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Transient α-helices in the disordered RPEL motifs of the serum response factor coactivator MKL1.

Mizuguchi M, Fuju T, Obita T, Ishikawa M, Tsuda M, Tabuchi A - Sci Rep (2014)

Bottom Line: Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions.The helix content is higher in the order of RPEL1, RPEL2, and RPEL3.The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

ABSTRACT
The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

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CD spectra of RPEL1 (solid line), RPEL2 (dotted line), and RPEL3 (dashed line) at pH 7.0 and 25°C in the far-UV region.
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f2: CD spectra of RPEL1 (solid line), RPEL2 (dotted line), and RPEL3 (dashed line) at pH 7.0 and 25°C in the far-UV region.

Mentions: We analyzed the secondary structure of RPELs using CD spectroscopy. The CD spectra of RPELs exhibited a strong negative band near 200 nm and a weak negative shoulder at 220 nm, which is characteristic of unfolded polypeptides21 (Figure 2). This is supported by the prediction of structural disorder by IUPred22, which indicates that RPEL1, RPEL2, and RPEL3 are disordered (Figure 1d). The negative band near 200 nm is stronger in the order of RPEL3 > RPEL2 > RPEL1 (Figure 2). Although free RPEL motifs are largely unstructured, analysis of the helix content shows that RPEL adopts a small but significant amount of α-helix conformation. The helix content is higher in the order of RPEL1 > RPEL2 > RPEL3 (Table 1).


Transient α-helices in the disordered RPEL motifs of the serum response factor coactivator MKL1.

Mizuguchi M, Fuju T, Obita T, Ishikawa M, Tsuda M, Tabuchi A - Sci Rep (2014)

CD spectra of RPEL1 (solid line), RPEL2 (dotted line), and RPEL3 (dashed line) at pH 7.0 and 25°C in the far-UV region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048911&req=5

f2: CD spectra of RPEL1 (solid line), RPEL2 (dotted line), and RPEL3 (dashed line) at pH 7.0 and 25°C in the far-UV region.
Mentions: We analyzed the secondary structure of RPELs using CD spectroscopy. The CD spectra of RPELs exhibited a strong negative band near 200 nm and a weak negative shoulder at 220 nm, which is characteristic of unfolded polypeptides21 (Figure 2). This is supported by the prediction of structural disorder by IUPred22, which indicates that RPEL1, RPEL2, and RPEL3 are disordered (Figure 1d). The negative band near 200 nm is stronger in the order of RPEL3 > RPEL2 > RPEL1 (Figure 2). Although free RPEL motifs are largely unstructured, analysis of the helix content shows that RPEL adopts a small but significant amount of α-helix conformation. The helix content is higher in the order of RPEL1 > RPEL2 > RPEL3 (Table 1).

Bottom Line: Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions.The helix content is higher in the order of RPEL1, RPEL2, and RPEL3.The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

ABSTRACT
The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

Show MeSH
Related in: MedlinePlus