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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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The 1H-15N HSQC spectrum of RPEL1 at pH 7.0 and 15°C.The backbone resonance assignments are indicated. Numbering is based on GenBank accession number BAN82605.1.
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f3: The 1H-15N HSQC spectrum of RPEL1 at pH 7.0 and 15°C.The backbone resonance assignments are indicated. Numbering is based on GenBank accession number BAN82605.1.

Mentions: The conformational properties of RPEL1 in the free state were investigated by NMR spectroscopy (Figure 3). NMR spectra were measured at 15°C to reduce hydrogen exchange. Backbone resonance assignments were obtained with standard triple-resonance NMR experiments. The 1H-15N HSQC spectrum of RPEL1 shows the backbone amide resonances within 8.0–8.6 ppm in the 1H dimension (Figure 3). The narrow chemical shift dispersion in the 1H dimension is characteristic of intrinsically disordered proteins (IDPs)2324.


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)

The 1H-15N HSQC spectrum of RPEL1 at pH 7.0 and 15°C.The backbone resonance assignments are indicated. Numbering is based on GenBank accession number BAN82605.1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The 1H-15N HSQC spectrum of RPEL1 at pH 7.0 and 15°C.The backbone resonance assignments are indicated. Numbering is based on GenBank accession number BAN82605.1.
Mentions: The conformational properties of RPEL1 in the free state were investigated by NMR spectroscopy (Figure 3). NMR spectra were measured at 15°C to reduce hydrogen exchange. Backbone resonance assignments were obtained with standard triple-resonance NMR experiments. The 1H-15N HSQC spectrum of RPEL1 shows the backbone amide resonances within 8.0–8.6 ppm in the 1H dimension (Figure 3). The narrow chemical shift dispersion in the 1H dimension is characteristic of intrinsically disordered proteins (IDPs)2324.

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