Crystal Structure of a “Nonfoldable” Insulin

• Published in: The Journal of biological chemistry Vol. 284; no. 50; p. 35259
• Main Authors: Ming Liu, Zhu-li Wan, Ying-Chi Chu, Hassan Aladdin, Birgit Klaproth, Meredith Choquette, Qing-xin Hua, Robert B. Mackin, J. Sunil Rao, Pierre De Meyts, Panayotis G. Katsoyannis, Peter Arvan, Michael A. Weiss
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 11.12.2009
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.046888

Protein evolution is constrained by folding efficiency (“foldability”) and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with β-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (Leu A16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, Val A16 blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. Val A16 destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike Val A16 , preserve folding efficiency. We propose that Leu A16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of Cys A20 and Cys B19 , thus surmounting their wide separation in sequence. Although Val A16 is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [Val A16 ]insulin and [Val A16 ]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved.