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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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Related in: MedlinePlus

ΔδCα - ΔδCβ secondary chemical shifts of the (a) L94P and (b) L105P mutant of RPEL1.The positions of helices α1 and α2 are indicated in each panel. The positions of the mutations are indicated by arrows.
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f5: ΔδCα - ΔδCβ secondary chemical shifts of the (a) L94P and (b) L105P mutant of RPEL1.The positions of helices α1 and α2 are indicated in each panel. The positions of the mutations are indicated by arrows.

Mentions: We confirmed the transient α-helix formation of RPEL1 using proline mutagenesis. Proline mutation unfolds or greatly destabilizes the protein structure when inserted in the middle of secondary structures2728. Mutation of Leu94 to Pro reduced the α-helical propensity in the helix α1 region, indicating the helix formation. However, the mutation of Leu94 to Pro had little effect on the helical propensity in the α2 region (Figure 5a). In addition, the mutation of Leu105 to Pro reduced the α-helical propensity in the α2 region of RPEL1 but had little effect on the helical propensity in the α1 region (Figure 5b). These results indicate that the transient α-helices in free RPEL1 are independently formed without helix-helix interactions.


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)

ΔδCα - ΔδCβ secondary chemical shifts of the (a) L94P and (b) L105P mutant of RPEL1.The positions of helices α1 and α2 are indicated in each panel. The positions of the mutations are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: ΔδCα - ΔδCβ secondary chemical shifts of the (a) L94P and (b) L105P mutant of RPEL1.The positions of helices α1 and α2 are indicated in each panel. The positions of the mutations are indicated by arrows.
Mentions: We confirmed the transient α-helix formation of RPEL1 using proline mutagenesis. Proline mutation unfolds or greatly destabilizes the protein structure when inserted in the middle of secondary structures2728. Mutation of Leu94 to Pro reduced the α-helical propensity in the helix α1 region, indicating the helix formation. However, the mutation of Leu94 to Pro had little effect on the helical propensity in the α2 region (Figure 5a). In addition, the mutation of Leu105 to Pro reduced the α-helical propensity in the α2 region of RPEL1 but had little effect on the helical propensity in the α1 region (Figure 5b). These results indicate that the transient α-helices in free RPEL1 are independently formed without helix-helix interactions.

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