uncovered that Ak E71 (designated E75 in that report) is critical for 11-5

uncovered that Ak E71 (designated E75 in that report) is critical for 11-5.2 mAb binding, and demonstrated that introduction of this residue into I-Ab (Ab G71E) results in 11-5.2 binding (Landais et al., 1986). between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain name pairing, 11-5.2 binding is influenced directly by chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by chain Arg-52 and chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. using CNS searching of helix interactions (CHI) as previously reported (Dixon et al., 2014; King and Dixon, 2010), and two low energy solutions were obtained in which the GxxxG chain motif is paired with either the MCOPPB 3HCl chain M1 motif or M2 motif (Physique 2D). These results Mouse monoclonal to CD3 confirm that human HLA-DQ class II TM domains can undergo differential GxxxG-driven TM domain name pairing as is seen in human HLA-DR (King MCOPPB 3HCl and Dixon, 2010) and mouse class II molecules (Dixon et al., 2014) and reveal that this pairing can impact the conformation of the class II extracellular domain name. Open in a separate window Physique 2 Alternative Pairing of Human HLA Class II Transmembrane DomainsPanel A: Alignment of the TM domains of mouse I-A and human HLA-DR and DQ MCOPPB 3HCl (GxxxG motifs are highlighted). Panel B: Binding of the 11-5.2-FITC and 10-3.6-PE mAbs to the indicated MHC class II molecules expressed in CIITA 293T cells (pairing of mouse and chimeric class II with the endogenous human class II chains is appreciable in this system). I-AkCDQ and I-AkCDR are chimeric molecules possessing an I-Ak extracellular domain name and human HLA-DQ and HLA-DR TM domain name. Panel C: Wild type (WT) or the indicated GxxxG AxxxA (G A) mutant mouse I-Ak or I-AkCDQ were expressed in CIITA 293T cells and the cells stained with 11-5.2-FITC and 10-3.6-PE. The dot plots show the staining of the indicated mutant vs. WT class II. After gating on cells with detectable 10-3.6-PE binding, the ratio of 11-5.2-FITC (FL1) to 10-3.6-PE (FL2) binding (as a reflection of M1 paired class II to total class II expression) was determined on a cell-by-cell bases. Those results are plotted in the adjacent histograms. Comparison of the FL1:FL2 ratio of M1 mutant to WT cells by a student’s t test gave a p value of 0.001 for both mouse and chimeric class II. Shown are representative results from one of three impartial experiments (panels B and C). Panel D: CHI molecular modeling of HLA-DQ TM domain name interactions as carried out as previously reported (Dixon et al., 2014; King and Dixon, 2010). Shown are examples of the obtained M1 and M2 paired low energy solutions. Modeling of HLA-DR interactions (which can adopt both patterns of pairing) has already been reported (King and Dixon, 2010). 3.2 Immunological Analysis of Soluble I-Ak Class II Molecules Given the importance of the 11-5.2 mAb in the study of the M1 paired class II conformers, it is critical to understand the determinants of 11-5.2 binding. Moreover, improved understanding of the molecular basis of reactivity of the 11-5.2 mAb will provide insight into the mechanism by which shifts in TM domain name GxxxG motif pairing have an allosteric effect on the conformation of the class II extracellular domain name and thus class II function. While it is possible that structures corresponding to the extracellular domains of M1 and M2 paired class II may already exist in the NCBI structure database, systematic comparison of the deposited class II structures has failed to reveal two readily distinguishable families of class II conformers. However, this is not overly surprising since all of the reported class II crystal structures are derived from the analysis of class II molecules that lack a TM domain MCOPPB 3HCl name, which we have shown to be important for the formation of the M1 paired class II conformer. In addition, many of the deposited structures are derived from the analysis of class II with a chain tethered peptide, the presence of which we have also shown to ablate 11-5.2 binding (Busman-Sahay et al., 2011). Nevertheless, X-ray crystallography of an.