High‐resolution structure of a partially folded insulin aggregation intermediate

Abstract

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details-the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

Mechanistic Studies of the Stabilization of Insulin Helical Structure by Coomassie Brilliant Blue

“…56 Previous studies have also indicated that the amyloid fibril formation for globular proteins, such as HI, occurs via partially unfolded intermediates that gradually associate to form the oligomers, eventually into well-ordered mature fibrils. 9,14,23,27,33,47,57,58 A partial unfolding of the monomeric globular protein produces a molten globule like structure that is comparable to the amyloidogenic protein intermediates, acting as the nucleating form. 29,50,55,[59][60][61] In the absence of zinc at physiological pH, HI is primarily present as a conformationally stable dimer;…”

“…Coarsely identical to the initial monomeric structure of the protein in buffer solution, these monomeric structures produce the lesser known nucleus en route to HI fibrillation. 9,14,23,27,33,34,47,57 The nucleation step has been identified with the C-terminal end of the B chain helix turning away from the C-terminal end of the A chain helix. This causes a partial loss of helicity on the C-terminal of the A-chain, accompanied by a compromise of the intra-chain hydrophobic contacts between Leu 16A on A chain C-terminal helix and Leu 15B and Val 18B on the B chain helix.…”

“…The consequences of the loss of these contacts is an opening up of the structure that creates a hydrophobic cavity in the centre of the protein comprised of the Phe 24B , Phe 25B , Tyr 26B , Val 12B , Val 3A , and Tyr 19A residues that may serve as a nucleation site for the very early step of fibrillation. 57 The NMR analysis defined several residues in the α-helical folds of the A chain along with other residues from either terminal segments of both the A and B chains that partake in producing the hydrophobic cavity, prompting the oligomerization events. This atomicresolution characterization has been imperative in enabling us to define efficient pharmaceutical excipients to arrest the aggregation-pro conformer.…”

“…Interestingly, CBBG addition at 60 °C also showed close proximity (NOE contacts) to the Glu 13B of the HI B chain, which has been suggested to partake in the dimerization of the domain. 57 Insulin B chain has been shown to adopt a β-hairpin topology wherein the N and C-terminal ends are held together by hydrophobic contacts and form a homodimer being held together by Glu 13B . This dimeric β-hairpin topology allows the B chain to interact with A chain residues in the native folds of HI.…”

Amyloid Prefibrillar Oligomers: The Surprising Commonalities in Their Structure and Activity

“…Recently, Ratha et al, characterized, at high-resolution, the structure of a partially folded intermediate of insulin, suggesting that this state may represent the starting point of the aggregation process. The authors observed slight structural changes in this intermediate compared to the monomer, claiming that these changes lead to the formation of a hydrophobic cavity in the center of the protein that acts as a nucleation center for oligomer formation [ 78 ].…”