Examples of uTOPE™ analysis graphical output

The following examples of some uTOPE™ graphical displays show one small protein, a bacterial porin.

NOTE: Click to toggle the list of bullet points below and view the uTOPE™ analysis graphics and information.
      • MHC Binding Predictions

MHC Hierarchical Predicted Binding Affinity

X axis shows sequential 9-mer (for MHC-I) or 15-mer (for MHC-II) peptides indexed to their N terminal positions; each pixel indicates the peptide starting at that index position. Pixels are colored by the predicted MHC binding affinity for that peptide to the MHC indicated on the Y-axis.

Thermometer shows the predicted binding affinity in standard deviation units. A ln(ic50) below the average indicates high binding; blue coloration shows highest predicted binding; red coloration have poor binding affinity. Values <-1 σ are indicative of peptides in top 15%

Y axis indicates MHC (human and murine) and shows hierarchical groupings of MHC which have similar binding patterns for this protein.

Baseline bar shows regions of highest binding affinity of permuted average of all MHC as ribbons indicating the top 10% affinity binding; Red=MHC-I, Blue=MHC-II. Orange ribbons indicate high probability B-cell binding (top 25%). Black bars show coincident epitope groups (CEGs) where MHC high affinity binding and B-cell epitope high probability overlap or lie within 3 amino acids. Background color shows which peptides are in the transmembrane domain (green), extra-membrane (yellow) or intra-membrane (pink). Signal peptides are white.

NOTE: Click a uTOPE™ analysis image below to enlarge. (Enlarged image will open in a new window.)

MHC-I
MHC-II
      • Permuted Population Plot

The figure below provides a representation of the overall immunome of the protein as seen by an “average” diverse population. It combines predicted B-cell linear epitopes, predicted MHC-I and MHC-II (for DR but not DP and DQ) binding affinities, and protein topology. It does not include the impact of cathepsin cleavage. As such it provides a good overview introduction to the immunologic features of a protein. The
permuted average of predicted MHC binding affinity is calculated for each sequential peptide. For MHC-I 9-mers are used, for MHC-II 15-mers, each with a single amino acid displacement. At each sequential peptide position we examine every possible heterozygous pair of HLA and mouse MHC alleles and determine which allele of the pair has a higher predicted binding affinity. For each of the higher binding alleles of each pair, the mean and standard deviation of predicted binding affinities is calculated. The predicted binding affinity is represented in standard deviation units relative to the mean for all the peptides in the protein as a whole. As the lines on the graphic show the permuted average, any individual allele affinity may differ from the average. X axis: Sequential peptides are arrayed and numbered N-C. Positions are for the index amino acid of each sequential peptide, with a single amino acid displacement.

The
red line shows the permuted average predicted MHC-IA and B (37 alleles) binding affinity by index position of sequential 9-mer peptides. The blue line shows the permuted average predicted MHC-II DRB allele (16 alleles) binding affinity by index position of sequential 15-mer peptides. Both are plotted in standard deviation units, shown on the Y axis. Orange lines show the predicted probability of B-cell binding (“BEPI”) for an amino acid centered in each sequential 9-mer peptide. Note that the standardized B-cell metric has been inverted (multiplied by -1) in order to overlay the standardized probability onto the same scale as the MHC data. Low numbers for MHC data represent high binding affinity, whereas low numbers equate to high BEPI contact probability. Ribbons (Red: MHC-I, Blue: MHC-II) indicate the 10% highest predicted affinity binding. Orange ribbons indicate the top 25% predicted probability B-cell binding. Background shading shows membrane (green) extramembrane (yellow), intramembrane (pink) location. Signal peptides have white background.

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Overlay Plot
      • Cross Presentation of MHC Binding

MHC Predicted Cross Presentation Plot

X axis shows sequential 15-mer peptides indexed to their N terminal positions; Y axis shows MHC-II alleles.

For each 15-mer having a predicted MHC-II binding <-1 σ, the number of 9-mer peptides with binding to any MHC-I with a predicted affinity of <-1 σ is computed. The number of such predicted MHC-I high binding peptides is indicated by the color, as shown in the thermometer. Hence red indicates a high probability of both MHC-I and MHC-II binding with high affinity to peptides at that location.

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MHC Predicted Cross Presentation Plot
      • Cathepsin Cleavage Predictions

Predicted Cleavage Sites for Cathepsin B, Cathepsin S and Cathepsin L

X axis shows the amino acid positions from N-C. Y axis shows the predicted probability of cleavage by one of three different endosomal cathepsins, top to bottom cathespin B (pink), cathepsin S (red) and cathepsin L (green). The bar indicates predicted cleavage probability on the C-terminal side of the amino acid at that position. Only probabilities > 0.2 are shown.

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Cathepsin Cleavage Predictions
      • Plots of Predicted High Affinity MHC-I and MHC-II Binding After Cleavage

Predicted high affinity MHC binding peptides following cleavage by cathepsin – MHC-I

X axis shows sequential peptide index positions from N-C. Y Axis shows MHC-I alleles. Typeface is colored by cluster but alleles are ranked alphanumerically. Murine alleles at the bottom.

Individual 9-mer peptides with high predicted binding affinity (<-1σ below the mean) for each allele which are also predicted to be excised by 1-3 cathepsins at their C-terminal are shown. Peptides released by cleavage by all three cathepsins (B, L, S) are shown in dark red, two of 3 in red and 1 of 3 in yellow.

The uniform background coloration indicates regions where peptides do not bind with an affinity <1σ, although there may be numerous potential cleavages predicted.

Baseline bar shows regions of highest binding affinity of permuted average of all MHC as ribbons indicating the top 10% affinity binding; Red=MHC-I, Blue-MHC-II. Orange ribbons indicate high probability B-cell binding (top 25%). Black bars show coincident epitope groups where MHC high affinity binding and B cell epitope have high probability of overlap or lie within 3 amino acids. Background color shows which peptides are in the transmembrane domain (green), extra-membrane (yellow) or intra-membrane (pink). Signal peptides are white.

For MHC-I we show the consolidated effect of the three cathepsins; plots for individual cathepsins typically show very sparse distribution of excised high affinity peptides; MHC-I epitopes are relatively rare but diffusely distributed.



Predicted high affinity MHC binding peptides following cleavage by cathepsin – MHC-II

X axis shows sequential peptide index positions from N-C. Y Axis shows MHC alleles. Typeface is colored by cluster but alleles are ranked alphanumerically. Murine alleles at the bottom.

Peptides with high predicted binding affinity (<-1σ below the mean) for each allele, that are between 15 and 20 amino acids in length and that are predicted to be excised by cathepsin cleavage are colored more intensely. The peptide pixel color intensity is indicative of the number of sites with > 0.5 probability of being cleaved i.e. the darkest colors might be cut in any one of six different positions yielding 15-20 mer peptides. As the MHC-II binding groove accommodates multiple lengths of peptide, any of the peptides is likely to bind.

The uniform background coloration indicates that there are no high affinity peptides in that region, although there may be numerous potential cleavages predicted. The difference in background colors are only to aid in identification of the operative peptidase.

Baseline bar shows regions of highest binding affinity of permuted average of all MHC as ribbons indicating the top 10% affinity binding; Red=MHC-I, Blue-MHC-II. Orange ribbons indicate high probability B-cell binding (top 25%). Black bars show coincident epitope groups where MHC high affinity binding and B-cell epitope high probability overlap or lie within 3 amino acids. Background color shows which peptides are in the transmembrane domain (green), extra-membrane (yellow) or intra-membrane (pink). Signal peptides are white.

NOTE: Click a uTOPE™ analysis image below to enlarge. (Enlarged image will open in a new window.)

MHC-I
MHC-II


Additional uTOPE™ analysis modules have been developed which are not shown here. The uTOPE™ platform also allows secondary analysis across many proteins. These include simulations of the impact of amino acid changes, cross correlations, analysis of T-cell recognition motifs, and many types of cluster analyses. For examples of some of these, see our publications.