Figure 1.
The Peptide Binding Region (PBR) of the Major Histocompatibility Complex Class II.
HLA-DRβ1*1501 molecule showing the α-chain as a pink ribbon and the β-chain as a light blue ribbon. (A) Frontal view and (B) top view. The amino acids defining each pocket are represented as balls according to their size in a different color: Pocket 1 (magenta), Pocket 4 (dark blue), Pocket 7 (gray) and Pocket 9 (green). Maximal Speed Molecular Surface representation (www.scripps.edu/̃sanner/html/msms_home.html) [37] (C) Frontal view evidencing the trough of the PBR and (D) Top view showing the deep pockets P1 and P9 whereas Pocket 4, 6 and 7, more superficial, lye towards the walls of the groove. These figures were made using the VMD software [24].
Table 1.
Amino acids defining each pocket and peptide sequence considered in the electrostatic analysis.
Figure 2.
Electrostatic landscape variations according to the TotDif.
(A) The magnitude of the total difference (TotDif) variable calculated for each of the pockets and with each of the occupying amino acids is shown. Large variations resulting from including each of the charged occupying amino acids into each pocket are evidenced. The magnitude of the electrostatic variation was higher for P1 and P9 compared to P4 and P7 in any system. (B) Cumulative variation of each multipolar moment where q Tot, d tot and C tot is the mean of total variations in multipolar moments for each pocket. The cumulative variation for Pocket 1 was high in all three multipolar moments although variation in the quadruple moment was the most prominent, while the dipolar electrostatic variation is the most notable in Pocket 9.
Figure 3.
Boxplot analysis of Saa for each amino acid defining a pocket.
The x-axis shows amino acids according to their position in the α and β-chain. The mean value of each data series are joined by a dashed line. The median value is the line into the box. Amino acids are classified according to their electrostatic effect which is represented by different colors: anchoring amino acids are shown in red; amino acids having a recognition effect in blue, amino acids having a dual effect are shown in green whereas those not having a notable electrostatic variation are shown in gray. The box plot analysis of Pocket 1 is illustrated in panel (A) while the analysis for Pocket 4 is illustrated in (B). The same analysis was performed for the remaining pockets (data not shown).
Figure 4.
Box plot analysis applied to distinguish amino acids according to their particular electrostatic behavior.
The box plot analysis allows separating data into high and low values in a quantitative-manner. High values were those lying above the third quartile and were further differentiated into outliers and upper-whisker values. (A) Summary of the Saa mean values of amino acids defining the four pockets allowing to differentiate amino acids having Saa outliers values, denoted as primary anchoring amino acids and those having upper-whisker values, named secondary anchoring amino acids. The higher anchoring effect occurred in α81H and β55E of pocket 1 (B) Summary of IQR values obtained from the box plot analysis of Saa allowing differentiating amino acids having IQR outliers values, denoted as fine differentiation amino acids and those having upper-whisker values, named coarse differentiation amino acids. The higher differentiation effect was seen in α26F, α13R and α28D residues.
Table 2.
Amino acids in each pocket being critical for peptide-HLA groove interaction according to their electrostatic behavior.
Figure 5.
Maximal Speed Molecular Surface representations (www.scripps.edu/̃sanner/html/msms_home.html) [37] of each of the four pockets showing the position of primary anchoring amino acids within pocket 1 and 9, while amino acids indicated in pocket 4 and 7 correspond to amino acids being assign a recognition electrostatic effect (A,B). The antigenic fragment fitting into the pocket is shown in yellow sticks with the occupying amino acid highlighted in purple. (A) Empty pockets. (B) Occupied pocket. (C) Stick representation showing the disposition of anchoring amino acids (in red), recognition amino acids (in green) and amino acids having a dual effect are shown (in blue) inside pockets buried inside the PBR formed by residues lying in the α-chain (shown in pink) and the β-chain (light blue). It can clearly seen that the α55E and β81H primary anchoring amino acids of P1 are located towards the outermost portion of the binding groove (see also Fig. 1) and that α24F lies in the farthest portion of the pocket near to P1.
Figure 6.
Occupying amino acid comparison with reference amino acid in MBP.
Comparison of the RefDif between each occupying amino acid and the native occupying amino acid in the MPB peptide (V in P1; F in P4; I in P7; T in P9) allowing to detect those amino acids more closely resembling to the reference amino acid in terms of global variations in the electrostatic landscape. (E) Logo displaying the experimentally recognized binding residues and motifs of HLA-DRβ1*1501 where the height of each column represents the information content (in bits) at the given position in the binding motif. The height of each letter within a column is proportional to the “frequency of the corresponding amino acid at that position”. The experimental data compelled in this graph was taken as reference of each pocket's preferential binding ability [33]. Download: www.cbs.dtu.dk/researchgroups/immunology/supertypes.php. Amino acids experimentally proven to have a preferential binding in each pocket according to the logo data are underlined in figures B, C, D and E, indicating that the theoretical analysis is in high agreement with the experimental data.