work used 3JH,H and RDC measurements on a Hp tetrasaccharide of well-defined chemical structure (Jin et al., 2009). After sophisticated structural refinement and NMR-restrained MD simulations together with RDC-based calculations to obtain the principal axis frame respective to the order tensors, conformations were generated (Fig. 4). Note that only small changes on the overall conformation of the tetrasaccharides can be seen even considering the two IdoA ring conformers (1C4 at Fig, 4A versus 2S0 at Fig. 4B).

Fig. 4. Orientation of the principal axis frame of the order tensor related to the structure of the Hp tetrasaccharide [DUA-(1/4)-GlcNS6S-(1/4)-IdoA2S-(1/4)GlcNS6S, labeled as A to D from the reducing end] considering the IdoA2S (ring B) conformations (A) 2S0, and (B) 1C4 The relative length of the principal axis corresponds to the sizes of the principal order parameters obtained in the reference. Reproduced with permission (Jin et al., 2009).

V.H. Pomin / Progress in Biophysics and Molecular Biology 114 (2014) 61e68

This work strongly supports the findings of Mulloy et al. (1993 and Mulloy and Forster, 2000). 5. Hyaluronan In the publication of Holmbeck et al. (1994), the investigators have studied the solution conformation of HA octasaccharide on the basis of NOE data and restrained MD calculations. The distance of 2.6  A between GlcA H1 and H3 as observed by X-ray diffraction data (Winter et al., 1975) was used to relatively calculate the other NOE-based distances. These NOE-based 1He1H inter-nuclear distances were further used as conformational restraints to obtain dihedral angle (F and J) values from the MD simulated conformations. Based on these values, a helical propensity to the HA derivative was raised (Holmbeck et al., 1994). The helical 3D-structural shape of HA was also pointed in the work of Almond and coworkers (2006). From this work based on NOE signals, J-coupling values, and computational simulation by MD, conformations for unreduced HA tetrasaccharide, hexasaccharide and octasaccharides were generated (Fig. 5). From these conformations, a contracted left-handed 4-fold helix was noted for the HA fragments in solution. Another work supporting the left-handed helix conformational shape of HA is the recent publication of Gargiulo et al. (2010). In this work, the authors have used RDC measurements, and RDC-restrained MD simulations for a HA decasaccharide [/4-GlcA-1 / 3-GlcNAc-1/]5 (Gargiulo et al., 2010). From this study, the authors were able to assess two different arrangements for the HA decasaccharide. They were described as three- or four-folded left-handed helical structures. The former structural conformation seems to occur within higher percentage than the latter. Nonetheless, this reference again heavily supports the left-handed helical shape for HA molecules like the two

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previous references (Holmbeck et al., 1994; Almond et al., 2006). Based unanimously on these three reports, it is unquestionable the left-handed helical solution conformation for HA. However, a debate about how really contracted (three- or four-folded) is the helical conformation of HA molecules can still raise. Conflict of interest Although this document comprises a review in which most of the information sources are coming from already published materials, the use of the current information was totally done under legal procedures regarding the law of copyrights, reprints and permission. The author states that he is not aware of any authorship, affiliations, memberships, funding, or financial holdings that might be perceived as damaged or as affecting the objectivity of the content of this material. The author declares no conflict of interest by any part. Acknowledgments The author acknowledges the Brazilian financial agencies FAPERJ and CNPq for the respective grants E-26/110.961/2013, and Universal-14/2013-[470330/2013-9]. The content of this work is solely the responsibility of the author and does not necessarily represent the official views of the funding agencies. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pbiomolbio.2014.01.001 References

Fig. 5. Overlays of 40 structures extracted from simulations of HA (A) tetrasaccharide, (B) hexasaccharide and (D) octasaccharide in aqueous solution (b-anomers). (C) The distribution of conformers present in hexasaccharide from a dynamic structural characterization made on the basis of NOE measurements for comparison with (B). Modified with permission (Almond et al., 2006).

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Solution NMR conformation of glycosaminoglycans.

Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structura...
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