For TP5, it contains Val and Tyr residues which belong to basic anchor motifs, providing multiple interaction sites. Especially, TP5 possesses a linear peptide whose structural flexibility may allow it to associate with different receptors through conformational changes. According to the rule of set of allowed anchor residues, TP5 is theoretically expected to bind HLA-DR. As far as MHC-binding motifs are concerned, anchors can be divided into two different types: type 1 anchors that contribute to the binding energy of the peptide, and type 2 anchors that contribute to the peptide conformation. Here, we performed molecular modeling evaluations to analyze the interaction between the anchors in the frame of TP5 and the binding sites within the groove of HLA-DR. The alanine substitutions of TP5 were tested for AZD6244 HLA-DR binding affinity. From the binding energies presented in Table 2, it can be observed that the substitution at positions 1 and 4 had a significant XAV939 effect on the binding affinity; the substitution at other positions resulted in only partial loss of binding affinity. It implied that the substitution of TP5 at positions 1 and 4 formed instable complexes with HLA-DR. Further, the anchor at positions 1 and 4 seemed to be obligatory as type 1 anchors due to decreasing the binding affinity by Ala substitution. It was also noteworthy that the substitution at positions 2 and 4 neither bound to the same binding sites containing Glu11 and Asn62 nor stretched into the binding groove. This observation suggested that anchors at positions 2 and 4 mainly exerted an effect on peptide conformation, and further implied that the conformation of DR-bound peptide should be very similar. With regard to positions 3 and 5, the substitution by Ala resulted in a moderate binding affinity decrease and variant binding sites containing Asn62 without Glu11, suggesting that peptides with several anchors in frame were more likely to bind to a particular binding site than those lacking partial anchors in frame. The variation of binding affinity showed that anchors at positions 1, 3 and 5 in the frame of TP5 actually interacted with the receptor HLA-DR; however, anchor at position 2 primarily involved in steric constraints. As for the Val at position 4, although the amino acid served as type 2 anchors identified in previous study, the contribution as type 1 anchors is more in the present study. This observation exhibits an obligatory role for Val anchor which is essential for HLA-DR/peptide interaction. The functional analysis of TP5 is basically consistent with previous study on the role of each amino acid in frame other than supplementing an important discovery to the effect of Val on TP5 binding receptor as type 1 anchors. The alanine-substituted peptide variants bound less well to HLA-DR than TP5 did, suggesting that Ala in the substitutions exerted its effect indirectly by inducing a conformational change.