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Identification of the rheumatoid arthritis shared epitope binding site on calreticulin.

Ling S, Cheng A, Pumpens P, Michalak M, Holoshitz J - PLoS ONE (2010)

Bottom Line: The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method.We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT.The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America. songlin@med.unich.edu

ABSTRACT

Background: The rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRbeta chain. The molecular mechanisms by which the SE affects susceptibility to--and severity of--RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT.

Principal findings: Surface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217-224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu(217) and Glu(223)--and to a lesser extent residue Asp(220)--in cell-free SPR-based binding and signal transduction assays.

Significance: We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

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Proposed CRT-SE interaction model.A. A SE-positive HLA-DR4 molecule (PDB ID: 2SEB) interacting with the SE-binding site in the CRT P-domain. The HLA-DRα chain is shown in green, HLA-DRβ chain is in yellow, the SE is in cyan, CRT P-domain is in purple, and the groove peptide is shown in orange. B. A closer view of the interactions between CRT Glu217 and SE Gln70 and between CRT Glu223 and SE Arg72.
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pone-0011703-g005: Proposed CRT-SE interaction model.A. A SE-positive HLA-DR4 molecule (PDB ID: 2SEB) interacting with the SE-binding site in the CRT P-domain. The HLA-DRα chain is shown in green, HLA-DRβ chain is in yellow, the SE is in cyan, CRT P-domain is in purple, and the groove peptide is shown in orange. B. A closer view of the interactions between CRT Glu217 and SE Gln70 and between CRT Glu223 and SE Arg72.

Mentions: Based on the critical role of CRT residues Glu217 and Glu223 shown here, on the one hand and the previously determined functional role of individual SE residues on the other, Figure 5 depicts a proposed model of CRT-SE interaction. According to this model - and consistent with docking Model A discussed above -, CRT Glu217 interacts with SE Gln70, while CRT Glu223 interacts with SE Arg72. This model is supported by several considerations: 1. Model A has been assigned the most significant docking score (−232 kcal/mole); 2. Different from the other docking models considered here, it implicates Glu217, a residue that shows the most significant impact on both binding and signaling; 3. It involves Gln70 on the SE side, a residue that has been previously shown to play a critical role in RA pathogenesis [34], [35]; 4. It involves the SE Arg72, a residue that is common to all the SE variants (QKRAA, QRRAA and RRRAA). 5. This model allows for engagement of both the CRT P-domain and the groove peptide without spatial interference (Fig. 5). In order to examine this model, we are presently gearing towards experiments with HLA-DR tetramers carrying site-specific mutations in the SE (residues 70–74). It should be mentioned, however that while point mutation analyses on both sides will help clarifying the spatial confines of SE-CRT interaction, more definitive data might be obtained from co-crystallization studies. Unfortunately, such studies may be technically challenging, given that the crystal structure of CRT has not been resolved to this date.


Identification of the rheumatoid arthritis shared epitope binding site on calreticulin.

Ling S, Cheng A, Pumpens P, Michalak M, Holoshitz J - PLoS ONE (2010)

Proposed CRT-SE interaction model.A. A SE-positive HLA-DR4 molecule (PDB ID: 2SEB) interacting with the SE-binding site in the CRT P-domain. The HLA-DRα chain is shown in green, HLA-DRβ chain is in yellow, the SE is in cyan, CRT P-domain is in purple, and the groove peptide is shown in orange. B. A closer view of the interactions between CRT Glu217 and SE Gln70 and between CRT Glu223 and SE Arg72.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908537&req=5

pone-0011703-g005: Proposed CRT-SE interaction model.A. A SE-positive HLA-DR4 molecule (PDB ID: 2SEB) interacting with the SE-binding site in the CRT P-domain. The HLA-DRα chain is shown in green, HLA-DRβ chain is in yellow, the SE is in cyan, CRT P-domain is in purple, and the groove peptide is shown in orange. B. A closer view of the interactions between CRT Glu217 and SE Gln70 and between CRT Glu223 and SE Arg72.
Mentions: Based on the critical role of CRT residues Glu217 and Glu223 shown here, on the one hand and the previously determined functional role of individual SE residues on the other, Figure 5 depicts a proposed model of CRT-SE interaction. According to this model - and consistent with docking Model A discussed above -, CRT Glu217 interacts with SE Gln70, while CRT Glu223 interacts with SE Arg72. This model is supported by several considerations: 1. Model A has been assigned the most significant docking score (−232 kcal/mole); 2. Different from the other docking models considered here, it implicates Glu217, a residue that shows the most significant impact on both binding and signaling; 3. It involves Gln70 on the SE side, a residue that has been previously shown to play a critical role in RA pathogenesis [34], [35]; 4. It involves the SE Arg72, a residue that is common to all the SE variants (QKRAA, QRRAA and RRRAA). 5. This model allows for engagement of both the CRT P-domain and the groove peptide without spatial interference (Fig. 5). In order to examine this model, we are presently gearing towards experiments with HLA-DR tetramers carrying site-specific mutations in the SE (residues 70–74). It should be mentioned, however that while point mutation analyses on both sides will help clarifying the spatial confines of SE-CRT interaction, more definitive data might be obtained from co-crystallization studies. Unfortunately, such studies may be technically challenging, given that the crystal structure of CRT has not been resolved to this date.

Bottom Line: The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method.We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT.The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America. songlin@med.unich.edu

ABSTRACT

Background: The rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRbeta chain. The molecular mechanisms by which the SE affects susceptibility to--and severity of--RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT.

Principal findings: Surface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217-224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu(217) and Glu(223)--and to a lesser extent residue Asp(220)--in cell-free SPR-based binding and signal transduction assays.

Significance: We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

Show MeSH
Related in: MedlinePlus