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Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

ABSTRACT: Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

SUBMITTER: Vikova J

PROVIDER: S-EPMC4726324 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

Viková Jitka J Collinsová Michaela M Kletvíková Emília E Buděšínský Miloš M Kaplan Vojtěch V Žáková Lenka L Veverka Václav V Hexnerová Rozálie R Tarazona Aviñó Roberto J RJ Straková Jana J Selicharová Irena I Vaněk Václav V Wright Daniel W DW Watson Christopher J CJ Turkenburg Johan P JP Brzozowski Andrzej M AM Jiráček Jiří J

Scientific reports 20160121

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipol ...[more]

PMID: 26792393

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Project description:Understanding the basis of the DNA-binding specificity of transcription factors (TFs) has been of long-standing interest. Despite extensive efforts to map millions of putative TF binding sequences, identifying the critical determinants for DNA binding specificity remains a major challenge. The coevolution of residues in proteins occurs due to a shared evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically collected publicly available data sets from multiple large-scale high-throughput TF-DNA interaction screening experiments for the major TF families with large numbers of TF members. These families included the Homeobox, HLH, bZIP_1, Ets, HMG_box, ZF-C4, and Zn_clus TFs. We detected TF subclass-determining sites (TSDSs) and showed that the TSDSs were more likely to coevolve with other TSDSs than with non-TSDSs, particularly for the Homeobox, HLH, Ets, bZIP_1, and HMG_box TF families. By in silico modeling, we showed that mutation of the highly coevolving residues could significantly reduce the stability of the TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence that coevolved residues relate to transcriptional regulation and provided insights into the potential application of engineered DNA-binding domains and proteins. IMPORTANCE While unraveling DNA-binding specificity of TFs is the key to understanding the basis and molecular mechanism of gene expression regulation, identifying the critical determinants that contribute to DNA binding specificity remains a major challenge. In this study, we provided evidence showing that coevolving residues in TF domains contributed to DNA binding specificity. We demonstrated that the TSDSs were more likely to coevolve with other TSDSs than with non-TSDSs. Mutation of the coevolving residue pairs (CRPs) could significantly reduce the stability of THE TF-DNA complex, and even the distant residues from the DNA interface contribute to TF-DNA binding activity. Collectively, our study expands our knowledge of the interactions among coevolved residues in TFs, tertiary contacting, and functional importance in refined transcriptional regulation. Understanding the impact of coevolving residues in TFs will help understand the details of transcription of gene regulation and advance the application of engineered DNA-binding domains and protein.

Dataset Information

Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

Publications

Rational steering of insulin binding specificity by intra-chain chemical crosslinking.

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