Structural basis of insulin fibrillation

Abstract

Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and forming cross-β fibers. Fibrillation complicates insulin storage and therapeutic application. Molecular details of insulin fibrillation remain unclear, hindering efforts to prevent fibrillation process. Here, we characterized insulin fibrils using cryo-electron microscopy (cryo-EM), showing multiple forms that contain one or more of the protofilaments containing both the A and B chains of insulin linked by disulfide bonds. We solved the cryo-EM structure of one of the fibril forms composed of two protofilaments at 3.2-Å resolution, which reveals both the β sheet conformation of the protofilament and the packing interaction between them that underlie the fibrillation. On the basis of this structure, we designed several insulin mutants that display reduced fibrillation while maintaining native IR signaling activity. These designed insulin analogs may be developed into more effective therapeutics for type 1 diabetes.

Fig. 1.. Cryo-EM structure of the insulin fibril.

(A) Cryo-EM maps of type I, II, and III insulin fibrils. Type I fibril is composed of one protofilament, while type II and III fibrils are composed of two protofilaments, which are arranged antiparallelly and parallelly, respectively. Only the cryo-EM of type II fibril was resolved at sufficient resolution for model building. (B) The model of type II fibril, shown in two views. The A and B chains of insulin are colored in blue and green, respectively. (C) Cryo-EM map of type II insulin fibril and the model fitted into the map. (D) The schematic of one protofilament from the type II fibril.

Fig. 2.. Structure-based design of insulin mutants resistant to fibrillization.

(A) Thioflavin T (ThT) assay of the fibrillation of insulin wild-type (WT) and the mutants designed to reduce the ability in forming fibril. (B) Negative-stain EM images of insulin WT and the mutants.

Fig. 3.. IR activation by designed insulin fibrillization-resistant mutants.

(A) IR autophosphorylation (pY IR) induced by the indicated concentration of insulin WT or insulin mutants for 10 min in 293FT cells expressing IR-WT. (B) Quantification of the Western blot data shown in (A). n = 4 independent experiments for all data points. Means ± SEM. The levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 10 nM insulin WT-treated cells. P value: WT versus ThrA8R (100 nM), ***P = 0.000319; WT versus IleA10R (100 nM), *P = 0.022828; WT versus AsnA18Q (100 nM), *P = 0.014585. (C) pY IR treated with 10 nM insulin WT or insulin mutants for the indicated time points in 293FT cells expressing IR-WT. (D) Quantification of the Western blot data shown in (C). IleA10R 10 min, n = 3; all other data points, n = 4 independent experiments. Means ± SEM. The levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 10 nM insulin WT-treated cells. P value: WT versus AsnA18Q (30 min), *P = 0.025973 (E) pY IR induced by 100 nM insulin WT or insulin mutants for 10 min in 293FT cells expressing IR-WT. The insulin stocks are incubated at room temperature for 3 days. (F) Quantification of the Western blot data shown in (E). n = 6 independent experiments for all data points. Means ± SD. The levels of pY IR were normalized to total IR levels and shown as intensities relative to that of IR in 100 nM insulin WT-treated cells.