Solution structure of proinsulin: connecting domain flexibility and prohormone processing.
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.
Yang
Yanwu
Y
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA. yanwu.yang@case.edu
Hua
Qing-Xin
QX
Liu
Jin
J
Shimizu
Eri H
EH
Choquette
Meredith H
MH
Mackin
Robert B
RB
Weiss
Michael A
MA
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
D004220:Disulfides; D011384:Proinsulin; D045664:Proprotein Convertase 1