Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain

Rozálie Hexnerová, Květoslava Křížková, Milan Fábry, Irena Sieglová, Kateřina Kedrová, Michaela Collinsová, Pavlína Ullrichová, Pavel Srb, Christopher Williams, Matthew P Crump, Zdeněk Tošner, Jiří Jiráček, Václav Veverka, Lenka Žáková

Research output: Contribution to journalArticle (Academic Journal)peer-review

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Abstract

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions, with insulin becoming a key regulator of metabolism, while IGF-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and IGF-I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity towards IR-A, particularly the analogs with Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and Ser29Asn point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for a cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domain.
Original languageEnglish
Pages (from-to)21234-21245
Number of pages22
JournalJournal of Biological Chemistry
Volume291
Issue number40
Early online date10 Aug 2016
DOIs
Publication statusPublished - 30 Sep 2016

Keywords

  • insulin
  • NMR
  • Structural Biology
  • Insulin-like growth factor
  • structure-function
  • insulin receptor

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