Contribution of TyrB26 to the Function and Stability of Insulin

Abstract

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr B26 , a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, nonaromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakestbinding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu B26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr B26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.Insulin, a small globular protein critical to the maintenance of metabolic homeostasis (1), provides a classical model for studies of protein structure and self-assembly (2) with longstanding application to human therapeutics (3). Insulin contains two polypeptide chains, designated A and B, that are linked by two disulfide bridges (cystines A7-B7 and A20-B19); the A chain is further stabilized by an intrachain bridge (cystine A6-A11). In pancreatic ␤-cells, insulin is stored within glucoseregulated secretory granules as zinc-coordinated hexamers (Fig. 1A). The hormone binds to a receptor-tyrosine kinase, designated the insulin receptor (IR). 5 The product of a single gene, the IR precursor is processed in the trans-Golgi network into its final disulfide-linked (␣␤) 2 homodimer conformation. The extracellular ␣ subunit contains hormone-binding elements, whereas the transmembrane ␤ subunit contains the intracellular tyrosine kinase domain (4). Alternative splicing leads to two receptor isoforms, IR-A and IR-B (5). The threedimensional structure of the holoreceptor has been visualized only at low resolution (Ͼ20 Å), but these findings have been inconclusive (for review, see Ref. 6). How extracellular binding of insulin alters the structure of the receptor, leading in turn to activation of the intracellular tyrosine kinase domains, is a major unsolved problem (7).Dissection of the IR into discrete domains has enabled crystallographic analysis of its parts. The relevant structural biology is as follows. (i) Structures of the intracellular tyrosine kinase domains at 1.9 Å resolution have been o...

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

“…To test whether the minor changes in structure indicated by both the NMR and X‐ray diffraction studies might affect biological activity of Se‐insulin[C6U A , C11U A ], we characterized the affinity of our analogue for the isolated insulin receptor (IR) . The binding assay employed detergent‐solubilized and lectin‐purified IR (isoform B) as immobilized in a plate assay . A control was provided with a semisynthetic version of insulin, Orn B29 insulin, which is known to have the same activity as WT‐insulin.…”

“…Analogue affinities for detergent‐solubilized IR‐B holoreceptor were measured in a competitive‐displacement assay . Successive dilutions of analogues were incubated overnight with WGA‐SPA beads (PerkinElmer Life Sciences ® ), receptor, and radiolabeled tracer before counting .…”

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding

“…This mode of binding, anticipated based on studies of anomalous insulin analogs (23,24) and residue-specific photo-crosslinking (25), defined the binding surfaces in the IR for insulin's conserved triplet of aromatic residues: Phe B24 , Phe B25 , and Tyr B26 . The side chain of Tyr B26 , although important for insulin self-assembly (6), packs at a solvated edge of the IR (15) and can be substituted by Ala, Ser, or Glu without loss of affinity (26).…”

Computational study of the activity, dynamics, energetics and conformations of insulin analogues using molecular dynamics simulations: Application to hyperinsulinemia and the critical residue B26

“…Due to the rapid rise in the number of diabetic people, there is great deal of interest in the development of insulin analogues with enhanced activity and affinity, which can be used for therapeutic purposes. Extensive mutagenesis and structural studies revealed the significant role of the BC-CT—and in particular the triplet of the B-chain aromatic residues F24 B , F25 B and Y26 B —in the activation process of insulin and the binding interface [16] , [22] , [23] , [24] , [25] , [26] , [29] , [30] , [31] , [46] , [47] , [48] , [49] . The F24 B residue has been shown to act as a hinge as the BC-CT opens during the activation of insulin (BC-CT opening) [23] , [25] , [26] , [30] , [46] , while the Y26 B residue has been shown to be a critical residue for the activation of insulin [26] , [30] , [31] , [47] , [48] , [49] .…”

“…Although a significant number of experimental studies focus on insulin and its complex structure with the IR [16] , [17] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [31] , [32] , [33] , [46] , [47] , [48] , [49] , [50] , as well as on mutations on the critical residues of insulin for predicting potential therapeutic candidates [23] , [25] , [26] , [27] , [31] , [46] , [47] , [48] , [49] , there are still a lot of key features that cannot be solved or are hard to explain using only experimental approaches. There have been relatively few computational studies investigating the conformational changes and dynamics of insulin [25] , [30] , [31] , [51] , [52] , and even less focusing on insulin mutations [25] , [31] , [53] , [54] .…”