Fig. 2

Structural overlays aligning the mini-proinsulin analogs with the insulin receptor to clarify probable binding mode differs. Panel A illustrates the native insulin-receptor (INS-IR) binding mode, obtained from the PDB structure (ID: 6VEP). Panel B shows a docking co-complex between the insulin receptor (extracted from the 6VEP structure) and mini-proinsulin (M2PI-IR) derived from the 1EFE PDB structure. This complex shows an alternate binding conformation, likely due to the presence of the C-peptide. Panel C presents another docking co-complex between the insulin receptor (6VEP) and a novel designer mini-proinsulin (nM2PI-IR) modeled using the AlphaFold tool, which demonstrates significant conformational changes in receptor binding. Panel D compares the superposition of INS-IR and M2PI-IR co-complexes, revealing slight differences in the binding of Chain A (ChA). Panel E shows a superposition of INS-IR and nM2PI-IR, highlighting substantial differences in both Chain A (ChA) and Chain B (ChB) conformations. Panel F compares the M2PI-IR and nM2PI-IR co-complexes, demonstrating pronounced differences in the ChA and ChB peptides. Panel G overlays INS-IR, M2PI-IR, and nM2PI-IR co-complexes, showing that both chains of nM2PI-IR exhibit significant distinctions compared to the other co-complexes. Panel H focuses on Chain A (IR binding site 1: G1A, I2A, V3A, E4A, Y19A, and N21A) of the M2PI analog, which exhibits lower binding affinity than native insulin. Panel I highlights that Chain B (IR binding site 1: GB8, SB9, LB11, VB12, YB16, FB24, FB25, YB26) of M2PI demonstrates strong binding affinity to the insulin receptor, comparable to native insulin’s Chain B. Panel J indicates that residues G1A, I2A, V3A, E4A, and Y19A of Chain A in nM2PI are localized on the left side of the co-complex relative to native insulin, resulting in a strong affinity for residues P716, R717, and P718 in the aCT domain of the insulin receptor. Panel K shows that, except for residue L11B, the other IR-binding residues of Chain B in nM2PI exhibit strong affinity to both the αCT and L1 domains of the insulin receptor, albeit with distinct binding patterns compared to native insulin. Panel L and N focuses on Chain A (IR binding site 2: T8A, I10A, S12A, E13A, and L17A) of the M2PI and nM2PI analogs, respectively, which those residues in both forms exhibit similar localization on IR compared to native insulin. Panel M and O highlights that Chain B (IR binding site 2: H10B, E13B, and L17B) of M2PI and nM2PI forms demonstrate those residues of both forms shows close proximity to αCT domain of IR.