title: Solution structure of proinsulin: connecting domain flexibility and prohormone processing.
abstract: The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic beta-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {(1)H}-(15)N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit alpha-helical-like features. Relative to canonical alpha-helices, however, segmental (13)C(alpha/beta) chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA. yanwu.yang@case.edu
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Yang
authors.forename: Qing-Xin
authors.initials: QX
authors.lastname: Hua
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.forename: Eri H
authors.initials: EH
authors.lastname: Shimizu
authors.forename: Meredith H
authors.initials: MH
authors.lastname: Choquette
authors.forename: Robert B
authors.initials: RB
authors.lastname: Mackin
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
mesh_terms: D003922:Diabetes Mellitus, Type 1 / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D050417:Insulin-Secreting Cells / Q000502:physiology*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011384:Proinsulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045664:Proprotein Convertase 1 / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004220:Disulfides; D011384:Proinsulin; D045664:Proprotein Convertase 1