Limits...
Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL.

Mouilleron S, Guettler S, Langer CA, Treisman R, McDonald NQ - EMBO J. (2008)

Bottom Line: Characterisation of the RPEL(MAL):G-actin interaction by fluorescence anisotropy and cell reporter-based assays validates the significance of actin-binding residues for proper MAL localisation and regulation in vivo.We identify important differences in G-actin engagement between the two RPEL(MAL) structures.Comparison with other actin-binding proteins reveals an unexpected similarity to the vitamin-D-binding protein, extending the G-actin-binding protein repertoire.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, Cancer Research UK, London Research Institute, London, UK.

ABSTRACT
Serum response factor transcriptional activity is controlled through interactions with regulatory cofactors such as the coactivator MAL/MRTF-A (myocardin-related transcription factor A). MAL is itself regulated in vivo by changes in cellular actin dynamics, which alter its interaction with G-actin. The G-actin-sensing mechanism of MAL/MRTF-A resides in its N-terminal domain, which consists of three tandem RPEL repeats. We describe the first molecular insights into RPEL function obtained from structures of two independent RPEL(MAL) peptide:G-actin complexes. Both RPEL peptides bind to the G-actin hydrophobic cleft and to subdomain 3. These RPEL(MAL):G-actin structures explain the sequence conservation defining the RPEL motif, including the invariant arginine. Characterisation of the RPEL(MAL):G-actin interaction by fluorescence anisotropy and cell reporter-based assays validates the significance of actin-binding residues for proper MAL localisation and regulation in vivo. We identify important differences in G-actin engagement between the two RPEL(MAL) structures. Comparison with other actin-binding proteins reveals an unexpected similarity to the vitamin-D-binding protein, extending the G-actin-binding protein repertoire.

Show MeSH
Structure of an RPEL peptide bound to G-actin. (A) Sequence alignment of individual RPEL motifs from murine MAL, myocardin (transcript variant A) and Phactr1. RPEL2MAL secondary structure and features discussed in text are shown above the sequence. Selected conserved residues are highlighted. (B) Two views of the RPEL2MAL:G-actin complex, related by a 90° rotation around the horizontal axis. Right-hand panel is the classical view of the ‘front' surface of actin (white with subdomains labelled 1–4). RPEL2MAL is drawn in green (cartoon) with highly conserved RPEL residues that interact with actin shown as sticks. The hydrophobic cleft and the subdomain 3 ledge of actin are indicated by red dashed circles. (C) Stereo view of the RPEL2MAL (green cartoon) interaction with G-actin. Actin surface is drawn as per (B) with selected RPEL-interacting residues shown as grey sticks and key hydrogen bonds are indicated as dashed lines. Two glycerol molecules, used as a cryoprotectant, are shown in yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2583105&req=5

f1: Structure of an RPEL peptide bound to G-actin. (A) Sequence alignment of individual RPEL motifs from murine MAL, myocardin (transcript variant A) and Phactr1. RPEL2MAL secondary structure and features discussed in text are shown above the sequence. Selected conserved residues are highlighted. (B) Two views of the RPEL2MAL:G-actin complex, related by a 90° rotation around the horizontal axis. Right-hand panel is the classical view of the ‘front' surface of actin (white with subdomains labelled 1–4). RPEL2MAL is drawn in green (cartoon) with highly conserved RPEL residues that interact with actin shown as sticks. The hydrophobic cleft and the subdomain 3 ledge of actin are indicated by red dashed circles. (C) Stereo view of the RPEL2MAL (green cartoon) interaction with G-actin. Actin surface is drawn as per (B) with selected RPEL-interacting residues shown as grey sticks and key hydrogen bonds are indicated as dashed lines. Two glycerol molecules, used as a cryoprotectant, are shown in yellow.

Mentions: For our structural analyses, we assembled purified skeletal muscle G-actin bound to latrunculin B (LatB) and ATP with individual 32-residue RPEL peptides from murine MAL. These peptides corresponded to RPEL1MAL, RPEL2MAL and RPEL3MAL and are known to bind actin efficiently in vitro (Guettler et al, 2008). High-resolution structures of the RPEL1MAL:LatB–actin:ATP and RPEL2MAL:LatB–actin:ATP complexes (hereafter shortened to RPELMAL:G-actin complexes) were subsequently determined and refined, but we were unable to crystallise the RPEL3MAL:G-actin complex (Supplementary Table 1). When bound to actin, RPEL1MAL and RPEL2MAL each contain two helices (α1 and α2) connected by a short loop and end with a short C-terminal capping (C-cap) region (Ermolenko et al, 2002) (Figure 1A). The observed helical contents of RPEL1MAL and RPEL2MAL are consistent with secondary structure predictions (56, 65 and 47%, respectively, for each of the three MAL RPEL peptides). However, circular dichroism (CD) experiments revealed that each RPEL peptide is largely unstructured in solution (α-helical content of 5.2, 4 and 4.3%, respectively) (Supplementary Figure S1), indicating that the observed RPEL peptide secondary structure is induced on binding actin.


Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL.

Mouilleron S, Guettler S, Langer CA, Treisman R, McDonald NQ - EMBO J. (2008)

Structure of an RPEL peptide bound to G-actin. (A) Sequence alignment of individual RPEL motifs from murine MAL, myocardin (transcript variant A) and Phactr1. RPEL2MAL secondary structure and features discussed in text are shown above the sequence. Selected conserved residues are highlighted. (B) Two views of the RPEL2MAL:G-actin complex, related by a 90° rotation around the horizontal axis. Right-hand panel is the classical view of the ‘front' surface of actin (white with subdomains labelled 1–4). RPEL2MAL is drawn in green (cartoon) with highly conserved RPEL residues that interact with actin shown as sticks. The hydrophobic cleft and the subdomain 3 ledge of actin are indicated by red dashed circles. (C) Stereo view of the RPEL2MAL (green cartoon) interaction with G-actin. Actin surface is drawn as per (B) with selected RPEL-interacting residues shown as grey sticks and key hydrogen bonds are indicated as dashed lines. Two glycerol molecules, used as a cryoprotectant, are shown in yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2583105&req=5

f1: Structure of an RPEL peptide bound to G-actin. (A) Sequence alignment of individual RPEL motifs from murine MAL, myocardin (transcript variant A) and Phactr1. RPEL2MAL secondary structure and features discussed in text are shown above the sequence. Selected conserved residues are highlighted. (B) Two views of the RPEL2MAL:G-actin complex, related by a 90° rotation around the horizontal axis. Right-hand panel is the classical view of the ‘front' surface of actin (white with subdomains labelled 1–4). RPEL2MAL is drawn in green (cartoon) with highly conserved RPEL residues that interact with actin shown as sticks. The hydrophobic cleft and the subdomain 3 ledge of actin are indicated by red dashed circles. (C) Stereo view of the RPEL2MAL (green cartoon) interaction with G-actin. Actin surface is drawn as per (B) with selected RPEL-interacting residues shown as grey sticks and key hydrogen bonds are indicated as dashed lines. Two glycerol molecules, used as a cryoprotectant, are shown in yellow.
Mentions: For our structural analyses, we assembled purified skeletal muscle G-actin bound to latrunculin B (LatB) and ATP with individual 32-residue RPEL peptides from murine MAL. These peptides corresponded to RPEL1MAL, RPEL2MAL and RPEL3MAL and are known to bind actin efficiently in vitro (Guettler et al, 2008). High-resolution structures of the RPEL1MAL:LatB–actin:ATP and RPEL2MAL:LatB–actin:ATP complexes (hereafter shortened to RPELMAL:G-actin complexes) were subsequently determined and refined, but we were unable to crystallise the RPEL3MAL:G-actin complex (Supplementary Table 1). When bound to actin, RPEL1MAL and RPEL2MAL each contain two helices (α1 and α2) connected by a short loop and end with a short C-terminal capping (C-cap) region (Ermolenko et al, 2002) (Figure 1A). The observed helical contents of RPEL1MAL and RPEL2MAL are consistent with secondary structure predictions (56, 65 and 47%, respectively, for each of the three MAL RPEL peptides). However, circular dichroism (CD) experiments revealed that each RPEL peptide is largely unstructured in solution (α-helical content of 5.2, 4 and 4.3%, respectively) (Supplementary Figure S1), indicating that the observed RPEL peptide secondary structure is induced on binding actin.

Bottom Line: Characterisation of the RPEL(MAL):G-actin interaction by fluorescence anisotropy and cell reporter-based assays validates the significance of actin-binding residues for proper MAL localisation and regulation in vivo.We identify important differences in G-actin engagement between the two RPEL(MAL) structures.Comparison with other actin-binding proteins reveals an unexpected similarity to the vitamin-D-binding protein, extending the G-actin-binding protein repertoire.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, Cancer Research UK, London Research Institute, London, UK.

ABSTRACT
Serum response factor transcriptional activity is controlled through interactions with regulatory cofactors such as the coactivator MAL/MRTF-A (myocardin-related transcription factor A). MAL is itself regulated in vivo by changes in cellular actin dynamics, which alter its interaction with G-actin. The G-actin-sensing mechanism of MAL/MRTF-A resides in its N-terminal domain, which consists of three tandem RPEL repeats. We describe the first molecular insights into RPEL function obtained from structures of two independent RPEL(MAL) peptide:G-actin complexes. Both RPEL peptides bind to the G-actin hydrophobic cleft and to subdomain 3. These RPEL(MAL):G-actin structures explain the sequence conservation defining the RPEL motif, including the invariant arginine. Characterisation of the RPEL(MAL):G-actin interaction by fluorescence anisotropy and cell reporter-based assays validates the significance of actin-binding residues for proper MAL localisation and regulation in vivo. We identify important differences in G-actin engagement between the two RPEL(MAL) structures. Comparison with other actin-binding proteins reveals an unexpected similarity to the vitamin-D-binding protein, extending the G-actin-binding protein repertoire.

Show MeSH