The transcription factor CP2 regulates various biological systems at diverse tissues and cells. However, none of the four CP2 isoforms has been solved in structure yet. In particular, two different regions of the CP2b isoform have been characterized to interact with the PIAS1 in nucleus to regulate ...
The transcription factor CP2 regulates various biological systems at diverse tissues and cells. However, none of the four CP2 isoforms has been solved in structure yet. In particular, two different regions of the CP2b isoform have been characterized to interact with the PIAS1 in nucleus to regulate the ${\alpha}$-globin gene expression. Among them, in this study, the region encompassing residues 251-309 of CP2b was prepared as a recombinant protein and its solution structure was characterized by NMR spectroscopy. The results indicated that the CP2b(251-309) fold belongs to typical IDRs (intrinsically disordered regions), likely to facilitate promiscuous interactions with various target proteins. Unfortunately, however, its interaction with the N-terminal domain of PIAS1 (residues 1-70), which has been identified as one of the CP2b-binding sites, was not observed in the NMR-based titration experiments. Therefore, it could be postulated that the 251-309 region of CP2b would not contact with the PIAS1(1-70), but alternatively interact with another CP2b-binding region that encompasses residues 400-651 of PIAS1.
The transcription factor CP2 regulates various biological systems at diverse tissues and cells. However, none of the four CP2 isoforms has been solved in structure yet. In particular, two different regions of the CP2b isoform have been characterized to interact with the PIAS1 in nucleus to regulate the ${\alpha}$-globin gene expression. Among them, in this study, the region encompassing residues 251-309 of CP2b was prepared as a recombinant protein and its solution structure was characterized by NMR spectroscopy. The results indicated that the CP2b(251-309) fold belongs to typical IDRs (intrinsically disordered regions), likely to facilitate promiscuous interactions with various target proteins. Unfortunately, however, its interaction with the N-terminal domain of PIAS1 (residues 1-70), which has been identified as one of the CP2b-binding sites, was not observed in the NMR-based titration experiments. Therefore, it could be postulated that the 251-309 region of CP2b would not contact with the PIAS1(1-70), but alternatively interact with another CP2b-binding region that encompasses residues 400-651 of PIAS1.
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대상 데이터
Nuclear magnetic resonance (NMR) spectroscopy- The conventional 2D-[1H/15N]-HSQC spectra of 0.6 mM [15N]-enriched CP2b(251-309), dissolved in the aforementioned standard buffer containing 7% D2O, were measured at 283 K, 298 K, and 313 K, on a Bruker Biospin Avance II 900 spectrometer equipped with a cryoprobe. To detect the molecular interaction with PIAS1(1-70), the isotopically non-labeled PIAS1(1-70) was added at 1:1 molar ratio to the [15N]CP2b(251-309).
A092006). This study made use of the NMR facility at the Korea Basic Science Institute, which is supported by the KBSI high-field NMR research program. We thank Korea Basic Science Institute (KBSI; Ochang, Chungbuk, Korea) for using CD, DLS, and NMR machines.
성능/효과
All those observations clearly support that the CP2b(251-309) undergoes a conformational exchange, thereby showing multiple resonances at lower temperatures, due to the slowly exchanging rate at an NMR time scale, whereas less peaks at higher temperate, due to the accelerated exchanging rate. Conclusively, the present results verify that the 251-309 region of CP2b could be appreciated as a typical IDR by adopting a mostly unfolded conformation that is also undergoing a conformational exchange. Such a peculiar conformation and molecular dynamics would be thermodynamically favorable for its dynamic interactions with various target partners, by reducing energy barriers to adopt or to select an appropriate conformational ensemble.
In conclusion, the present results provide the first detailed structural characterization of the CP2-family proteins and revealed that the 251-309 region of CP2b constitutes a typical IDR, probably to facilitate promiscuous interactions with other proteins. Particularly, its known interaction with the PIAS1 is expected to be mediated by the C-terminal region of PIAS1, rather than the N-terminal region.
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