| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 49, 49261-49270, December 5, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
¶**
From the
MRC Immunochemistry Unit, the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and the ||Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
Received for publication, August 29, 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-1,3-N-acetylglucosamine--1,4-; up to 107 Da) that, unlike other glycosaminoglycans, is neither attached to a core protein nor sulfated. This functional complexity is thought to arise from the interaction of HA with a large number of specific HA-binding proteins (7), which can form structurally diverse complexes (see Ref. 8). The majority of these "hyaladherins" belong to a superfamily of proteins that share a common 
100 amino acid domain, termed a Link module, that mediates the interaction with HA.
Previously we have determined the solution structure of the Link module from human TSG-6 (the protein product of the tumor necrosis factor-stimulated gene-6 (9)), thereby defining the consensus fold for this superfamily (10). In TSG-6, a 35-kDa secreted protein composed mainly of contiguous Link and CUB modules, the Link module is sufficient to mediate a high affinity interaction with HA (10, 11); this has been termed a "type A" HA-binding domain (7). The HA receptor CD44, which has an important role in mediating lymphocyte migration, however, requires N- and C-terminal extensions to its Link module for correct folding and functional activity of its type B interaction domain. Most other members of the superfamily, such as link proteins and chondroitin-sulfate proteoglycans (critical for extracellular matrix organization; see Ref. 12), have larger HA-binding domains containing two tandem Link modules (7).
TSG-6 is not constitutively expressed in normal adult tissues but is produced during inflammatory disease (13), e.g. in the joint tissues of arthritis patients (14, 15). Recently it has been found that TSG-6 protects against cartilage matrix destruction (16, 17) and has anti-inflammatory activities (18) in mouse models of arthritis; the Link module alone is a potent inhibitor of neutrophil migration in vivo (19). These studies suggest that TSG-6 is an endogenous component of a negative feedback loop capable of down-regulating the inflammatory response (13). TSG-6 is also expressed in inflammation-like processes such as ovulation (20) and deletion of the TSG-6 gene (4), or decreased TSG-6 expression (21) cause female infertility in mice.
Significant progress has been made in the characterization of the HA-binding properties of the TSG-6 Link module (termed Link_TSG6). Thermodynamic studies of the interaction between Link_TSG6 and defined oligomers of HA by isothermal titration calorimetry (ITC) indicated that an octasaccharide (HA8) was close to the minimum length that bound optimally to the protein (11). NMR spectroscopy on Link_TSG6, in the absence and presence of HA8 (11), and site-directed mutagenesis (22) have been used to identify the position of the HA-binding site. However, to date it has only been possible to map these data on the structure of the free Link module (10), which does not provide a clear picture of how HA is recognized by the protein.
Here we have determined the structure of the TSG-6 Link module in its HA8-bound conformation. This has revealed a well defined HA-binding groove containing all the amino acids implicated previously in binding. Comparison with a de novo calculated structure of the free protein demonstrates that a small but significant ligand-induced conformational change occurs on interaction with HA, switching the Link module from a closed to an open state. The minimum length of HA that can be accommodated within the binding site and its polarity in the groove have been identified. These structural studies provide valuable new insights into the function of TSG-6 and the Link module superfamily in general.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
NMR Data Collection—All NMR experiments were performed at 25 °C on spectrometers operating at 500, 600, or 750 MHz. Assignment used the spectra recorded previously (1H,15N-HSQC, HCCH-TOCSY, HNCA, HN(CO)CA, and CBCA(CO)NH; see Ref. 11) supplemented with HNCO, C(CO)NH, HC(CO)NH and 26 and 13 ms constant time 1H,13CHSQC spectra of the aromatic region, acquired on the same samples (i.e. 2.8 mM13C,15N-Link_TSG6, 2 mM 13C,15N-Link_TSG6/HA8). Two-dimensional 1H,1H-TOCSY spectra were recorded on 1 mM samples in D2O at 750 MHz (mixing time of 46 ms). The nitrogen carrier frequency was routinely set to 119.0 ppm.
NOESY data sets were recorded on samples of free and HA8-bound Link_TSG6. 1H,15N-NOESY-HSQC (600 MHz; with an NOE mixing time of 60 ms) and 1H,15N-HSQC-NOESY-HSQC (750 MHz; 70 ms mixing time) spectra were acquired on samples of 2 mM15N-Link_TSG6 (with and without HA8). 1H,13C-NOESY-HSQC data sets were recorded at 600 MHz on the 13C,15N-labeled samples described above, and 1H,1HNOESY spectra were collected at 750 MHz on the 15N-labeled samples (2 mM in water or 1 mM in D2O) with mixing times of 80 ms. The observation of amide exchange was performed at 500 MHz on 0.4 mM 15N-Link_TSG6 samples (±HA8; buffered with 2 mM MES, pH 6.75) with 1H,15N-HSQC experiments recorded at 105-min intervals after the addition of D2O to the lyophilized material.
1H,15N-HSQC spectra were recorded (500 MHz) on 15N-Link_TSG6 in the absence and presence of equimolar concentrations of HA oligosaccharides (HA6,HA7,HA8, and HA10 at1mM;HA4 and HA5 at 0.4 mM because of limiting amounts of sugar). A data set with HA4 at a 10:1 ratio was recorded, and a comparison of the chemical shift perturbations from four resonances in fast exchange at the 1:1 and 10:1 ratios was used to estimate the Kb; the majority of resonances are in slow exchange in the Link_TSG6-HA4 complex, whereas all resonances are in slow exchange with the longer oligomers. This estimate is based on the assumption that in the latter case (10:1 ratio) all the protein was in the complexed state and that only one HA4 can bind per Link_TSG6. 1H,15N-HSQC experiments with 15N-labeled oligomers (15N-HA6 0.6 mM, 15N-HA8 0.3 mM) were performed (with 15N offset at 122.5 ppm) in the absence and presence of unlabeled protein (1:1 stoichiometry).
Data Processing and Structure Calculations—Data were processed using FELIX 2.3 (Biosym Inc.) and referenced and analyzed with XEasy as described previously (11). NOE intensities for each data set were calibrated using interproton distances in regions of secondary structure and converted into three distance restraint categories with limits of 2.7, 3.5, and 5.0 Å. These restraints, with 13C
and 13C chemical shift values, were used in the program CNS version 0.9 in the ab initio-simulated annealing protocol described previously (26); H-bonds were included as two restraints toward the end of the structure calculations. A total of 250 structures was calculated for both the free and HA8-bound Link_TSG6 by using identical protocols, and in each case the 30 with the lowest energy were refined using an additional cycle of simulated annealing, followed by extensive restrained energy minimization. The resulting 20 lowest energy structures were deposited at the Protein Data Bank with accession codes of 1o7b
[PDB]
and 1o7c
[PDB]
for free and HA8-bound Link_TSG6, respectively. Figures were prepared using MOLMOL, POV-Ray, and RASMOL.
Model Building—Molecular modeling calculations were performed using CHARMm version 28b2 (27). The coordinates of the lowest energy protein structure (in its HA8-bound conformation) were fixed throughout the docking simulation; Arg81 was replaced by an Ala residue because its side chain was not particularly well defined in the family and may have caused steric interference with ligand insertion. An HA8 molecule was built into the binding groove based on the inferred polarity and register (see below). Energy minimization of the HA8 molecule was performed on the basis of van der Waals contacts and internal energies calculated from the force field to obtain the final model. The glycosidic bond angles for the 8-mer were close to those predicted for free HA (28).
Isothermal Titration Calorimetry—The interactions between Link_TSG6 and HA oligomers of different lengths were investigated on a Microcal VP-ITC instrument at 25 °C in 5 mM MES, pH 6.0, as described previously (19, 22). Oligosaccharide solutions (ranging from 180 to 870 µM, determined on the basis of the accurately determined protein concentration and known stoichiometry as described previously (11)) were added in 5-µl injections (28 in total) to protein (ranging from 10.0 to 58.6 µM). Data were fitted to a one-site model by nonlinear least squares regression with the Origin software package, after subtracting the heats resulting from the addition of oligosaccharide into buffer alone. Affinities for the interaction with HA6 and HA8 were determined by averaging results from 5 or 10 experiments, respectively, whereas all other sugars were analyzed at least twice.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
25% of the total populations) were not included in the structure calculations.
Solution structures of Link_TSG6 in both its free and HA8-bound states were generated completely independently using NOE restraints, 13C/13C chemical shifts, and H-bonds identified on the basis of hydrogen exchange data (Fig. 1 and Table I). As can be seen from Fig. 2 (and Table I) the lowest energy structure families are well defined, with backbone r.m.s.d. values over amino acids 2–94 (i.e. excluding N- and C-terminal "tails") of 0.49 and 0.53 Å for free and HA8-bound protein, respectively.
|
|
|
The Link module fold, which is identical in both free and HA8-bound forms of Link_TSG6 determined here (see Fig. 2, B and D), is composed of two triple-stranded anti-parallel -sheets (SI (1, 2, and 6) and SII (3, 4, and 5)) and two -helices (1 and 2); disulfide bridges connect 1to 6 (Cys23–Cys92) and the irregular loop following 2 to the long loop between 4 and 5 (Cys47–Cys68). As can be seen from Fig. 3 the SI -sheet and 1-helix are identical to those described before (10). There are, however, subtle differences in the definition of the SII sheet (i.e. 3, 4, and 5 correspond to residues 49–52, 56–61, and 74–77, respectively, rather than 49–51, 56–60, and 75–81 (with a bulge at 77–79) reported previously (10)). In addition, the 2-helix was incorrectly orientated in our original structure and corresponds to residues 33–42 (instead of 36–41); slowly exchanging amides support the presence of caps at both N (Thr32–Gln35) and C termini (Schellman motif (29), Gly43). The proposed N-cap on 1 (Thr15–Glu18 (10)) has been confirmed, but the C-terminal Schellman motif does not appear to be present. Consistent with this, these three cap motifs are highly conserved across the Link module superfamily, whereas an 1 Schellman motif is not (see Fig. 3). Slowly exchanging amide protons were also observed for Trp88 and Leu14, which H-bond to each other in an antiparallel arrangement. Leu14 packs behind 1 and against the side of the 2-helix; a large hydrophobic residue is conserved at this position across the Link module superfamily (Fig. 3). These interactions orientate the 1/1 loop (which contains the HA-binding residues Lys11 and Tyr12 (22)) and could constitute an additional, short -element denoted here as "1a" (see Fig. 3 and Supplemental Material Fig. S1).
|
As shown in Fig. 4A, the majority of residues within Link_TSG6 has the same structure in free and bound states. The backbone of the secondary elements (defined above) overlay between the 20 free and 20 bound structures with a r.m.s.d. of 0.54 Å, barely greater than that of the families individually (see Table I), and critical core residues such as Tyr91 and Tyr93 (Fig. 4A) occupy identical positions in the overlaid 40 structures. Clearly, there is no gross alteration to the Link module structure on its interaction with HA.
|
1-strand and 1-helix (Fig. 4C), whereas Tyr59, Phe70, and Tyr78 are in an adjacent region comprising the 4 and 5 strands and the long loop connecting them (Fig. 4B). This face also contains Trp88, which has a different side chain orientation in the free and bound structures (Fig. 4A).
|
0.25 ppm was introduced for the aromatic carbons. When these shift perturbations are mapped onto the structure of Link_TSG6 (in its HA8-bound conformation), it can be seen that the corresponding nuclei line a shallow groove on the protein surface that contains the key functional residues determined by mutagenesis (Fig. 5).
As noted above, Lys11, Tyr12, Tyr59, Phe70, and Tyr78 have been identified as key HA-binding residues (22). For example, Lys11 has been hypothesized to form a salt bridge to a carboxylate group of glucuronic acid (mutation to Gln causes a 20-fold reduction in binding affinity (22)); consistent with this the C moves by 0.80 ppm on binding. Arg81, at the opposite end of the binding groove from Lys11 (see Fig. 5D), could also be directly involved in such an interaction because it experiences chemical shift perturbations only at N
(0.29 ppm), C (1.40 ppm), and C (2.90 ppm) nuclei (i.e. toward the end of the side chain). In this regard, ITC experiments performed at various NaCl concentrations indicate that Link_TSG6 makes between 1 and 2 salt bridges with HA8 (30). Given that other basic residues in the proximity of the binding site (e.g. Lys13 and Lys72) have already been shown to play no functional role (see Ref. 22), Lys11 and Arg81 are the best candidates. Unfortunately, it has not yet been possible to generate a folded protein with a mutation at Arg81 to test this hypothesis directly.
Slowly exchanging H
hydroxyl protons are observed on both Tyr12 and Tyr78 in HA8-bound Link_TSG6, although these are clearly in rapid exchange in the free protein. Given their apparent solvent-exposed position in the bound structure, it is likely that these hydroxyl protons are stabilized by making direct hydrogen bonds to the HA. This is consistent with the observation that mutation of Tyr12 or Tyr78 to phenylalanine reduces binding affinity by 100- and 16-fold, respectively (19). Replacement of Tyr59 with Phe also results in a large reduction in the binding constant (25-fold (22)). This residue, however, may be acting as a hydrogen bond acceptor as the hydroxyl proton has not been observed in the Link_TSG6-HA8 complex.
Discounting amino acids that appear to have chemical shift perturbations arising mainly from changes in backbone conformation (see below), rather than direct contact with the ligand, the HA-interaction surface is thus generated by Lys11, Tyr12, Val57, Tyr59, Pro60, Ile61, Phe70, Ile76, Tyr78, Arg81, and Trp88 (i.e. the residues that form the binding groove).
Evidence of HA-induced Conformational Change—The extensive nature of the perturbations throughout the 4-5 loop (i.e. the lobe with Phe70 at the top; see Fig. 5) is consistent with an HA-induced conformational change in this region (see Fig. 4, A and B). Although this loop (amino acids 62–73) is the least well defined part of the structures (see Fig. 1), there are sufficient distance restraints to define confidently the conformational change (i.e. an average of 7.3 and 9.3 NOEs per loop residue in the free and HA8-bound forms, respectively, with a total of 120 NOE differences between them in this region). The 1-1 loop also undergoes a subtle but significant rearrangement on HA binding (Fig. 4C).
The side chains of the key functional residues assume different positions in the free and bound structures (Fig. 4, B and C), as do other residues that line the binding groove (such as Trp88; Fig. 4A), due to distinct differences in observed NOEs (i.e. 14, 25, 45, 14, and 12 NOE differences for Lys11, Tyr12, Tyr59, Phe70, and Tyr78, respectively). For example, Lys11 not only changes its orientation but also becomes ordered on HA binding (Fig. 4C). Tyr59, which is extremely well resolved in both free and bound structures, lies flat against the protein surface in the bound state. In free Link_TSG6, only one chemical shift is observed for each of the H (6.54 ppm) and H (6.45 ppm) pairs of ring protons, due to rapid rotation of the ring averaging their chemical environments. On binding, however, both H and H chemical shifts are significantly perturbed, and the H protons become distinguishable (H1 6.26 ppm, H2 6.16 ppm), implying that the ring is no longer able to rotate (at least on the NMR time scale). This could be accounted for by a stacking interaction of Tyr59 against a sugar ring in HA, as has been observed in the crystal structures of hyaluronate lyases in complex with HA oligosaccharides (31, 32). This is also likely to be the case for Tyr78, which becomes significantly ordered on binding (Fig. 4B), lies flatter against the protein, and exhibits distinct shifts for its H protons (6.35 and 5.91 ppm).
Movement of the 4-5 Loop Opens the HA-binding Groove—As the aromatic rings of Tyr59 and Tyr78 become flat against the protein surface on HA8 binding, the 4-5 loop (containing Phe70) retracts away from them (see Fig. 4, A and B). These, and other rearrangements, such as the movement of Trp88 and ordering of Lys11, combine to open a previously closed groove on the surface of the protein (Fig. 6). The loop is effectively hinged at either end (Pro60 and Gly74) and is opened by a change in the geometry of the disulfide bridge between Cys47 and Cys68 (Fig. 6, C and D). Rotation occurs around the Cys47 
and 
1 bonds, and the side chain chemical shifts of this amino acid exhibit large differences in the free and bound states (C 3.14 ppm, H1 0.27 ppm, and H2 0.31 ppm). Dynamics experiments clearly indicate that the 4-5 loop is stabilized significantly on HA binding2; for example, the side chain of Asn67 becomes much less dynamic upon binding (11) even though it does not appear to be directly involved in the interaction with HA (22). Fig. 6 (E and F) shows a simple docking model illustrating that the open groove is of a size and shape (20 Å long, 10 Å wide, and 10 Å deep) that would allow good intermolecular van der Waals contacts and favorable glycosidic 
, angles in a bound HA molecule.
|
protons of Tyr12 and Tyr78 are visible in the Link_TSG6-HA7 complex but are not seen with the shorter oligomers.
|
/ anomerization). Because there is good evidence that several highly specific contacts are made between HA and Link_TSG6 (see above), it is likely that this glycosaminoglycan will only be able to bind in one orientation relative to the protein. Therefore, a series of NMR experiments was performed to determine the polarity of HA within the binding groove.
1H,15N-HSQC spectra were collected for 15N-labeled Link_TSG6 in the absence and presence of unlabeled HA oligosaccharides of different length (i.e. HA4, HA5, HA6, HA7, HA8, and HA10). The pattern of chemical shift perturbations caused by the interaction of HA and Link_TSG6 (i.e. the "shift map") is extremely similar for all of these oligosaccharides (see Supplemental Material Fig. S4A). This indicates that the various HA oligomers all bind into the same site on the protein surface and cause a similar conformational change in the 4-5 loop. However, there are some discrete differences in the perturbations seen for particular residues (see Fig. 7A) that are likely to be due to differences in the structures of the oligosaccharides or their register within the binding groove.
|
group of Arg81 has two resonances in the presence of HA10 (one at the HA8 position; see Supplemental Material Fig. S4B); this "split" population probably arises from HA10 binding in two different registers (see schematic model in Fig. 7B), where differential end effects (e.g. in dynamic mobility) may account for the observed differences in chemical shift. Thus, it seems likely that HA8 is able to completely fill the binding site, in agreement with the ITC data. Therefore, the HA8 shift map was used as a reference against which the other oligomers were compared.
The only difference seen between the 7- and 8-mers (which have similar binding affinities; Table II) is a larger perturbation of the side chain HN group of Arg81 (R81sc) in the presence of HA7 (see Fig. 7A, panel 1). Clearly, the eighth GlcNAc ring is absent in HA7, and thus its reducing terminus is now on the GlcUA ring 7. It seems likely that the difference in the perturbation of R81sc seen with HA7 results from the proximity of this terminal GlcUA and may be caused by its / anomerization (see Fig. 6B). As noted above, the perturbation of R81sc seen with HA8 (Fig. 6A) is probably caused by the formation of a salt bridge between the arginine and the carboxylate group of glucuronic acid. In addition HA5, which has a reducing terminal GlcUA, causes a larger perturbation of R81sc than HA8 (Fig. 7A). Therefore, in HA5 this terminal sugar ring is also likely to be proximal to Arg81. In contrast HA4 and HA6 do not cause significant perturbations of the R81sc from its position in the free protein, indicating that they both lie in the binding groove in such a way that a GlcUA ring is not close to Arg81 (i.e. they are unable to make a salt bridge); this might account for the weaker binding of HA6 to Link_TSG6 (7% of the HA8 affinity). However, HA4 and HA6 cause a large perturbation in the backbone amide of Tyr78 not seen with the other oligosaccharides (see Fig. 7A, panel 2). This indicates that the reducing terminal GlcNAc rings of these oligomers are proximal to the backbone NH of Tyr78 and thus cause its perturbation due to their / anomerization (Fig. 7B). In this regard, it can be seen from the spectra of 15N-labeled HA6 (Fig. 7A, panel 4) that the characteristic chemical shifts of the - and -anomers move from their free positions on binding Link_TSG6, showing that this terminal sixth ring is in intimate contact with the protein. However, in 15N-HA8 there are no perturbations of these resonances in the presence of protein (Fig. 7A, panel 5), indicating that the eighth ring does not make significant contacts with the Link module (Fig. 7B).
As described above, the differential perturbations seen for Tyr78 and Arg81 with the different lengths of HA oligomer can all be explained on the basis of the positions of the reducing terminal sugar rings. These amino acids are located at one end of the HA-binding groove (see Fig. 5) providing clear evidence for the polarity of HA relative to the Link module. This is illustrated in the schematic model shown in Fig. 7B, which also shows the registers of the various oligosaccharides. In this model Lys11, which is at the other end of the binding groove from Arg81, is positioned so that it can interact with ring 3 of HA8 (i.e. a glucuronic acid), which is reasonable given the separation of these residues (17.4 Å from Lys11 N
to Arg81 C, respectively, in the lowest energy bound structure) and the likely distance between the carboxylates on rings 3 and 7 (20 Å).
It has been noted previously that mutation of Glu6 (colored green on Fig. 6) to lysine causes 4-fold increase in the Link_TSG6 binding affinity for HA8 (19). This observation can now be explained in light of our alignment model (Fig. 7B); a lysine at this position could form an additional ionic interaction with the GlcUA at ring 1, effectively extending the binding site.
Based on the similarity of the shift maps, all the oligosaccharides tested can be concluded to cause a conformational alteration on binding to Link_TSG6 (Supplemental Material Fig. 4A). However, the extent of this ligand-induced conformational change may differ with the size of HA. As can be seen from Fig. 7A (panel 3), HA6, HA7, and HA8 all cause an identical large perturbation of the backbone amide resonance for Lys63, whereas HA4 and HA5 have a smaller effect; this is also apparent for other residues on the 4-5 loop (data not shown). Therefore, a 6-mer is the minimum size of HA that can induce a full conformational change in the protein, and it is likely that the smaller oligomers probably generate intermediate conformational states.
The Link Module Is Related to the C-type Lectin Domain—An automated search of the DALI data base (33) with the free Link_TSG6 coordinates identified 15 other structures with similar folds (Z score 2.0); the greatest similarities were seen for human eosinophil granule major basic protein (Protein Data Bank code 1h8u
[PDB]
, Z = 5.0), invasin (Protein Data Bank 1cwv
[PDB]
, Z = 4.5), intimin (Protein Data Bank 1f00
[PDB]
, Z = 4.1), E-selectin (Protein Data Bank 1esl
[PDB]
, Z = 3.9), and macrophage mannose receptor (Protein Data Bank 1egg
[PDB]
, Z = 3.7). The matching regions of these 15 structures all correspond to C-type lectin-like folds (in most cases from proteins known to interact with carbohydrate), confirming its structural similarity to the Link module noted previously (10, 34). The highest scoring match is with eosinophil granule major basic protein (EMBP), which is clearly a member of the C-type lectin superfamily (on the basis of sequence), but does not have a typical Ca2+/carbohydrate-binding site (35, 36). Interestingly, EMBP has been shown to interact with the sulfated glycosaminoglycan heparin, and the basic residues implicated in binding (35) are found on an equivalent face of the protein as the HA-binding site in the TSG-6 Link module. It is possible therefore that EMBP and TSG-6 constitute a subgroup of C-type lectins that interact with glycosaminoglycans in a Ca2+-independent manner. As noted previously (10), the Link module lacks the long Ca2+-binding loop found in classical C-type lectins, and this is also absent in invasin and intimin, cell adhesion molecules from enteropathogenic bacteria (37, 38).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
25% of the free energy of HA8 binding to Link_TSG6 comes from ionic interactions at physiological salt strengths (30). This is consistent with our data from mutagenesis suggesting that Tyr12, Tyr59, and Tyr78 have important roles in mediating HA binding (19, 22), and the finding here is that these highly conserved aromatic residues line the binding groove. Our NMR data provide evidence that Tyr59 and Tyr78 are likely to be involved in stacking interactions, in which the flat plane of the aromatic ring aligns with a hydrophobic face of a saccharide, as commonly seen in protein-carbohydrate complexes (41, 42). Such interactions could contribute to the precise positioning of the HA molecule within the binding groove as has been noted in the structures of the streptococcal hyaluronate lyases (31, 32, 40). Phe70, which is on the top of the long 4-5 loop that changes conformation on interaction with ligand, may also stack against a sugar ring, closing over the bound HA molecule and clamping it in place. However, not all of the contacts with aromatic residues are likely to be ring-stacking interactions because the orientation of Tyr12 appears to preclude this, and this residue (as well as Tyr59 and Tyr78) appears to hydrogenbond to the sugar. NMR and calorimetric studies are consistent with a 7-mer (with terminal GlcUA sugars) being the minimum size of HA oligosaccharide that can make a complete interaction network with the protein.
As noted above, the TSG-6 Link module has a distinct conformation in its free state compared with that of the Link_TSG6-HA8 complex, and these are interchanged by rotation around the Cys47–Cys68 disulfide bridge (with the concomitant movement of the 4-5 loop). This disulfide is found in all Link modules except KIA0527 (34), and the residues that provide the hinges on which the loop moves (Pro60 and Gly74) are very highly conserved (Fig. 3), indicating that the conformational change seen for TSG-6 is likely to occur in most members of the superfamily. This could provide a mechanism for regulation of HA binding and may be relevant in CD44, because this receptor can clearly exist in different activation states (7).
Recently it has become apparent that TSG-6 has a crucial role in mammalian ovulation and fertilization via its stabilization of the nascent HA-rich matrix formed during the cumulusoocyte complex (COC) expansion (4, 21). One of the mechanisms underlying this stabilization appears to be the formation of covalent complexes between TSG-6 and the heavy chains (HC) of inter--inhibitor (20). These HC-TSG-6 complexes can become firmly associated with HA and may function as matrix cross-links through HA binding to TSG-6. Furthermore, HCTSG-6 complexes also act as intermediates in the covalent transfer of the HCs to HA (4),3 which become associated via an ester linkage between the carboxylic acid group of C-terminal aspartic acids in the HC and a C-6 hydroxyl of GlcNAc residues in HA (43); it seems likely that HA binding to the TSG-6 Link module, in the context of the HC-TSG-6 complex, serves to orientate the HA in the correct position relative to the HC and may also activate the sugar, thus facilitating the transfer reaction (i.e. formation of the ester bond). Mice lacking these HC-HA or HC-TSG-6 complexes, due to impairment of either TSG-6 (4) or inter--inhibitor genes (44), are infertile because they are unable to incorporate HA into the COC extracellular matrix; the covalently linked HC may act as cross-links between HA chains. In addition to being produced during ovulation, HC-HA (and HC-TSG-6 (14)) complexes are also a feature of inflammation as they have been detected in the synovial fluids of patients with arthritis and may correlate with disease severity (45). It is clear therefore that the interaction of TSG-6 with HA has a fundamental role in both normal physiological and pathological processes.
The determination here of the structure of the TSG-6 Link module in its HA-bound state provides important new insights into the molecular basis of HA binding and will greatly facilitate further studies to determine the mechanism underlying the HC transfer reaction. This structure will also allow homology modeling of other Link module-containing proteins in their active conformations, thus aiding identification of important functional residues in their HA-binding sites.
Not all of the functions of TSG-6 are dependent on its interaction with HA; for example, its inhibition of neutrophil migration, an activity encoded in the Link module domain, does not appear to be associated with HA binding (19). In this regard, the Link module has been shown to interact with many other molecules (13). The determination of a refined structure for the free Link module, described here, will be valuable in understanding the molecular basis of these activities and in mapping the binding surfaces for its other ligands.
|
FOOTNOTES |
|---|
* This work was supported in part by Arthritis Research Campaign Grants D0525, D0569, and M0625. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains Figs. S1-S4.
¶ Recipient of a Yamanouchi Research Institute scholarship.
** To whom correspondence should be addressed: MRC Immunochemistry Unit, Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, UK. Tel.: 44 1865 275349; Fax: 44 1865 275729; E-mail: tony.day{at}bioch.ox.ac.uk.
1 The abbreviations used are: HA, hyaluronan; GlcUA, glucuronic acid; HAn, an n-mer of HA; ITC, isothermal titration calorimetry; Link_TSG6, the recombinant Link module from human TSG-6; TSG-6, tumor necrosis factor-stimulated gene-6; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; MES, 4-morpholineethanesulfonic acid; HC, heavy chains; r.m.s.d, root mean square deviations; EMBP, eosinophil granule major basic protein.
2 C. D. Blundell and A. J. Day, manuscript in preparation.
3 M. S. Rugg, A. C. Willis, D. Mukohpadhyay, V. C. Hascall, E. Fries, C. Fülöp, and A. J. Day, manuscript in preparation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
, residues 62–73), which has a lower than average number of NOEs per residue (due to its high percentage of glycines (i.e. amino acids 65, 69, and 71) and its protrusion from the rest of the structure), exhibits the highest local backbone r.m.s.d.. In the free protein this region displays significant flexibility as determined from a 15N-{1H} NOE experiment, whereas in the complex it is considerably less dynamic.2 The C-terminal tail (residues 95–98) is more flexible than the family of structures and local backbone r.m.s.d. would suggest because only the major resonance assignment gave rise to NOEs, and so only this conformation is represented.
