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Structural analysis of the interactions between paxillin LD motifs and alpha-parvin.

Lorenz S, Vakonakis I, Lowe ED, Campbell ID, Noble ME, Hoellerer MK - Structure (2008)

Bottom Line: Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker.Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations.Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, University of Oxford, Oxford OX1 3QU, United Kingdom.

ABSTRACT
The adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as alpha-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CH(C)) of alpha-parvin at 1.05 A resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

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NMR Titrations of α-Parvin-CHC with Paxillin LD PeptidesWeighted combined chemical shift perturbations extrapolated to saturating ligand concentrations along the sequence of α-parvin-CHC. Grey patches denote unassigned residues or residues broadened due to intermediate exchange (residues 253 and 268). Note that the overall higher amplitudes for LD4 might be due to the relatively high error in the extrapolation to full saturation. LD5 was excluded from this plot, since full saturation could not be achieved.
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fig2: NMR Titrations of α-Parvin-CHC with Paxillin LD PeptidesWeighted combined chemical shift perturbations extrapolated to saturating ligand concentrations along the sequence of α-parvin-CHC. Grey patches denote unassigned residues or residues broadened due to intermediate exchange (residues 253 and 268). Note that the overall higher amplitudes for LD4 might be due to the relatively high error in the extrapolation to full saturation. LD5 was excluded from this plot, since full saturation could not be achieved.

Mentions: On the basis of primary sequence comparison with other LD-binding proteins, such as FAK and vinculin, and mutation studies, the LD-binding site (or “paxillin binding subdomain” [PBS]) of α-parvin was mapped to residues 274–291 (Nikolopoulos and Turner, 2000) (i.e., the A/C-loop of α-parvin-CHC) (Figure 1B). However, we previously demonstrated that LD-binding to the FAT domain of FAK does not reside in a local peptide sequence such as the PBS (Hoellerer et al., 2003) and thus investigated the interaction of α-parvin-CHC with LD motifs using solution NMR. 1H-15N HSQC monitored titrations of 15N-enriched α-parvin-CHC were performed with peptides representing all five paxillin LD motifs. Each peptide was found to induce resonance-specific chemical shift perturbations (Figure 2 and data not shown), indicating an interaction with α-parvin-CHC. Global fitting of the resulting binding curves shows that the affinities of individual LD motifs for α−parvin-CHC differ substantially (Table 2; Figure S2) with LD1 being the highest affinity ligand followed by LD4 and LD2. All three bind with affinities in the micromolar range, while LD3 and LD5 bind in the millimolar range. The overall pattern of chemical shift perturbations induced by saturating concentrations of different LD peptides is very similar (Figure 2), suggesting that all five LD motifs interact with α-parvin-CHC in a similar fashion. Large chemical shift perturbations (δΔ1H15N) ≥ 0.2 ppm) map to the N-linker helix, helix A, and helix G, whereas the central region is less affected.


Structural analysis of the interactions between paxillin LD motifs and alpha-parvin.

Lorenz S, Vakonakis I, Lowe ED, Campbell ID, Noble ME, Hoellerer MK - Structure (2008)

NMR Titrations of α-Parvin-CHC with Paxillin LD PeptidesWeighted combined chemical shift perturbations extrapolated to saturating ligand concentrations along the sequence of α-parvin-CHC. Grey patches denote unassigned residues or residues broadened due to intermediate exchange (residues 253 and 268). Note that the overall higher amplitudes for LD4 might be due to the relatively high error in the extrapolation to full saturation. LD5 was excluded from this plot, since full saturation could not be achieved.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572193&req=5

fig2: NMR Titrations of α-Parvin-CHC with Paxillin LD PeptidesWeighted combined chemical shift perturbations extrapolated to saturating ligand concentrations along the sequence of α-parvin-CHC. Grey patches denote unassigned residues or residues broadened due to intermediate exchange (residues 253 and 268). Note that the overall higher amplitudes for LD4 might be due to the relatively high error in the extrapolation to full saturation. LD5 was excluded from this plot, since full saturation could not be achieved.
Mentions: On the basis of primary sequence comparison with other LD-binding proteins, such as FAK and vinculin, and mutation studies, the LD-binding site (or “paxillin binding subdomain” [PBS]) of α-parvin was mapped to residues 274–291 (Nikolopoulos and Turner, 2000) (i.e., the A/C-loop of α-parvin-CHC) (Figure 1B). However, we previously demonstrated that LD-binding to the FAT domain of FAK does not reside in a local peptide sequence such as the PBS (Hoellerer et al., 2003) and thus investigated the interaction of α-parvin-CHC with LD motifs using solution NMR. 1H-15N HSQC monitored titrations of 15N-enriched α-parvin-CHC were performed with peptides representing all five paxillin LD motifs. Each peptide was found to induce resonance-specific chemical shift perturbations (Figure 2 and data not shown), indicating an interaction with α-parvin-CHC. Global fitting of the resulting binding curves shows that the affinities of individual LD motifs for α−parvin-CHC differ substantially (Table 2; Figure S2) with LD1 being the highest affinity ligand followed by LD4 and LD2. All three bind with affinities in the micromolar range, while LD3 and LD5 bind in the millimolar range. The overall pattern of chemical shift perturbations induced by saturating concentrations of different LD peptides is very similar (Figure 2), suggesting that all five LD motifs interact with α-parvin-CHC in a similar fashion. Large chemical shift perturbations (δΔ1H15N) ≥ 0.2 ppm) map to the N-linker helix, helix A, and helix G, whereas the central region is less affected.

Bottom Line: Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker.Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations.Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, University of Oxford, Oxford OX1 3QU, United Kingdom.

ABSTRACT
The adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as alpha-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CH(C)) of alpha-parvin at 1.05 A resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

Show MeSH
Related in: MedlinePlus