Steric requirements at position B12 for high biological activity in insulin

Self Cite

The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal ␣-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the Nterminal ␣-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity.1 H NMR studies of a representative analog lacking invariant side chains Ile A2 and Val A3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise nativelike structure. That this and related partial folds retain efficient disulfide pairing suggests that the native Nterminal ␣-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Mechanism of Insulin Chain Combination

Hua

Chen

Jia

et al. 2002

Self Cite

“…Vertebrate insulin sequences exhibit broad conservation (17), including several invariant residues recently shown to pack at the primary hormone-receptor interface (4) (13,(55)(56)(57). Co-evolution of the hormone-receptor interface has presumably led to the strict conservation of such contact sites.…”

Section: Discussionmentioning

confidence: 99%

Contribution of TyrB26 to the Function and Stability of Insulin

Pandyarajan¹,

Phillips²,

Rege³

et al. 2016

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...

“…Wild-type S-sulfonate B-chain derivatives were obtained by oxidative sulfitolysis of insulin (23). Insulin analogs were prepared by chain combination and purified as described (22,23).…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type S-sulfonate B-chain derivatives were obtained by oxidative sulfitolysis of insulin (23). Insulin analogs were prepared by chain combination and purified as described (22,23). The paired His A-chain substitutions were also incorporated into engineered monomer DKP-insulin (His B10 3 Asp, Pro B28 3 Lys, and Lys B29 3 Pro) (24).…”

Section: Methodsmentioning

confidence: 99%

Supramolecular Protein Engineering

Phillips

Wan

Whittaker

et al. 2010

Bottom-up control of supramolecular protein assembly can provide a therapeutic nanobiotechnology. We demonstrate that the pharmacological properties of insulin can be enhanced by design of "zinc staples" between hexamers. Paired (i, i؉4) His substitutions were introduced at an ␣-helical surface. The crystal structure contains both classical axial zinc ions and novel zinc ions at hexamer-hexamer interfaces. Although soluble at pH 4, the combined electrostatic effects of the substitutions and bridging zinc ions cause isoelectric precipitation at neutral pH. Following subcutaneous injection in a diabetic rat, the analog effected glycemic control with a time course similar to that of long acting formulation Lantus. Relative to Lantus, however, the analog discriminates at least 30-fold more stringently between the insulin receptor and mitogenic insulin-like growth factor receptor. Because aberrant mitogenic signaling may be associated with elevated cancer risk, such enhanced specificity may improve safety. Zinc stapling provides a general strategy to modify the pharmacokinetic and biological properties of a subcutaneous protein depot.