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Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes.

Nurzia E, Narzi D, Cauli A, Mathieu A, Tedeschi V, Caristi S, Sorrentino R, Böckmann RA, Fiorillo MT - PLoS ONE (2012)

Bottom Line: Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding.This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells.Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology C Darwin, Sapienza University, Rome, Italy.

ABSTRACT
The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.

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Cell surface expression of R62A and R62K B*2705/09 mutants on Hela transfectants.A) Surface expression of R62A and R62K mutants within B*2705 (left panel) and B*2709 context (right panel) compared to that of wt molecules. Cells were stained with ME1 mAb and analysed by flow cytometry analysis. B) Surface expression of free heavy chains of R62A and R62K B*2705 mutants (left panel) and R62A and R62K B*2709 mutants (left panel) versus wt molecules analysed with HC10 mAb. Grey histograms represent wt HeLa cells (neg) used as controls. Mouse IgG negative controls have not been shown. One representative experiment is shown here. C) Comparison of the surface expression of wt (B*2705 on left panel; B*2709 on right panel) molecules with the relative mutants as folded heterodimers (white bars) and β2m-free heavy chains (black bars). Untransfected HeLa cells (HeLa in the figure) do not expressed unfolded heavy chains. The results are expressed as mean fluorescence ± SD of five/six independent experiments.
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pone-0032865-g003: Cell surface expression of R62A and R62K B*2705/09 mutants on Hela transfectants.A) Surface expression of R62A and R62K mutants within B*2705 (left panel) and B*2709 context (right panel) compared to that of wt molecules. Cells were stained with ME1 mAb and analysed by flow cytometry analysis. B) Surface expression of free heavy chains of R62A and R62K B*2705 mutants (left panel) and R62A and R62K B*2709 mutants (left panel) versus wt molecules analysed with HC10 mAb. Grey histograms represent wt HeLa cells (neg) used as controls. Mouse IgG negative controls have not been shown. One representative experiment is shown here. C) Comparison of the surface expression of wt (B*2705 on left panel; B*2709 on right panel) molecules with the relative mutants as folded heterodimers (white bars) and β2m-free heavy chains (black bars). Untransfected HeLa cells (HeLa in the figure) do not expressed unfolded heavy chains. The results are expressed as mean fluorescence ± SD of five/six independent experiments.

Mentions: In order to investigate the functional role of Arg62, we performed single amino acid substitutions in both B27 subtypes. In particular, we generated B*2705 and B*2709 mutants substituting Arg62 by Lys (R62K) that conserves the positive charge and by Ala (R62A) in which a non-polar residue replaces the positively charged residue. For functional experiments, we stably transfected the cDNAs encoding the four B27 mutants in HeLa cells. Afterwards, HeLa transfectants have been cloned and several different clones analysed by flow cytometry to assess the expression of B27 mutants on the cell surface. One clone for each B27 mutant was chosen for further experiments. The B27 mutants expression profile has been analysed with either ME1, a mAb recognizing the properly folded B27 molecules (heavy chain/β2m/peptide) or HC10 that reacts with the heavy chains dissociated from the β2m [49], [50]. The staining with the conformational dependent antibody ME1 showed an expression level of the folded R62K and R62A B27 mutants slightly lower and higher, respectively, than wt (Fig. 3A and C). On the opposite, the analysis with the HC10 mAb showed that both R62K and R62A mutants in the context of B*2709 and, even more, in that of B*2705 displayed an evident decreased amount of β2m-free heavy chains compared to wt molecules (Fig. 3B and C). The data suggest that these mutations do not induce dissociation of the three party complexes on the cell surface. No free heavy chains are detectable on the cell surface of untransfected HeLa cells (Fig. 3B).


Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes.

Nurzia E, Narzi D, Cauli A, Mathieu A, Tedeschi V, Caristi S, Sorrentino R, Böckmann RA, Fiorillo MT - PLoS ONE (2012)

Cell surface expression of R62A and R62K B*2705/09 mutants on Hela transfectants.A) Surface expression of R62A and R62K mutants within B*2705 (left panel) and B*2709 context (right panel) compared to that of wt molecules. Cells were stained with ME1 mAb and analysed by flow cytometry analysis. B) Surface expression of free heavy chains of R62A and R62K B*2705 mutants (left panel) and R62A and R62K B*2709 mutants (left panel) versus wt molecules analysed with HC10 mAb. Grey histograms represent wt HeLa cells (neg) used as controls. Mouse IgG negative controls have not been shown. One representative experiment is shown here. C) Comparison of the surface expression of wt (B*2705 on left panel; B*2709 on right panel) molecules with the relative mutants as folded heterodimers (white bars) and β2m-free heavy chains (black bars). Untransfected HeLa cells (HeLa in the figure) do not expressed unfolded heavy chains. The results are expressed as mean fluorescence ± SD of five/six independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293911&req=5

pone-0032865-g003: Cell surface expression of R62A and R62K B*2705/09 mutants on Hela transfectants.A) Surface expression of R62A and R62K mutants within B*2705 (left panel) and B*2709 context (right panel) compared to that of wt molecules. Cells were stained with ME1 mAb and analysed by flow cytometry analysis. B) Surface expression of free heavy chains of R62A and R62K B*2705 mutants (left panel) and R62A and R62K B*2709 mutants (left panel) versus wt molecules analysed with HC10 mAb. Grey histograms represent wt HeLa cells (neg) used as controls. Mouse IgG negative controls have not been shown. One representative experiment is shown here. C) Comparison of the surface expression of wt (B*2705 on left panel; B*2709 on right panel) molecules with the relative mutants as folded heterodimers (white bars) and β2m-free heavy chains (black bars). Untransfected HeLa cells (HeLa in the figure) do not expressed unfolded heavy chains. The results are expressed as mean fluorescence ± SD of five/six independent experiments.
Mentions: In order to investigate the functional role of Arg62, we performed single amino acid substitutions in both B27 subtypes. In particular, we generated B*2705 and B*2709 mutants substituting Arg62 by Lys (R62K) that conserves the positive charge and by Ala (R62A) in which a non-polar residue replaces the positively charged residue. For functional experiments, we stably transfected the cDNAs encoding the four B27 mutants in HeLa cells. Afterwards, HeLa transfectants have been cloned and several different clones analysed by flow cytometry to assess the expression of B27 mutants on the cell surface. One clone for each B27 mutant was chosen for further experiments. The B27 mutants expression profile has been analysed with either ME1, a mAb recognizing the properly folded B27 molecules (heavy chain/β2m/peptide) or HC10 that reacts with the heavy chains dissociated from the β2m [49], [50]. The staining with the conformational dependent antibody ME1 showed an expression level of the folded R62K and R62A B27 mutants slightly lower and higher, respectively, than wt (Fig. 3A and C). On the opposite, the analysis with the HC10 mAb showed that both R62K and R62A mutants in the context of B*2709 and, even more, in that of B*2705 displayed an evident decreased amount of β2m-free heavy chains compared to wt molecules (Fig. 3B and C). The data suggest that these mutations do not induce dissociation of the three party complexes on the cell surface. No free heavy chains are detectable on the cell surface of untransfected HeLa cells (Fig. 3B).

Bottom Line: Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding.This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells.Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology C Darwin, Sapienza University, Rome, Italy.

ABSTRACT
The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.

Show MeSH
Related in: MedlinePlus