Implications for the active form of human insulin based on the structural convergence of highly active hormone analogues

Abstract

Insulin is a key protein hormone that regulates blood glucose levels and, thus, has widespread impact on lipid and protein metabolism. Insulin action is manifested through binding of its monomeric form to the Insulin Receptor (IR). At present, however, our knowledge about the structural behavior of insulin is based upon inactive, multimeric, and storage-like states. The active monomeric structure, when in complex with the receptor, must be different as the residues crucial for the interactions are buried within the multimeric forms. Although the exact nature of the insulin's induced-fit is unknown, there is strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Here, we present the design and analysis of highly active (200-500%) insulin analogues that are truncated at residue 26 of the B-chain (B 26 ). They show a structural convergence in the form of a new β-turn at B 24 -B 26 . We propose that the key element in insulin's transition, from an inactive to an active state, may be the formation of the β-turn at B 24 -B 26 associated with a trans to cis isomerisation at the B 25 -B 26 peptide bond. Here, this turn is achieved with N-methylated L-amino acids adjacent to the trans to cis switch at the B 25 -B 26 peptide bond or by the insertion of certain D-amino acids at B 26 . The resultant conformational changes unmask previously buried amino acids that are implicated in IR binding and provide structural details for new approaches in rational design of ligands effective in combating diabetes.β-turn | diabetes | peptide bond isomerisation | protein | structure T he peptide hormone insulin regulates blood glucose levels with a widespread impact on lipid and protein metabolism. It is a molecule of major therapeutic importance in the treatment of diabetes. The mature form of insulin is formed by two chains "A" and "B" with a B chain running from Phe B1 -Thr B30 and an A chain Gly A1 -Asn A21 , stabilized by two inter and one intra chain disulphide bonds. Insulin's metabolic actions are expressed through binding as a monomer to the insulin receptor (IR). The structure of insulin, which has been known for four decades (1), has not provided insight into the mode of receptor binding and hormone activation. This is because detailed three-dimensional knowledge of insulin's complex structural behavior is limited to its inactive storage (hexameric, dimeric) states (2-4). The NMR structures of the monomeric form of insulin facilitated by mutations (5), applications of organic co-solvents (6) or truncation of the B-chain (7) merely confirm the conformations known from the inactive forms but also indicate intrinsic mobility of the N-and C termini of the B-chain. It has also been found that the N terminus of insulin can exist in so called T (extended) or R (helical) conformations, however, their role for insulin activation is still ambiguous (3, 4).It is widely acknowledged that insulin must therefore undergo induced-fit structural changes upon binding to the IR be...

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

“…Four of these analogues are novel molecules, with [PheB26]-insulin having been already reported by Gauguin et al (39). The crystal structure and binding affinity of [NMeTyrB26]-insulin was recently reported by us elsewhere (17). The first three analogues differ in the position of N-methylation of the peptide bond amide at the B24, B25, or B26 positions of human insulin.…”

“…Crystal Structure of [NMeTyrB26]-Insulin Hexamer-Although we already reported this structure elsewhere (17), the nature of the dimer interface and its R 6 hexamer structure was not discussed in that report. Remarkably, it can be crystallized both as a monomer and R 6 hexamer under appropriate conditions.…”

“…One of the main signatures of this so-called "active form" of insulin should be the exposure of the previously hidden amino acids Gly-A1, Ile-A2, and Val-A3, which are important for the interaction with IR (3). Recently, we described crystal structures of several shortened and full-length insulin analogues with modifications at the B26 position (17). The structural convergence of some of these highly active analogues (200 -400%) enabled us to postulate that the active form of human insulin is characterized by a formation of a new type II ␤-turn at positions B24 -B26.…”

“…Our previous studies on the structural and functional relevance of the B26 (17,25,26) and B25 (27) positions of insulin focused on modulation of hormone conformation by the incorporation of an N-methyl group on the amide nitrogen of the peptide bond in shortened and full-length insulin analogues. Subsequently, we investigated the selective application of this N-methylation within the B24 -B26 region of insulin by the systematic elimination of the inter/intramolecular hydrogen bonds, which are important for the stability and folding of the insulin monomer, dimer formation, and interaction with IR.…”

Protective hinge in insulin opens to enable its receptor engagement

“…The latter nonpolar side chains are exposed in C and D and so poised to engage αCT on receptor binding. ¶ Nonstandard analogs directing B26 (and, where present, residues beyond) away from the insulin core exhibit high affinity presumably due to general predisplacement of B26-B30 rather than specific mimicry of the receptor-bound state as originally envisioned (69).…”