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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) Amino acid sequences of RPEL motifs from rat MKL1. The positions of helices α1 and α2 of RPEL1 in complex with G-actin are indicated. The sequences of RPxxxEL and RRxxxEL are underlined. Numbering is based on GenBank accession number BAN82605.1. Asterisks indicate the essential residues for actin-binding. (b) Three-dimensional structure of RPEL2 bound to G-actin (PDB entry 2V52). The positions of helices α1 and α2 are indicated. (c) The interaction region between RPEL2 and G-actin (PDB entry 2V52). G-actin is shown in surface and cartoon representations. RPEL2 is shown in stick representations and essential residues for actin-binding are shown in red. The side chain of K139 in RPEL2 adopts two conformations, both interacting with G-actin20. (d) Prediction of disorder tendency of full-length MKL1 by the IUPred predictor22. Scores above a threshold value of 0.5 are considered to be disordered. The positions of RPEL1, RPEL2, and RPEL3 are indicated.
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f1: (a) Amino acid sequences of RPEL motifs from rat MKL1. The positions of helices α1 and α2 of RPEL1 in complex with G-actin are indicated. The sequences of RPxxxEL and RRxxxEL are underlined. Numbering is based on GenBank accession number BAN82605.1. Asterisks indicate the essential residues for actin-binding. (b) Three-dimensional structure of RPEL2 bound to G-actin (PDB entry 2V52). The positions of helices α1 and α2 are indicated. (c) The interaction region between RPEL2 and G-actin (PDB entry 2V52). G-actin is shown in surface and cartoon representations. RPEL2 is shown in stick representations and essential residues for actin-binding are shown in red. The side chain of K139 in RPEL2 adopts two conformations, both interacting with G-actin20. (d) Prediction of disorder tendency of full-length MKL1 by the IUPred predictor22. Scores above a threshold value of 0.5 are considered to be disordered. The positions of RPEL1, RPEL2, and RPEL3 are indicated.

Mentions: Rat MKL1 (GenBank accession number: BAN82605.1) is a 1038 amino acid protein that has an N-terminal actin-binding RPEL domain, basic boxes, a glutamine-rich domain, an SAP domain, a leucine zipper-like domain, and a transactivation domain1718. The RPEL domain consists of three RPEL motifs, each of which functions as an actin-binding element19. RPEL2 and RPEL3 each have a core sequence of RPxxxEL, while RPEL1 has a noncanonical RRxxxEL core sequence (Figure 1a). The crystal structures of RPEL1 and RPEL2 in complex with G-actin have been reported20. In these complexes, the RPEL motif adopts two α-helices (helices α1 and α2) and binds to the hydrophobic cleft and hydrophobic ledge of G-actin (Figure 1b). The complex structure also suggests that side chains of L136, K139, I140, R143, L149, I154, and L155 of RPEL2 are essential for the interaction with G-actin (Figure 1c)20.


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) Amino acid sequences of RPEL motifs from rat MKL1. The positions of helices α1 and α2 of RPEL1 in complex with G-actin are indicated. The sequences of RPxxxEL and RRxxxEL are underlined. Numbering is based on GenBank accession number BAN82605.1. Asterisks indicate the essential residues for actin-binding. (b) Three-dimensional structure of RPEL2 bound to G-actin (PDB entry 2V52). The positions of helices α1 and α2 are indicated. (c) The interaction region between RPEL2 and G-actin (PDB entry 2V52). G-actin is shown in surface and cartoon representations. RPEL2 is shown in stick representations and essential residues for actin-binding are shown in red. The side chain of K139 in RPEL2 adopts two conformations, both interacting with G-actin20. (d) Prediction of disorder tendency of full-length MKL1 by the IUPred predictor22. Scores above a threshold value of 0.5 are considered to be disordered. The positions of RPEL1, RPEL2, and RPEL3 are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: (a) Amino acid sequences of RPEL motifs from rat MKL1. The positions of helices α1 and α2 of RPEL1 in complex with G-actin are indicated. The sequences of RPxxxEL and RRxxxEL are underlined. Numbering is based on GenBank accession number BAN82605.1. Asterisks indicate the essential residues for actin-binding. (b) Three-dimensional structure of RPEL2 bound to G-actin (PDB entry 2V52). The positions of helices α1 and α2 are indicated. (c) The interaction region between RPEL2 and G-actin (PDB entry 2V52). G-actin is shown in surface and cartoon representations. RPEL2 is shown in stick representations and essential residues for actin-binding are shown in red. The side chain of K139 in RPEL2 adopts two conformations, both interacting with G-actin20. (d) Prediction of disorder tendency of full-length MKL1 by the IUPred predictor22. Scores above a threshold value of 0.5 are considered to be disordered. The positions of RPEL1, RPEL2, and RPEL3 are indicated.
Mentions: Rat MKL1 (GenBank accession number: BAN82605.1) is a 1038 amino acid protein that has an N-terminal actin-binding RPEL domain, basic boxes, a glutamine-rich domain, an SAP domain, a leucine zipper-like domain, and a transactivation domain1718. The RPEL domain consists of three RPEL motifs, each of which functions as an actin-binding element19. RPEL2 and RPEL3 each have a core sequence of RPxxxEL, while RPEL1 has a noncanonical RRxxxEL core sequence (Figure 1a). The crystal structures of RPEL1 and RPEL2 in complex with G-actin have been reported20. In these complexes, the RPEL motif adopts two α-helices (helices α1 and α2) and binds to the hydrophobic cleft and hydrophobic ledge of G-actin (Figure 1b). The complex structure also suggests that side chains of L136, K139, I140, R143, L149, I154, and L155 of RPEL2 are essential for the interaction with G-actin (Figure 1c)20.

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