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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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PRE Experiment with Spin-Labeled LD1(A) Details of the 1H-15N HSQC spectra of 230 μM 15N-enriched α-parvin-CHC and 250 μM PROXYL-labeled LD1 peptide in the absence (left) and presence (right) of 5 mM ascorbate. The latter serves to reduce the spin label, thereby eliminating PRE effects. The binding orientation of LD1 seen in the crystal structure is denoted “forward.”(B) Ribbon representation of α-parvin-CHC in gold and LD1 in green. The position of α-parvin residues 257 and 370 are highlighted in blue and red, respectively.
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fig5: PRE Experiment with Spin-Labeled LD1(A) Details of the 1H-15N HSQC spectra of 230 μM 15N-enriched α-parvin-CHC and 250 μM PROXYL-labeled LD1 peptide in the absence (left) and presence (right) of 5 mM ascorbate. The latter serves to reduce the spin label, thereby eliminating PRE effects. The binding orientation of LD1 seen in the crystal structure is denoted “forward.”(B) Ribbon representation of α-parvin-CHC in gold and LD1 in green. The position of α-parvin residues 257 and 370 are highlighted in blue and red, respectively.

Mentions: On the basis of the cocrystal structures of α-parvin-CHC with LD1 and LD4, we calculated theoretical PRE values for all protein NH resonances corresponding to either forward or backward binding modes, respectively. Interestingly, the experimentally derived PRE data resemble a mixture of the two simulated PRE profiles (Figure S5A). This phenomenon is also illustrated in Figure 5, using two diagnostic NMR signals as examples: the resonance originating from residue 257 is predicted to experience strong PRE in the forward mode (calculated distance from spin-label 10.7 Å) but should only be weakly affected in the backward mode (calculated distance 20.2 Å). The opposite behavior should apply to the resonance assigned to residue 370 (calculated distances of 23.3 Å in forward versus 14.1 Å in backward mode). However, our experimental data show that both resonances undergo significant broadening, suggesting that a simple unidirectional model may be insufficient to describe LD1 binding in solution. To quantify this observation, we calculated the linear correlation coefficients, R, between the experimental and the simulated PRE data for various ratios of forward-to-backward binding in a bidirectional mixture (Figure S5B). The resulting curve is bell shaped, indicating that the experimental data are more consistent with a bidirectional than a unidirectional binding model. The best correlation, with a value of ∼0.8, is generated by a model corresponding to a forward-to-backward ratio of ∼75%/25%. Although the improvement on R upon consideration of a bidirectional model is small, F-test analysis between the unidirectional forward and this optimal bidirectional model shows that the improvement in the later is statistically significant (p < 0.001), and argues for the presence of both binding orientations in solution. The observation that no model returns an R value of 1.0 may reflect both the neglect of peptide flexibility in the PRE simulation, in particular the dynamic process of LD1 helix formation on α-parvin-CHC, and/or nonspecific effects of the spin label. In conclusion, our analysis provides support for the hypothesis that both antiparallel binding modes are accessible to the LD1 peptide in solution.


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)

PRE Experiment with Spin-Labeled LD1(A) Details of the 1H-15N HSQC spectra of 230 μM 15N-enriched α-parvin-CHC and 250 μM PROXYL-labeled LD1 peptide in the absence (left) and presence (right) of 5 mM ascorbate. The latter serves to reduce the spin label, thereby eliminating PRE effects. The binding orientation of LD1 seen in the crystal structure is denoted “forward.”(B) Ribbon representation of α-parvin-CHC in gold and LD1 in green. The position of α-parvin residues 257 and 370 are highlighted in blue and red, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: PRE Experiment with Spin-Labeled LD1(A) Details of the 1H-15N HSQC spectra of 230 μM 15N-enriched α-parvin-CHC and 250 μM PROXYL-labeled LD1 peptide in the absence (left) and presence (right) of 5 mM ascorbate. The latter serves to reduce the spin label, thereby eliminating PRE effects. The binding orientation of LD1 seen in the crystal structure is denoted “forward.”(B) Ribbon representation of α-parvin-CHC in gold and LD1 in green. The position of α-parvin residues 257 and 370 are highlighted in blue and red, respectively.
Mentions: On the basis of the cocrystal structures of α-parvin-CHC with LD1 and LD4, we calculated theoretical PRE values for all protein NH resonances corresponding to either forward or backward binding modes, respectively. Interestingly, the experimentally derived PRE data resemble a mixture of the two simulated PRE profiles (Figure S5A). This phenomenon is also illustrated in Figure 5, using two diagnostic NMR signals as examples: the resonance originating from residue 257 is predicted to experience strong PRE in the forward mode (calculated distance from spin-label 10.7 Å) but should only be weakly affected in the backward mode (calculated distance 20.2 Å). The opposite behavior should apply to the resonance assigned to residue 370 (calculated distances of 23.3 Å in forward versus 14.1 Å in backward mode). However, our experimental data show that both resonances undergo significant broadening, suggesting that a simple unidirectional model may be insufficient to describe LD1 binding in solution. To quantify this observation, we calculated the linear correlation coefficients, R, between the experimental and the simulated PRE data for various ratios of forward-to-backward binding in a bidirectional mixture (Figure S5B). The resulting curve is bell shaped, indicating that the experimental data are more consistent with a bidirectional than a unidirectional binding model. The best correlation, with a value of ∼0.8, is generated by a model corresponding to a forward-to-backward ratio of ∼75%/25%. Although the improvement on R upon consideration of a bidirectional model is small, F-test analysis between the unidirectional forward and this optimal bidirectional model shows that the improvement in the later is statistically significant (p < 0.001), and argues for the presence of both binding orientations in solution. The observation that no model returns an R value of 1.0 may reflect both the neglect of peptide flexibility in the PRE simulation, in particular the dynamic process of LD1 helix formation on α-parvin-CHC, and/or nonspecific effects of the spin label. In conclusion, our analysis provides support for the hypothesis that both antiparallel binding modes are accessible to the LD1 peptide in solution.

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