Deciphering the hidden informational content of protein sequences: foldability of proinsulin hinges on a flexible arm that is dispensable in the mature hormone.
Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe(B1) in placental mammals) is variably positioned within crystal structures and exhibits (1)H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1-B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.
Liu
Ming
M
Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Hua
Qing-xin
QX
Hu
Shi-Quan
SQ
Jia
Wenhua
W
Yang
Yanwu
Y
Saith
Sunil Evan
SE
Whittaker
Jonathan
J
Arvan
Peter
P
Weiss
Michael A
MA
D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008956:Models, Chemical; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
D011384:Proinsulin