AYAN, E.; Nguyen, H.; Demirci, H.; Haliloglu, T.; Bahar, I.

Elucidating the structure and dynamics of insulin and its analogs has been of broad interest, while presenting challenges due to the unique structural dynamics of insulin (composed itself of two multiply cross-linked peptides A and B) and its ability to assemble in a variety of oligomeric structures under physiological conditions. Here, we present two distinct X-ray crystallographic structures of the long-acting human insulin analog detemir (INSD) resolved in hexameric and dodecameric (or dihexameric) states at 2.85 [A] and 2.70 [A] resolution, respectively, using diffraction data collected under ambient temperature conditions. Characterization of the collective dynamics of these oligomers using the Gaussian Network Model (GNM) reveals several key features: (i) Oligomerization imparts high cooperativity in structural dynamics evidenced by dissection of the crosscorrelations at various hierarchical levels; (ii) detemir monomers conformational flexibility is highly suppressed within oligomeric constructs, the effect being particularly strong in the dihexamer due to the asymmetric packing of the hexamers and the presence of myristoyl groups at B peptides termini whose interactions imparts further heterogeneities; and (iii) a number of key residues retain, however, their intrinsic dynamics, to be deployed upon release from the oligomers. We distinguish in particular residues serving as hinge sites that mediates the conformational dynamics of the asymmetric units (dimers) and monomers (I2A-V3A and Y19A-C20A, and L11B-L15B and Y26B of the respective peptides A and B), or as anchors supporting structural stability (disulfide-bridge forming cysteines, plus selected residues such as L16A, G8B and R22B-F24B. Overall, this study provides a structural-dynamic framework for gaining new insights into the dynamics of long-acting analog INSD and helps identify actionable sites for modulating insulin (analogs) dynamics toward designing more effective therapeutics.