HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available
Figure 1
Cooperative effect on peptide dissociation from HLA-DR1 in presence of DM is evidenced at the level of the exchange peptide.
(a) Dissociation rate of peptides from HLA-DR1 in the presence of DM was measured as described in Results. Data is plotted as the fraction of DR1/labeled peptide complex remaining relative to t = 0. Reactions were performed in triplicate, and data points represent one of two independent experiments. Lines fit the data to a single exponential decay function. (b) Natural log (ln) plot of cooperativity (expected/observed t1/2) vs. DM-mediated (solid line) and intrinsic (dashed line) dissociation rate for each DR1/peptide complex tested. To facilitate the comparison, data points were plotted on different scales for t1/2 values. Top x-axis scale refers to intrinsic dissociation rate. Bottom x-axis scale refers to DM-mediated off rate. Since we defined cooperativity C as the ratio of the expected to observed values for Δt1/2, and t1/2 is directly proportional to stability, the cooperative effect is positive if 0≤C<1, while if C>1 the cooperative effect is negative. In the ln plot, positive cooperativity in stability is indicated on the y-axis by values <0 and negative cooperativity by values >0. Horizontal error bars represent the SD of the t1/2 measurement. Vertical error bars represent the error of cooperativity as calculated through SE propagation. Lines indicate the fit of the data to a linear regression. (c) DM-mediated dissociation of the HAS peptide from DR1. The nature of the competing peptide present in excess during the reaction is identified in the legend. Data points represent the mean of two independent experiments, and lines represent the fit of the data to a two or three parameter single exponential decay function. (d) Natural log (ln) plot of cooperativity (expected/observed t1/2) vs. dissociation rate of DR1/HAS complex for each multiple substituted exchange peptide tested. Error bars are as in panel B. The line indicates the fit of the data to a linear regression. (e) Intrinsic dissociation of the HAS peptide from DR1. The nature of the competing peptide present in excess during the reaction is identified in the legend. Data points represent the mean of two independent experiments, and lines represent the fit of the data to a two or three parameter single exponential decay function. (f) Natural log (ln) plot of cooperativity (expected/observed t1/2) vs. dissociation rate of DR1/HAS complex for each multiple substituted exchange peptide tested. Error bars are as above. The line indicates the fit of the data to a linear regression. (g) The ratio of t1/2 for the DR1/HAS complex measured in the presence of different exchange peptides and DM as compared to the t1/2 measured in the presence of HAS is plotted as function of the natural log of exchange peptide KD. The line indicate the fit of the data to an exponential function (r2 = 0.98). (h) The ratio of t1/2 for the DR1/HAS complex measured in the presence of different exchange peptides and in absence of DM as compared to the t1/2 measured in the presence of HAS is plotted as function of the natural log of exchange peptide KD. The line indicate the fit of the data to an exponential function (r2 = 0.97).