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Crystal structure of LIR-2 (ILT4) at 1.8 A: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18.

Willcox BE, Thomas LM, Chapman TL, Heikema AP, West AP, Bjorkman PJ - BMC Struct. Biol. (2002)

Bottom Line: To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76-84 loop mainchain is displaced 11 A with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation.In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA. b.willcox@bham.ac.uk

ABSTRACT

Background: Leukocyte Immunoglobulin-like Receptor-1 (LIR-1) and LIR-2 (also known as ILT2 and ILT4 respectively) are highly related cell surface receptors that bind a broad range of class I MHC molecules with low (microM) affinities. Expressed on monocytic cells and macrophages, both molecules transmit inhibitory signals after binding ligands. In addition to binding host class I MHC, the LIR-1 molecule, which is also expressed on lymphoid tissues, binds with a high (nM) affinity to UL18, a class I MHC homolog encoded by Human Cytomegalovirus (HCMV). In comparison, LIR-2 binds UL18 only weakly (microM KD). To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.

Results: Recombinant LIR-2 (domains 1 and 2) was produced in E. coli and crystallized using streak seeding to optimize the crystal morphology. A data set complete to 1.8 A was collected at 100 K from a single crystal in the P4(1)2(1)2 spacegroup. The structure was solved by molecular replacement, using a search model based on the LIR-1 structure.

Conclusions: The overall structure of LIR-2 D1D2 resembles both LIR-1, and Killer Inhibitory Receptors, in that the A strand in each domain forms hydrogen bonds to both beta sheets, and there is a sharp angle between the two immunoglobulin-like domains. However, differences from LIR-1 are observed in each domain, with two key changes apparent in the ligand-binding domain, D1. The region corresponding to the residue 44-57 helix of LIR-1 adopts a topology distinct from that of both LIR-1 and the KIR structures, involving a shortened 310 helix. Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76-84 loop mainchain is displaced 11 A with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation. In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.

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Alteration of the UL18-binding site in LIR-2 D1. A. Structure of LIR-2 D1. The region of LIR-2 analogous to the proposed UL18-binding site in LIR-1. Main chain regions of the 76–84 F-G loop and Tyr 38, both of which implicated in LIR-1/UL18 interaction, are indicated in red. Side chains for LIR-2 residues 38 (Tyr), 76 (Gln), 80 (Arg) and 86 (Leu) are also shown. B. Conformation of Tyr 38 in LIR-1. Likely hydrogen bonds in (B) and (C) are shown as dotted lines. C. Conformation of Tyr 38 in LIR-2. The Ser (87) to Leu (86) change would cause a steric clash with the LIR-1 conformation of Tyr 38, and forces a side chain reorientation towards Gln 76. In LIR-1, substitution of Gln 76 for Tyr would similarly prevent adoption of the LIR-2 Tyr 38 orientation due to steric hindrance. D. Conformational shift of the 76–84 loop region. The LIR-1 loop (dark blue) protrudes from the side of the domain
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Figure 3: Alteration of the UL18-binding site in LIR-2 D1. A. Structure of LIR-2 D1. The region of LIR-2 analogous to the proposed UL18-binding site in LIR-1. Main chain regions of the 76–84 F-G loop and Tyr 38, both of which implicated in LIR-1/UL18 interaction, are indicated in red. Side chains for LIR-2 residues 38 (Tyr), 76 (Gln), 80 (Arg) and 86 (Leu) are also shown. B. Conformation of Tyr 38 in LIR-1. Likely hydrogen bonds in (B) and (C) are shown as dotted lines. C. Conformation of Tyr 38 in LIR-2. The Ser (87) to Leu (86) change would cause a steric clash with the LIR-1 conformation of Tyr 38, and forces a side chain reorientation towards Gln 76. In LIR-1, substitution of Gln 76 for Tyr would similarly prevent adoption of the LIR-2 Tyr 38 orientation due to steric hindrance. D. Conformational shift of the 76–84 loop region. The LIR-1 loop (dark blue) protrudes from the side of the domain

Mentions: A mutagenesis study of LIR-1 identified residues in the membrane-distal tip of the A'CC'FG face of D1 as important for UL18-binding [12]. This region (Figure 3A) is distant from the D1D2 elbow region that KIRs use to contact class I MHC molecules [13,23]. Mutation of LIR-1 Tyr 38 to Ala reduced UL18 binding affinity by ~18-fold, and a triple mutant (Y76A/D80A/R84A) showed a similar (~20-fold) reduction in affinity [12]. The analogous residues in LIR-2 (Tyr 38, Gln 76, Arg 80 and Trp 83) either exhibit an altered conformation, a different side chain, or both. Although Tyr 38 is conserved in both structures, the sidechain positions differ: in LIR-1, Tyr 38 is a solvent-exposed residue on the C strand, which points towards the D1 RSESS motif connecting the F and G strands and is within hydrogen-bonding distance of the hydroxyl oxygen of Ser 87 (Figure 3B). In LIR-2 the residue equivalent to Ser 87 is a leucine, and as a result Tyr 38 reorients such that it points towards the N-terminus of the C strand with its hydroxyl group within hydrogen-bonding distance of the amino group of Gln 76 (Figure 3C). The other residues implicated in LIR-1 binding to UL18 (equivalent to LIR-2 residues 76, 80, and 83) are located in the F to G turn (residues 76–84). In LIR-2, this region contains a one residue deletion relative to LIR-1, and two glycine to non-glycine changes (G78 and G83, corresponding to Y78 and R82 in LIR-2), and undergoes a significant conformational shift relative to LIR-1. Whereas this region protrudes from the A'CC'FG face of D1 in LIR-1, in LIR-2 it is oriented closer to residues 28–32 connecting the B and C strands (Figure 3D). In each case the conformation is stabilized by three main chain hydrogen bonds within the turn region, and for LIR-2 the shift is accompanied by an additional hydrogen bond between the carbonyl oxygen of Tyr 78 and the main chain NH group of Gln 33. The conformational shift in this region culminates at residue 80, where the Cα positions are greater than 11 Å apart (Figure 3D).


Crystal structure of LIR-2 (ILT4) at 1.8 A: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18.

Willcox BE, Thomas LM, Chapman TL, Heikema AP, West AP, Bjorkman PJ - BMC Struct. Biol. (2002)

Alteration of the UL18-binding site in LIR-2 D1. A. Structure of LIR-2 D1. The region of LIR-2 analogous to the proposed UL18-binding site in LIR-1. Main chain regions of the 76–84 F-G loop and Tyr 38, both of which implicated in LIR-1/UL18 interaction, are indicated in red. Side chains for LIR-2 residues 38 (Tyr), 76 (Gln), 80 (Arg) and 86 (Leu) are also shown. B. Conformation of Tyr 38 in LIR-1. Likely hydrogen bonds in (B) and (C) are shown as dotted lines. C. Conformation of Tyr 38 in LIR-2. The Ser (87) to Leu (86) change would cause a steric clash with the LIR-1 conformation of Tyr 38, and forces a side chain reorientation towards Gln 76. In LIR-1, substitution of Gln 76 for Tyr would similarly prevent adoption of the LIR-2 Tyr 38 orientation due to steric hindrance. D. Conformational shift of the 76–84 loop region. The LIR-1 loop (dark blue) protrudes from the side of the domain
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Figure 3: Alteration of the UL18-binding site in LIR-2 D1. A. Structure of LIR-2 D1. The region of LIR-2 analogous to the proposed UL18-binding site in LIR-1. Main chain regions of the 76–84 F-G loop and Tyr 38, both of which implicated in LIR-1/UL18 interaction, are indicated in red. Side chains for LIR-2 residues 38 (Tyr), 76 (Gln), 80 (Arg) and 86 (Leu) are also shown. B. Conformation of Tyr 38 in LIR-1. Likely hydrogen bonds in (B) and (C) are shown as dotted lines. C. Conformation of Tyr 38 in LIR-2. The Ser (87) to Leu (86) change would cause a steric clash with the LIR-1 conformation of Tyr 38, and forces a side chain reorientation towards Gln 76. In LIR-1, substitution of Gln 76 for Tyr would similarly prevent adoption of the LIR-2 Tyr 38 orientation due to steric hindrance. D. Conformational shift of the 76–84 loop region. The LIR-1 loop (dark blue) protrudes from the side of the domain
Mentions: A mutagenesis study of LIR-1 identified residues in the membrane-distal tip of the A'CC'FG face of D1 as important for UL18-binding [12]. This region (Figure 3A) is distant from the D1D2 elbow region that KIRs use to contact class I MHC molecules [13,23]. Mutation of LIR-1 Tyr 38 to Ala reduced UL18 binding affinity by ~18-fold, and a triple mutant (Y76A/D80A/R84A) showed a similar (~20-fold) reduction in affinity [12]. The analogous residues in LIR-2 (Tyr 38, Gln 76, Arg 80 and Trp 83) either exhibit an altered conformation, a different side chain, or both. Although Tyr 38 is conserved in both structures, the sidechain positions differ: in LIR-1, Tyr 38 is a solvent-exposed residue on the C strand, which points towards the D1 RSESS motif connecting the F and G strands and is within hydrogen-bonding distance of the hydroxyl oxygen of Ser 87 (Figure 3B). In LIR-2 the residue equivalent to Ser 87 is a leucine, and as a result Tyr 38 reorients such that it points towards the N-terminus of the C strand with its hydroxyl group within hydrogen-bonding distance of the amino group of Gln 76 (Figure 3C). The other residues implicated in LIR-1 binding to UL18 (equivalent to LIR-2 residues 76, 80, and 83) are located in the F to G turn (residues 76–84). In LIR-2, this region contains a one residue deletion relative to LIR-1, and two glycine to non-glycine changes (G78 and G83, corresponding to Y78 and R82 in LIR-2), and undergoes a significant conformational shift relative to LIR-1. Whereas this region protrudes from the A'CC'FG face of D1 in LIR-1, in LIR-2 it is oriented closer to residues 28–32 connecting the B and C strands (Figure 3D). In each case the conformation is stabilized by three main chain hydrogen bonds within the turn region, and for LIR-2 the shift is accompanied by an additional hydrogen bond between the carbonyl oxygen of Tyr 78 and the main chain NH group of Gln 33. The conformational shift in this region culminates at residue 80, where the Cα positions are greater than 11 Å apart (Figure 3D).

Bottom Line: To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76-84 loop mainchain is displaced 11 A with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation.In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA. b.willcox@bham.ac.uk

ABSTRACT

Background: Leukocyte Immunoglobulin-like Receptor-1 (LIR-1) and LIR-2 (also known as ILT2 and ILT4 respectively) are highly related cell surface receptors that bind a broad range of class I MHC molecules with low (microM) affinities. Expressed on monocytic cells and macrophages, both molecules transmit inhibitory signals after binding ligands. In addition to binding host class I MHC, the LIR-1 molecule, which is also expressed on lymphoid tissues, binds with a high (nM) affinity to UL18, a class I MHC homolog encoded by Human Cytomegalovirus (HCMV). In comparison, LIR-2 binds UL18 only weakly (microM KD). To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.

Results: Recombinant LIR-2 (domains 1 and 2) was produced in E. coli and crystallized using streak seeding to optimize the crystal morphology. A data set complete to 1.8 A was collected at 100 K from a single crystal in the P4(1)2(1)2 spacegroup. The structure was solved by molecular replacement, using a search model based on the LIR-1 structure.

Conclusions: The overall structure of LIR-2 D1D2 resembles both LIR-1, and Killer Inhibitory Receptors, in that the A strand in each domain forms hydrogen bonds to both beta sheets, and there is a sharp angle between the two immunoglobulin-like domains. However, differences from LIR-1 are observed in each domain, with two key changes apparent in the ligand-binding domain, D1. The region corresponding to the residue 44-57 helix of LIR-1 adopts a topology distinct from that of both LIR-1 and the KIR structures, involving a shortened 310 helix. Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76-84 loop mainchain is displaced 11 A with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation. In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.

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