Ligand Receptor Interface

Table I summarizes the interactions between IL-10 and sIL-10R1 in the range of 3.7A. It is obvious that most of the contacts have polar nature. The ligand/receptor interface is formed by the residues coming from helix A, interhelical loop AB, and helix F of the IL-10, and from loops L2-L6 of the sIL-10R1. The interface can be divided into two interacting sites, Ia and Ib.49

The site Ia includes C-terminal part of helix A, loop AB and the middle part of helix F on the IL-10 side, and loops L2-L4 of the receptor, while site Ib includes the N-terminal and

Figure 2. Stereo diagram of 1:2 IL-10/sIL-10R1 complex. IL-10 subunits are shown in violet and green, receptors are red, important receptor loops are labeled as L2-L6, N- and C-terminal domains of the receptor molecules as D1 and D2.

middle part of helix A, C-terminal part of helix F of the IL-10, and loops L5-L6 of the receptor (Table 1). Site Ia is certainly the primary binding site accounting for about 67% of the total buried surface of the interface.49 It is centered around receptor residues Tyr43, Arg76 and Arg96, which make the majority of the interactions with IL-10. Burying of Tyr43 shields the largest surface area compared to any residues forming this interface.49 Its side chain forms hydrogen bonds with the side chains of Lys138, Glu142 and main chain carbonyl oxygen of Asn45 through its hydroxyl group, while its aromatic ring penetrates a hydrophobic cavity made by side chains of Leu46 and Ile145 of IL-10, and the aliphatic parts of Arg76 and Arg96 of the receptor (Fig. 3). The guanidino groups of Arg76 and Arg96 make extensive hydrogen bonds with the ligand. The side chain of Arg76 adopts two alternative conformations; in the first one it interacts with Asp44 and Gln42, whereas in the second one it interacts with the main chain carbonyl oxygen of Gln38, and, through a bridging water Wat103, with Gln42. NH1 atom of Arg96 makes hydrogen bonds with Gln38 and the carbonyl oxygen of Ser141, while its NH2 interacts with the carboxyl group of Asp144, and through a bridging water Wat71, with Gln38 and Lys34. There is also a bridging water Wat88 in the site Ia that mediates interactions of IL-10 Lys34 with the carbonyl oxygen of Arg96, main chain nitrogen, and the hydroxyl group of Ser98 of the receptor. It is interesting to note that out of three water molecules involved in forming the ligand/receptor interface site Ia, Wat88 and Wat103 certainly

Figure 3. Interactions of one domain (green) of the IL-10 with the sIL-10R1 (red). Only residues included in Table 1 are shown, one letter amino acid residue code is used, not all residues are labeled. Water molecules 71, 88 and 103 found in the ligand/receptor interface are blue, only waters w88 and w103 are labeled. IL-10 helices are labeled as A-D and E'-F', | -strands of the receptor N- and C-terminal domains D1 and D2 are labeled as A-C and C'-G.

Figure 3. Interactions of one domain (green) of the IL-10 with the sIL-10R1 (red). Only residues included in Table 1 are shown, one letter amino acid residue code is used, not all residues are labeled. Water molecules 71, 88 and 103 found in the ligand/receptor interface are blue, only waters w88 and w103 are labeled. IL-10 helices are labeled as A-D and E'-F', | -strands of the receptor N- and C-terminal domains D1 and D2 are labeled as A-C and C'-G.

belong to the receptor molecule, while Wat71 is likely to belong to free IL-10. The latter water has a counterpart in Wat260 of IL-10 (pdb entry 2ILK) - the distance between the positions of Wat71 and Wat260 is only 1.7A when free and receptor-bound IL-10 molecules are superimposed.

Site Ib may be considered to be secondary and it is centered around Arg27 and Glu151 of IL-10. The guanidinium group ofArg27 makes hydrogen bonds with the side chains ofAsn148 and Glu151 of IL-10 and with the carbonyl oxygen of Ser190 of the receptor. In addition, the carboxyl group of Glu151 interacts with the hydroxyl group of Ser190 and with the side chain of Arg191 on the receptor side. The side chain of Arg24 of IL-10 also makes a hydrogen bond through its NE atom with the main chain carbonyl of Arg191. Hydrophobic contacts occur between the aromatic ring of the receptor Phe143 sandwiched between Pro20 and Ile158 (Fig. 3), the letter amino acid residue is not shown in Table 1 since the distance between its side chain and Phe143 is around 4 A what is acceptable for hydrophobic interactions.

Therefore, even though most of the interactions in the ligand/receptor interface have polar nature, Tyr43 and Phe143 of the receptor are two hydrophobic residues, located at the top and bottom of the ligand/receptor interface (Fig. 3) and playing the role of hydrophobic locks that are likely to keep molecules together after they first recognized each other by long-range ionic interactions.

It is interesting to note that EBV IL-10, having very high identity (85%) toward human IL-10,18-20 has about 1000-fold lower affinity toward IL-10R1.3 Crystal structure of the IL-10/ sIL-10R1 complex appears to explain this phenomenon. The hydrophobic pocket made by IL-10 residues Pro20 and Ile158 for the receptor side chain of Phe143 does not exist in EBV IL-10, in which residues 17-20 (human IL-10 numbering scheme) are deleted, and Ile158 is substituted by Ala. Because of the deletions at the N-terminus, the conformation of the main

Table 1. Amino acid residues of IL-10 and sIL-10R1 (pdb entry 1J7V49) in the range 3.7 D from each other. Secondary structure elements are given in accordance with ref. 37 for IL-10 and ref. 49 for sIL-10R1

IL-10

sIL10R1

Amino Acid

Amino Acid

Residue

Location

Residue

Location

Type of Interaction

Site Ia

Lys34

Helix A

Arg96

Loop L4

Hydrogen bond through Wat71

Arg96

Loop L4

Hydrogen bond through Wat88

Ser98

Loop L4

Hydrogen bond through Wat88

Thr35

Helix A

Thr95

Loop L4

Close contact

Gln38

Helix A

Asn94

Loop L4

Close contact

Thr95

Loop L4

Close contact

Arg96

Loop L4

Hydrogen bond

Arg96

Loop L4

Hydrogen bond through Wat71

Met39

Helix A

Asn94

Loop L4

Close contact

Gln42

Loop AB

Arg76

Loop L3

Close contact

Arg76

Loop L3

Hydrogen bond through Wat103

Asp44

Loop AB

Gly44

Loop L2

Hydrogen bond

Ile45

Loop L2

Hydrogen bond

Arg76

Loop L2

Hydrogen bond

Asn45

Loop AB

Tyr43

Loop L2

Hydrogen bond

Glu46

Loop L2

Close contact

Leu46

Loop AB

Tyr43

Loop L2

Hydrophobic

Asn73

Loop L3

Close contact

Lys138

Helix F

Tyr43

Loop L2

Hydrogen bond

Ser141

Helix F

Arg96

Loop L4

Hydrogen bond

Glu142

Helix F

Tyr43

Loop L2

Hydrogen bond

Asp144

Helix F

Arg96

Loop L4

Hydrogen bond

Ile145

Helix F

Asn73

Loop L3

Close contact

Arg96

Loop L4

Close contact

Site Ib

Pro20

Helix A

Phe143

Loop LS

Hydrophobic

Ala189

Loop L6

Close contact

Ser190

Loop L6

Close contact

Arg24

Helix A

Ala189

Loop L6

Close contact

Ser190

Loop L6

Close contact

Arg191

Loop L6

Hydrogen bond

Arg27

Helix A

Ser190

Loop L6

Hydrogen bond

Arg191

Loop L6

Close contact

Asp28

Helix A

Ser192

Loop L6

Close contact

Glu151

Helix F

Ser190

Loop L6

Hydrogen bond

Arg191

Loop L6

Hydrogen bond

Arg159

Helix F

His142

Loop LS

Close contact

chain in this area is quite different and in order for Phel43 of the receptor to get into even weak hydrophobic contacts with IL-10, some local conformational changes on the ligand side are necessary. In addition, the conformation of the loop AB involved in the formation of the site Ia is also different from what was found in the structure of EBV IL-10.27 In other words, this would also require conformational change to take place, at the cost of additional energy.

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