The A-chain of Insulin Contacts the Insert Domain of the Insulin Receptor

Abstract

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val A3 , which projects within a crevice between the A-and B-chains. Engineered receptor ␣-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap B25 -insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and ␣-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly A3 and Ser A3 ) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba A3 and allo-Thr A3 analogs are reduced at least 10-fold; the activity of Thr A3 -DKPinsulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba A3 ) or by altered polarity (allo-Thr A3 and Thr A3 ). Our results provide evidence that Val A3 , a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the ␣-subunit in a hormone-receptor complex.Binding of insulin to its receptor is central to the hormonal control of metabolism. Although recent crystallographic studies have provided insights into the structure of the free receptor ectodomain (1), mechanisms underlying hormone-receptor recognition remain elusive. Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues), derived from proinsulin (2). In the pancreatic ␤-cell insulin is stored as Zn 2ϩ -stabilized hexamers within secretory granules (2). The hexamers dissociate on secretion into the portal circulation, enabling the hormone to function as a Zn 2ϩ -free monomer. In this study we investigate structure-function relationships at position Val A3 , a site of mutation associated with diabetes mellitus (3) located within a crevice between A-and B-chains (Fig. 1A).Diabetes-associated mutations have been identified at three invariant sites as follows: Val A3 3 Leu A3 (insulin Wakayama), Phe B24 3 Ser (insulin Los Angeles), and Phe B25 3 Leu (insulin Chicago). These variants exhibit impaired receptor binding but to different extents (4). The most severely affected is Leu A3 -insulin, whose receptor binding is reduced by 500-fold. Such a marked impairment, unusual among mutant insulins (5-7), suggests either nonlocal structural perturb...

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

“…The decrease in free energy (⌬⌬G u 0.8 Ϯ 0.1 kcal/mol) is less than that observed in studies of an Ala B5 analog (⌬⌬G u 1.7 Ϯ 0.1 kcal/mol) (22). The decreased stability of Arg B5 -insulin is more modest than that observed on substitution of Val A3 by Thr (⌬⌬G u 1.3 Ϯ 0.1 kcal/ mol) in which a polar ␤-OH makes unfavorable interactions within an analogous nonpolar crevice elsewhere in the protein (48).…”

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

“…4B). Insulin structures have been determined both by x-ray crystallography and NMR, and our IDE-bound insulin structure deviates significantly from structures of wild type insulin and insulin analogs at three discrete regions (41). By binding to the exosite of IDE, the N-terminal end of insulin A chain is converted from an ␣-helix to a ␤-strand.…”

Introduction of mutations in insulin molecule: positive and negative mutations

“…Первая мутация уменьшает сродство к рецептору в 500 раз, вторая -6-кратно, третья -100-кратно [6]. Кроме того, существует ряд мутаций, вызывающих диабет MODY (maturity-onset diabetes of the young, сахарный диабет взрослого типа у молодых), PNDM (permanent neonatal diabetes mellitus, персистирующий сахарный диабет), гиперинсулинемию и гиперпроинсулинемию (табл.…”

“…Интересно отметить, что точечные замены на D-или L-аминокислоты в различных частях молекулы сохраняют природоподобную "инсулиновую укладку" молекулы [23]. Замены ValA3 на Thr и allo-Thr приводят к уменьшению стабильности молекулы и ослабляют связывание с рецептором 4-20 кратно [6,24].…”