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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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(a) SSP scores of RPEL1. (b) ΔδCα - ΔδCβ secondary chemical shifts of RPEL1. The positions of helices α1 and α2 are indicated in each panel.
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f4: (a) SSP scores of RPEL1. (b) ΔδCα - ΔδCβ secondary chemical shifts of RPEL1. The positions of helices α1 and α2 are indicated in each panel.

Mentions: NMR chemical shifts can be used to probe the propensity of proteins to adopt an α-helical conformation and extended (β-strand) conformation at the residue level. In order to investigate the conformational properties of RPEL, we utilized the secondary structure propensity (SSP) program25. The SSP program combines chemical shifts from different nuclei into a single score indicating the secondary structure propensity. A positive SSP score indicates a propensity for α-helical conformation, and a negative score indicates a propensity for extended (β-strand) conformation (Figure 4a). The SSP scores represent the expected fraction of α-helical or extended (β-strand) conformation at a given residue25. For instance, an SSP score of 0.5 indicates that 50% of the conformers in the disordered state ensemble adopt an α-helical conformation at that particular position. In order to examine the positions of helices, we utilized the difference between 13Cα secondary shifts and 13Cβ secondary shifts (ΔδCα - ΔδCβ) (Figure 4b). Consecutive positive values of ΔδCα - ΔδCβ indicate a propensity to adopt an α-helical structure, and consecutive negative values indicate an extended (β-strand) structure25. Since appropriate random coil chemical shifts are important to obtain reliable secondary structure propensities, we used the random coil chemical shifts suitable for IDPs26.


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)

(a) SSP scores of RPEL1. (b) ΔδCα - ΔδCβ secondary chemical shifts of RPEL1. The positions of helices α1 and α2 are indicated in each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: (a) SSP scores of RPEL1. (b) ΔδCα - ΔδCβ secondary chemical shifts of RPEL1. The positions of helices α1 and α2 are indicated in each panel.
Mentions: NMR chemical shifts can be used to probe the propensity of proteins to adopt an α-helical conformation and extended (β-strand) conformation at the residue level. In order to investigate the conformational properties of RPEL, we utilized the secondary structure propensity (SSP) program25. The SSP program combines chemical shifts from different nuclei into a single score indicating the secondary structure propensity. A positive SSP score indicates a propensity for α-helical conformation, and a negative score indicates a propensity for extended (β-strand) conformation (Figure 4a). The SSP scores represent the expected fraction of α-helical or extended (β-strand) conformation at a given residue25. For instance, an SSP score of 0.5 indicates that 50% of the conformers in the disordered state ensemble adopt an α-helical conformation at that particular position. In order to examine the positions of helices, we utilized the difference between 13Cα secondary shifts and 13Cβ secondary shifts (ΔδCα - ΔδCβ) (Figure 4b). Consecutive positive values of ΔδCα - ΔδCβ indicate a propensity to adopt an α-helical structure, and consecutive negative values indicate an extended (β-strand) structure25. Since appropriate random coil chemical shifts are important to obtain reliable secondary structure propensities, we used the random coil chemical shifts suitable for IDPs26.

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