PDBsum entry 1o7c

Cell adhesion

PDB id: 1o7c

Contents

Protein chain

98 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The link module from ovulation- And inflammation-Associated protein tsg-6 changes conformation on hyaluronan binding.

Authors

C.D.Blundell, D.J.Mahoney, A.Almond, P.L.Deangelis, J.D.Kahmann, P.Teriete, A.R.Pickford, I.D.Campbell, A.J.Day.

Ref.

J Biol Chem, 2003, 278, 49261-49270. [DOI no: 10.1074/jbc.M309623200]

PubMed id

12972412

Abstract

The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations. This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand. The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions. This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily. NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.

Figure 2.
FIG. 2. Solution structures of the TSG-6 Link module in its free (A and B) and HA[8]-bound states (C and D). A and C, stereoviews of backbone traces for the family of 20 structures superimposed on the backbone heavy atoms in the secondary structure elements. B and D, secondary structure organization of the Link module, shown on the lowest energy structure of each family. The fold consists of two antiparallel -sheets SI (light blue; residues 2-6 ( 1), 29-31 ( 2), and 89-93 ( 6)) and SII (dark blue; residues 49-52 ( 3), 56-61 ( 4), and 74-77 ( 5)), connected in a parallel arrangement by two H-bonds between strands 3 and 6 (see Supplemental Material Fig. S1) and two helices (residues 16-25 ( 1) and 33-42 ( 2)) shown in red.

Figure 6.
FIG. 6. The interaction of HA with the TSG-6 Link module induces the opening of the binding groove. A and B, atomic spheres depiction of the lowest energy free (closed) and HA[8]-bound (open) structures, in the same orientation, with the bottom portion of each structure are shown in a ribbon representation. The conformational change of the 4- 5 loop opens a groove, exposing the key HA-binding residues (red); the binding site can be extended by mutation of Glu6 (green) to Lys, resulting in a higher affinity interaction with HA. The closed (A) and open (B) states differ principally in the geometry of the disulfide bridge (sulfur atoms in yellow) linking the 4- 5 loop (Cys68) to the rigid connection between 2 and 4 (Cys47), as shown by sticks in C and D. E and F, the open groove, which is lined with atoms that experience significant shift perturbations on ligand binding (red), can accommodate an HA octasaccharide (blue sticks and green atomic spheres) in a favorable geometry without serious steric clashes; one possible conformation of HA is shown. The polarity and register were determined as described in text (see Fig. 7). F is rotated 90° toward the reader around the horizontal axis relative to E.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 49261-49270) copyright 2003.

Added reference #1^*

Title

Towards a structure for a TSG-6.hyaluronan complex by modeling and NMR spectroscopy: insights into other members of the link module superfamily.

Authors

C.D.Blundell, A.Almond, D.J.Mahoney, P.L.DeAngelis, I.D.Campbell, A.J.Day.

Ref.

J Biol Chem, 2005, 280, 18189-18201. [DOI no: 10.1074/jbc.M414343200]

PubMed id

15718240

Figure 1.
FIG. 1. Models of HA[8] built into the binding groove of Link_TSG6. A, HA[8] constructed into the lowest energy protein structure; B, overlay of the family of 11 selected models, comparing the range of HA[8] conformations against the lowest energy protein structure. Left, protein shown in spheres, with HA[8] shown as blue sticks; right, solvent-accessible surface for both molecules (1.4-Å radius). Key binding residues on the protein are shown in red (aromatic) and green (basic).

Figure 2.
FIG. 2. A, the HA-binding groove of Link_TSG6 contains two hydrophobic pockets (I and II), which were found to accommodate the bulky GlcNAc acetamido side chains on rings 4 and 6, respectively, in the HA[8]-bound models. The solvent-accessible surface area of the lowest energy protein structure is shown, with the binding residues colored red and green and the heavy atoms of the HA[8] molecule shown as sticks. B, stereo view of residues forming pocket I (with bonds shown as green sticks and the disulfide bridge in yellow) shown clustered around the GlcNAc side chain of ring 4 (spheres). The heavy atoms of the HA[8] molecule and the two tyrosine residues forming stacking interactions (Tyr59 and Tyr78) are shown as blue and red sticks, respectively; hydroxyl groups on these tyrosines are colored cyan. The orientation of HA is similar to that in A, but rotated around a vertical axis by 60° toward the reader.

The above figures are reproduced from the cited reference with permission from the ASBMB

^*Note, "added" references are those not in the PDB file but which have either been obtained from the journal or suggested by the author(s).

Secondary reference #1

Title

The link module from human tsg-6 inhibits neutrophil migration in a hyaluronan- And inter-Alpha -Inhibitor-Independent manner.

Authors

S.J.Getting, D.J.Mahoney, T.Cao, M.S.Rugg, E.Fries, C.M.Milner, M.Perretti, A.J.Day.

Ref.

J Biol Chem, 2002, 277, 51068-51076. [DOI no: 10.1074/jbc.M205121200]

PubMed id

12401803

Figure 3.
Fig. 3. Link_TSG6 inhibits PMN accumulation in IL-1 -inflamed air pouch model. Mice were treated either intravenously (i.v.) in the tail vein or locally in the air pouch (a.p.) with 100 µl of PBS alone or PBS containing 1 µg Link_TSG6 15 min before local injection of IL-1 (10 ng in 0.5% (w/v) CMC in PBS); the control group (Control) received local IL-1 only. The number of PMN in the air pouch was measured 4 h after the inflammatory challenge. Administration of 0.5% (w/v) CMC in PBS alone caused mild inflammation with an influx of 2.0 ± 0.1 × 10^6 PMN per mouse (n = 4). Data are the mean ± S.E. of n = 8 mice per group. *, p < 0.05 versus the respective PBS group.

Figure 4.
Fig. 4. Link_TSG6 reduces PMN accumulation in a skin model of inflammation. Mice were given intradermal injections of IL-1 or fMLP alone or in combination with 1 µg Link_TSG6. Control skin sites were injected with sterile PBS alone. Neutrophil accumulation was determined 4 h later by myeloperoxidase activity. Data are the mean ± S.E. of n = 6 mice per group; *, p < 0.05 versus control group.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #2

Title

Hyaluronan binding properties of a cd44 chimera containing the link module of tsg-6.

Authors

J.Lesley, N.M.English, I.Gál, K.Mikecz, A.J.Day, R.Hyman.

Ref.

J Biol Chem, 2002, 277, 26600-26608. [DOI no: 10.1074/jbc.M201068200]

PubMed id

12011075

Figure 1.
Fig. 1. Schematic diagram of the HA-binding domains of wild-type CD44 and the CD44/TSG-6 chimera. The Link module of mouse CD44 (in blue) was replaced with the Link module of human TSG-6 (in green) to create the CD44/TSG-6 chimera. The regions of CD44 N- and C-terminal to the Link module that contain additional residues implicated in HA binding ( 23) are colored yellow. Amino acid (aa) numbering is from human CD44 (4). The positions of the five N-glycosylation sites N1-N5 in CD44 are labeled. The CD44/TSG-6 chimera includes the N1 site of CD44 and the single N-glycosylation site of human Link_TSG6. Circles represent the positions of amino acids shown to be important in HA binding. In wild-type CD44, these critical amino acids are Arg29 (23); Lys38, Arg41, Tyr42, Lys68, Arg78, Tyr79, Asn100, His101 (Asn in human), and Tyr105 (22); and Arg150, Arg154, Lys158, and Arg162 (23). Critical residues for HA binding in the TSG-6 Link module include Lys11, Tyr12, Tyr59, Phe^70, and Tyr78 (data and amino acid numbering from Ref. 34). The open circle (Tyr78) represents the amino acid critical for recognition of Link_TSG6 by mAbs A6, A38, and A68. The asterisks indicate the location of the mAb IM7-binding epitope, just outside of the Link domain.

Figure 5.
Fig. 5. mAb A38 blocks HA binding to TSG-6 and recognizes the HA-binding site. A, recombinant human TSG-6 (rhuTSG-6) attached to a microtiter plate bound HA, as shown by binding of FL-HA detected with anti-fluorescein antibody. The addition of mAb A38 hybridoma supernatant completely blocked HA binding, whereas mAb Q75 supernatant had no significant effect. Data are the means ± S.E. of triplicate samples. B, wild-type Link_TSG6 and mutants with single amino acid substitutions were immunoblotted with mAb A38 supernatant. The Y78F mutant, previously characterized as defining an epitope important for HA binding (34), was not recognized by mAb A38.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #3

Title

Mapping the hyaluronan-Binding site on the link module from human tumor necrosis factor-Stimulated gene-6 by site-Directed mutagenesis.

Authors

D.J.Mahoney, C.D.Blundell, A.J.Day.

Ref.

J Biol Chem, 2001, 276, 22764-22771. [DOI no: 10.1074/jbc.M100666200]

PubMed id

11287417

Figure 7.
Fig. 7. Alignment of Link module sequences. Residues of TSG-6 and CD44 which have been demonstrated by mutagenesis to interact with HA are colored as in Fig. 6A; amino acids that are not involved in HA binding are shown in lowercase. Asterisks denote sequence positions that can contribute to HA binding in TSG-6 and/or CD44 (numbered 1-11 as in Fig. 6). These are colored (as in Fig. 6B) to indicate whether the sequence position is functionally TSG-6-specific, CD44-specific, or utilized by both proteins. This color coding is also used to indicate whether an amino acid capable of making an interaction with HA (i.e. salt bridges or hydrogen bonds) is found at these positions in the Link modules from other members of the Link module superfamily. Residues are underlined if they are identical to, or a conservative replacement of, functional amino acids in TSG-6 or CD44. Residues shown in green in Lp2 may also be involved in HA binding (see Fig. 8 and "Results and Discussion").

Figure 8.
Fig. 8. Putative HA-binding site on human link protein. Lp1 and Lp2 were modeled on the basis of the Link_TSG6 coordinates. The models are shown in the same orientation (on the basis of secondary structure elements) as for Link_TSG6 and CD44 in Fig. 6. Amino acids that could participate in HA binding are colored (as in Fig. 6B and Fig. 7) to indicate whether the sequence position at which they are found is TSG-6-like (red), CD44-like (dark or light blue), or common (purple). In Lp2, additional amino acids can be identified (green), which could contribute to HA binding and are in close proximity to the consensus residues.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #4

Title

Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.

Authors

D.J.Mahoney, R.T.Aplin, A.Calabro, V.C.Hascall, A.J.Day.

Ref.

Glycobiology, 2001, 11, 1025-1033.

PubMed id

11805075

Secondary reference #5

Title

Localization and characterization of the hyaluronan-Binding site on the link module from human tsg-6.

Authors

J.D.Kahmann, R.O'Brien, J.M.Werner, D.Heinegârd, J.E.Ladbury, I.D.Campbell, A.J.Day.

Ref.

Structure, 2000, 8, 763-774. [DOI no: 10.1016/S0969-2126(00)00163-5]

PubMed id

10903951

Figure 2.
Figure 2. Correlation of thermodynamic parameters (D[b]G, D[b]H and TD[b]S) with oligosaccharide length at 25°C.

The above figure is reproduced from the cited reference with permission from Cell Press

Secondary reference #6

Title

Method for quantitative refolding of the link module from human tsg-6.

Authors

J.D.Kahmann, R.Koruth, A.J.Day.

Ref.

Protein Expr Purif, 1997, 9, 315-318.

PubMed id

9126602

Secondary reference #7

Title

Overexpression, Purification, And refolding of link module from human tsg-6 in escherichia coli: effect of temperature, Media, And mutagenesis on lysine misincorporation at arginine aga codons.

Authors

A.J.Day, R.T.Aplin, A.C.Willis.

Ref.

Protein Expr Purif, 1996, 8, 1. [DOI no: 10.1006/prep.1996.0068]

PubMed id

8812829

Secondary reference #8

Title

Solution structure of the link module: a hyaluronan-Binding domain involved in extracellular matrix stability and cell migration.

Authors

D.Kohda, C.J.Morton, A.A.Parkar, H.Hatanaka, F.M.Inagaki, I.D.Campbell, A.J.Day.

Ref.

Cell, 1996, 86, 767-775. [DOI no: 10.1016/S0092-8674(00)80151-8]

PubMed id

8797823

Figure 3.
Figure 3. The Secondary Structure Organization of the Link Module, Which Consists of Two Helices (α1 and α2) and Two Antiparallel β SheetsThe SI sheet is composed of strands β1, β2, and β6 and the SII sheet of β3, β4, and β5. The β5 strand contains a bulge (residues 77–79), which is indicated by no shading. This figure, in which the structure is in the same orientation as in Figure 2, was made using the program MOLSCRIPT ( [34]).

Figure 6.
Figure 6. Putative HA-Binding Surface of Link ModuleThe solvent-accessible surface of the protein is shown, where residues that comprise the predicted HA-binding site are highlighted: aromatics in yellow (Tyr-12, Tyr-59, Tyr-78, and Trp-88), basics in blue (Lys-11, Lys-72, and Arg-81), and acidic residues in red (Asp-77 and Glu-86). The HA[12], i.e., six [D-glucuronic acid (β1→3) N-acetyl- Image -glucosamine (β1→4)] disaccharide units, is shown in green, with the carboxyl oxygens of D-glucuronic acid and the nitrogen atom of N-acetyl- Image -glucosamine in red and blue, respectively. The N- and C-termini and Lys-11 of the protein are indicated. From the figure it can be seen that, if a single Link module does interact with an HA[10], then contacts could occur with regions of the protein outside the predicted binding surface.

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #9

Title

A novel secretory tumor necrosis factor-Inducible protein (tsg-6) is a member of the family of hyaluronate binding proteins, Closely related to the adhesion receptor cd44.

Authors

T.H.Lee, H.G.Wisniewski, J.Vilcek.

Ref.

J Cell Biol, 1992, 116, 545-557.

PubMed id

1730767