Contribution of residue B5 to the folding and function of insulin and IGF-I: Constraints and fine-tuning in the evolution of a protein family

Youhei Sohma, Qing Xin Hua, Ming Liu, Nelson B. Phillips, Shi Quan Hu, Jonathan Whittaker, Linda J. Whittaker, Aubree Ng, Charles T. Roberts, Peter Arvan, Stephen B.H. Kent, Michael A. Weiss

    Research output: Contribution to journalArticlepeer-review

    19 Scopus citations


    Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6-A11, A7-B7, and A20-B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7-A11, A6-B7, and A20-B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis.

    Original languageEnglish (US)
    Pages (from-to)5040-5055
    Number of pages16
    JournalJournal of Biological Chemistry
    Issue number7
    StatePublished - Feb 12 2010

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology


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