Crystal structure of a "nonfoldable" insulin. Impaired folding efficiency despite native activity

Ming Liu, Zhu Ii Wan, Ying Chi Chu, Hassan Aladdin, Birgit Klaproth, Meredith Choquette, Qing Xin Hua, Robert Mackin, J. Sunil Rao, Pierre De Meyts, Panayotis G. Katsoyannis, Peter Arvan, Michael A. Weiss

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 (LeuA16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, ValA16 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. ValA16 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 ValA16, preserve folding efficiency. We propose that LeuA16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of CysA20 and CysB19, thus surmounting their wide separation in sequence. Although ValA16 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 [ValA16]insulin and [ValA16]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved.

Original languageEnglish
Pages (from-to)35259-35272
Number of pages14
JournalJournal of Biological Chemistry
Volume284
Issue number50
DOIs
StatePublished - Dec 11 2009

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Proinsulin
Disulfides
Crystal structure
Insulin
Substitution reactions
Poisons
Medical problems
Thermodynamics
Cell culture
Endoplasmic Reticulum
Diabetes Mellitus
Proteins
Cell Culture Techniques
Degradation
Peptides
Kinetics
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Liu, M., Wan, Z. I., Chu, Y. C., Aladdin, H., Klaproth, B., Choquette, M., ... Weiss, M. A. (2009). Crystal structure of a "nonfoldable" insulin. Impaired folding efficiency despite native activity. Journal of Biological Chemistry, 284(50), 35259-35272. https://doi.org/10.1074/jbc.M109.046888

Crystal structure of a "nonfoldable" insulin. Impaired folding efficiency despite native activity. / Liu, Ming; Wan, Zhu Ii; Chu, Ying Chi; Aladdin, Hassan; Klaproth, Birgit; Choquette, Meredith; Hua, Qing Xin; Mackin, Robert; Rao, J. Sunil; De Meyts, Pierre; Katsoyannis, Panayotis G.; Arvan, Peter; Weiss, Michael A.

In: Journal of Biological Chemistry, Vol. 284, No. 50, 11.12.2009, p. 35259-35272.

Research output: Contribution to journalArticle

Liu, M, Wan, ZI, Chu, YC, Aladdin, H, Klaproth, B, Choquette, M, Hua, QX, Mackin, R, Rao, JS, De Meyts, P, Katsoyannis, PG, Arvan, P & Weiss, MA 2009, 'Crystal structure of a "nonfoldable" insulin. Impaired folding efficiency despite native activity', Journal of Biological Chemistry, vol. 284, no. 50, pp. 35259-35272. https://doi.org/10.1074/jbc.M109.046888
Liu, Ming ; Wan, Zhu Ii ; Chu, Ying Chi ; Aladdin, Hassan ; Klaproth, Birgit ; Choquette, Meredith ; Hua, Qing Xin ; Mackin, Robert ; Rao, J. Sunil ; De Meyts, Pierre ; Katsoyannis, Panayotis G. ; Arvan, Peter ; Weiss, Michael A. / Crystal structure of a "nonfoldable" insulin. Impaired folding efficiency despite native activity. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 50. pp. 35259-35272.
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