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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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LIR-2 D1D2 Crystal structure. A: Ribbon diagram of the structure LIR-2 D1D2 and comparison with the LIR-1 D1D2 structure. Dashed lines indicate disordered loops. Arrows indicate the single turn of 310 helix observed in LIR-2 D1 (residues 52–55), in comparison to the longer 310 helical segment (residues 44–57) in the same region of LIR-1 D1. 410 and 310 helices are shown in red, Polyproline II helices in green. The root mean square deviation between the two structures is 3.05 Å for domain 1 and 2.31 Å for D2 for all carbon alpha atoms. B: Conformational change in D2 146–154 loop relative to LIR-1. The LIR-2 loop is shown in ball-and-stick representation. The LIR-1 loop is shown in blue as a ribbon diagram. The conformational difference is maximal at residue Ala 150 (equivalent to Ala 151 in LIR-1), where the Cα positions differ by 14 Å. C: Crystal contacts involving the D2 146–154 loop for LIR-2 and LIR-1 (147–155). Both LIR-1 (left panel) and LIR-2 (right panel) are shown in cyan with the loop region highlighted in red, and the equivalent neighboring molecule shown in grey.
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Figure 2: LIR-2 D1D2 Crystal structure. A: Ribbon diagram of the structure LIR-2 D1D2 and comparison with the LIR-1 D1D2 structure. Dashed lines indicate disordered loops. Arrows indicate the single turn of 310 helix observed in LIR-2 D1 (residues 52–55), in comparison to the longer 310 helical segment (residues 44–57) in the same region of LIR-1 D1. 410 and 310 helices are shown in red, Polyproline II helices in green. The root mean square deviation between the two structures is 3.05 Å for domain 1 and 2.31 Å for D2 for all carbon alpha atoms. B: Conformational change in D2 146–154 loop relative to LIR-1. The LIR-2 loop is shown in ball-and-stick representation. The LIR-1 loop is shown in blue as a ribbon diagram. The conformational difference is maximal at residue Ala 150 (equivalent to Ala 151 in LIR-1), where the Cα positions differ by 14 Å. C: Crystal contacts involving the D2 146–154 loop for LIR-2 and LIR-1 (147–155). Both LIR-1 (left panel) and LIR-2 (right panel) are shown in cyan with the loop region highlighted in red, and the equivalent neighboring molecule shown in grey.

Mentions: The crystal structure of LIR-2 D1D2 was determined in the space group P41212 to 1.8 Å by molecular replacement using the LIR-1 D1D2 structure [12]. Overall, the structure closely resembles that of LIR-1 in that it consists of two Ig-like domains related by an approximately orthogonal angle, forming a highly bent structure (Figure 2A). As with LIR-1, each domain is composed primarily of β strands arranged into two antiparallel sheets, with a KIR-like folding topology, whereby the first strand of each domain forms hydrogen bonds to both the B and G strands, thereby bridging the two β sheets [12].


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)

LIR-2 D1D2 Crystal structure. A: Ribbon diagram of the structure LIR-2 D1D2 and comparison with the LIR-1 D1D2 structure. Dashed lines indicate disordered loops. Arrows indicate the single turn of 310 helix observed in LIR-2 D1 (residues 52–55), in comparison to the longer 310 helical segment (residues 44–57) in the same region of LIR-1 D1. 410 and 310 helices are shown in red, Polyproline II helices in green. The root mean square deviation between the two structures is 3.05 Å for domain 1 and 2.31 Å for D2 for all carbon alpha atoms. B: Conformational change in D2 146–154 loop relative to LIR-1. The LIR-2 loop is shown in ball-and-stick representation. The LIR-1 loop is shown in blue as a ribbon diagram. The conformational difference is maximal at residue Ala 150 (equivalent to Ala 151 in LIR-1), where the Cα positions differ by 14 Å. C: Crystal contacts involving the D2 146–154 loop for LIR-2 and LIR-1 (147–155). Both LIR-1 (left panel) and LIR-2 (right panel) are shown in cyan with the loop region highlighted in red, and the equivalent neighboring molecule shown in grey.
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Related In: Results  -  Collection

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Figure 2: LIR-2 D1D2 Crystal structure. A: Ribbon diagram of the structure LIR-2 D1D2 and comparison with the LIR-1 D1D2 structure. Dashed lines indicate disordered loops. Arrows indicate the single turn of 310 helix observed in LIR-2 D1 (residues 52–55), in comparison to the longer 310 helical segment (residues 44–57) in the same region of LIR-1 D1. 410 and 310 helices are shown in red, Polyproline II helices in green. The root mean square deviation between the two structures is 3.05 Å for domain 1 and 2.31 Å for D2 for all carbon alpha atoms. B: Conformational change in D2 146–154 loop relative to LIR-1. The LIR-2 loop is shown in ball-and-stick representation. The LIR-1 loop is shown in blue as a ribbon diagram. The conformational difference is maximal at residue Ala 150 (equivalent to Ala 151 in LIR-1), where the Cα positions differ by 14 Å. C: Crystal contacts involving the D2 146–154 loop for LIR-2 and LIR-1 (147–155). Both LIR-1 (left panel) and LIR-2 (right panel) are shown in cyan with the loop region highlighted in red, and the equivalent neighboring molecule shown in grey.
Mentions: The crystal structure of LIR-2 D1D2 was determined in the space group P41212 to 1.8 Å by molecular replacement using the LIR-1 D1D2 structure [12]. Overall, the structure closely resembles that of LIR-1 in that it consists of two Ig-like domains related by an approximately orthogonal angle, forming a highly bent structure (Figure 2A). As with LIR-1, each domain is composed primarily of β strands arranged into two antiparallel sheets, with a KIR-like folding topology, whereby the first strand of each domain forms hydrogen bonds to both the B and G strands, thereby bridging the two β sheets [12].

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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