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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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Structure of α-Parvin-CHC(A) Schematic of the domain structure of α-parvin according to SMART (Schultz et al., 1998) and the crystallized fragment α-parvin-CHC.(B) Superposition of the ribbon representations of α-parvin-CHC in blue and the type-1 CH domain of α-actinin 3 (residues 42 to 149 of 1WKU) in red. Canonical secondary structural elements include helices αA (258–279), αC (294–304), αE (320–336), αF (346–350), and αG (354–368). Features unique to α-parvin, such as the N-linker helix (249–256) and the C/E-loop containing the 3-residue insertion (313–315), are highlighted.(C) Sequence alignment of α-parvin-CHC with type-5 CH domains of other members of the parvin family. Secondary structure elements are indicated as above. Note that αD (310–312) is only found in 1 of 2 molecules in the asymmetric unit.
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fig1: Structure of α-Parvin-CHC(A) Schematic of the domain structure of α-parvin according to SMART (Schultz et al., 1998) and the crystallized fragment α-parvin-CHC.(B) Superposition of the ribbon representations of α-parvin-CHC in blue and the type-1 CH domain of α-actinin 3 (residues 42 to 149 of 1WKU) in red. Canonical secondary structural elements include helices αA (258–279), αC (294–304), αE (320–336), αF (346–350), and αG (354–368). Features unique to α-parvin, such as the N-linker helix (249–256) and the C/E-loop containing the 3-residue insertion (313–315), are highlighted.(C) Sequence alignment of α-parvin-CHC with type-5 CH domains of other members of the parvin family. Secondary structure elements are indicated as above. Note that αD (310–312) is only found in 1 of 2 molecules in the asymmetric unit.

Mentions: We initially attempted to solve the crystal structure of full-length human α-parvin, but no crystals could be obtained. However, limited proteolysis led to the identification of a stable fragment, α-parvin-CHC, that readily crystallized. This fragment includes residues 242–372 and thereby spans the entire C-terminal CH domain as defined by SMART (Schultz et al., 1998) and a portion of the inter-CH domain linker (Figure 1A). The crystal structure of α-parvin-CHC at 1.05 Å resolution was determined by molecular replacement using an ensemble of homologous type-1 CH domain structures as search model. Data collection and refinement statistics are summarized in Table 1. The refined structural model includes α-parvin residues 246–372 (molecule A) or 247–372 (molecule B) and represents the first high-resolution crystal structure of a type-5 CH domain (Figure 1B). In spite of very low levels of sequence conservation (≤26% identity) compared to canonical type-1 CH domains (see Figure S1 available online), α-parvin-CHC exhibits a typical CH domain core composed of four main α-helices (αA, αC, αE, and αG; nomenclature from Djinovic Carugo et al., 1997), which are connected by loops and shorter helical elements (αD and αF). Secondary structure matching (Krissinel and Henrick, 2004) of α-parvin-CHC with its closest homolog, the type-1 CH domain of α-actinin 3 (PDB: 1WKU), yields an RMSD of 1.19 Å in 103 equivalent Cα-positions. Atypically, however, the N-terminal boundary of α-parvin-CHC is extended by a short α-helix comprising residues 249 to 256. This “N-linker helix” tightly associates with helices A and G of the canonical CH domain through electrostatic interactions (of residues D248, D251, and D255 with K355 and R359 in αG) and hydrophobic contacts (of residues F250, L253, and F254 with L354, K355, L358, and R359 of αG and K260 and L261 of αA). We therefore conclude that the N-linker helix forms an integral part of the type-5 CH domain of α-parvin, which is in agreement with its resistance to proteolysis. Another feature peculiar to this type-5 CH domain is the loop between helices C and E, which contains a 3 (or 4)-residue insertion (relative to type-1 CH domains) that is conserved throughout the parvin family (Figures 1C and S1). However, conformational differences in this region may not be significant, since the C/E-loop structure varies between α-parvin-CHC molecules both in the same and different crystal forms and is involved in crystal packing (data not shown).


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)

Structure of α-Parvin-CHC(A) Schematic of the domain structure of α-parvin according to SMART (Schultz et al., 1998) and the crystallized fragment α-parvin-CHC.(B) Superposition of the ribbon representations of α-parvin-CHC in blue and the type-1 CH domain of α-actinin 3 (residues 42 to 149 of 1WKU) in red. Canonical secondary structural elements include helices αA (258–279), αC (294–304), αE (320–336), αF (346–350), and αG (354–368). Features unique to α-parvin, such as the N-linker helix (249–256) and the C/E-loop containing the 3-residue insertion (313–315), are highlighted.(C) Sequence alignment of α-parvin-CHC with type-5 CH domains of other members of the parvin family. Secondary structure elements are indicated as above. Note that αD (310–312) is only found in 1 of 2 molecules in the asymmetric unit.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Structure of α-Parvin-CHC(A) Schematic of the domain structure of α-parvin according to SMART (Schultz et al., 1998) and the crystallized fragment α-parvin-CHC.(B) Superposition of the ribbon representations of α-parvin-CHC in blue and the type-1 CH domain of α-actinin 3 (residues 42 to 149 of 1WKU) in red. Canonical secondary structural elements include helices αA (258–279), αC (294–304), αE (320–336), αF (346–350), and αG (354–368). Features unique to α-parvin, such as the N-linker helix (249–256) and the C/E-loop containing the 3-residue insertion (313–315), are highlighted.(C) Sequence alignment of α-parvin-CHC with type-5 CH domains of other members of the parvin family. Secondary structure elements are indicated as above. Note that αD (310–312) is only found in 1 of 2 molecules in the asymmetric unit.
Mentions: We initially attempted to solve the crystal structure of full-length human α-parvin, but no crystals could be obtained. However, limited proteolysis led to the identification of a stable fragment, α-parvin-CHC, that readily crystallized. This fragment includes residues 242–372 and thereby spans the entire C-terminal CH domain as defined by SMART (Schultz et al., 1998) and a portion of the inter-CH domain linker (Figure 1A). The crystal structure of α-parvin-CHC at 1.05 Å resolution was determined by molecular replacement using an ensemble of homologous type-1 CH domain structures as search model. Data collection and refinement statistics are summarized in Table 1. The refined structural model includes α-parvin residues 246–372 (molecule A) or 247–372 (molecule B) and represents the first high-resolution crystal structure of a type-5 CH domain (Figure 1B). In spite of very low levels of sequence conservation (≤26% identity) compared to canonical type-1 CH domains (see Figure S1 available online), α-parvin-CHC exhibits a typical CH domain core composed of four main α-helices (αA, αC, αE, and αG; nomenclature from Djinovic Carugo et al., 1997), which are connected by loops and shorter helical elements (αD and αF). Secondary structure matching (Krissinel and Henrick, 2004) of α-parvin-CHC with its closest homolog, the type-1 CH domain of α-actinin 3 (PDB: 1WKU), yields an RMSD of 1.19 Å in 103 equivalent Cα-positions. Atypically, however, the N-terminal boundary of α-parvin-CHC is extended by a short α-helix comprising residues 249 to 256. This “N-linker helix” tightly associates with helices A and G of the canonical CH domain through electrostatic interactions (of residues D248, D251, and D255 with K355 and R359 in αG) and hydrophobic contacts (of residues F250, L253, and F254 with L354, K355, L358, and R359 of αG and K260 and L261 of αA). We therefore conclude that the N-linker helix forms an integral part of the type-5 CH domain of α-parvin, which is in agreement with its resistance to proteolysis. Another feature peculiar to this type-5 CH domain is the loop between helices C and E, which contains a 3 (or 4)-residue insertion (relative to type-1 CH domains) that is conserved throughout the parvin family (Figures 1C and S1). However, conformational differences in this region may not be significant, since the C/E-loop structure varies between α-parvin-CHC molecules both in the same and different crystal forms and is involved in crystal packing (data not shown).

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