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Protective hinge in insulin opens to enable its receptor engagement.

ABSTRACT: Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in ? cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (?-subunit domains L1 and ?CT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 ?-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 ?-strand away from the hormone core to lie antiparallel to the receptor's L1-?2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.

SUBMITTER: Menting JG

PROVIDER: S-EPMC4143003 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Protective hinge in insulin opens to enable its receptor engagement.

Menting John G JG Yang Yanwu Y Chan Shu Jin SJ Phillips Nelson B NB Smith Brian J BJ Whittaker Jonathan J Wickramasinghe Nalinda P NP Whittaker Linda J LJ Pandyarajan Vijay V Wan Zhu-li ZL Yadav Satya P SP Carroll Julie M JM Strokes Natalie N Roberts Charles T CT Ismail-Beigi Faramarz F Milewski Wieslawa W Steiner Donald F DF Chauhan Virander S VS Ward Colin W CW Weiss Michael A MA Lawrence Michael C MC

Proceedings of the National Academy of Sciences of the United States of America 20140804 33

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes ...[more]

PMID: 25092300

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Project description:Cellular levels of estrogen receptor-alpha (ERalpha) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERalpha and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERalpha, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERalpha-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERalpha or ERalpha with lysine-to-alanine substitutions at K302 and K303 (ERalpha-AA). In the unliganded state, ERalpha-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERalpha, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17beta-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERalpha via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERalpha-AA was less efficiently degraded. Furthermore, ERalpha-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERalpha-AA in the unliganded state and the increased stability of ERalpha-AA in the liganded state were concordant with reporter gene assays demonstrating that ERalpha-AA has lower basal activity but higher E2 inducibility than wtERalpha. These data provide the first evidence that K302/303 protect ERalpha from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.

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Protective hinge in insulin opens to enable its receptor engagement.

Publications

Protective hinge in insulin opens to enable its receptor engagement.

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