Abstract
β-Parvin is a cytoplasmic adaptor protein that localizes to focal adhesions where it interacts with integrin-linked kinase and is involved in linking integrin receptors to the cytoskeleton. It has been reported that despite high sequence similarity to α-parvin, β-parvin does not bind paxillin, suggesting distinct interactions and cellular functions for these two closely related parvins. Here, we reveal that β-parvin binds directly and specifically to leucine-aspartic acid repeat (LD) motifs in paxillin via its C-terminal calponin homology (CH2) domain. We present the co-crystal structure of β-parvin CH2 domain in complex with paxillin LD1 motif to 2.9 Å resolution and find that the interaction is similar to that previously observed between α-parvin and paxillin LD1. We also present crystal structures of unbound β-parvin CH2 domain at 2.1 Å and 2.0 Å resolution that show significant conformational flexibility in the N-terminal α-helix, suggesting an induced fit upon paxillin binding. We find that β-parvin has specificity for the LD1, LD2, and LD4 motifs of paxillin, with K(D) values determined to 27, 42, and 73 μM, respectively, by surface plasmon resonance. Furthermore, we show that proper localization of β-parvin to focal adhesions requires both the paxillin and integrin-linked kinase binding sites and that paxillin is important for early targeting of β-parvin. These studies provide the first molecular details of β-parvin binding to paxillin and help define the requirements for β-parvin localization to focal adhesions.
Highlights
The parvins (␣, , and ␥) are a family of calponin homology (CH)4 domain-containing adaptor proteins that localize to
We have used biochemical, biophysical, and crystallographic techniques to demonstrate for the first time that -parvin directly binds paxillin and provide evidence that this interaction is important for early recruitment of -parvin to focal adhesions
This binding surface is extremely well conserved through evolution and among parvin family members (Fig. 3), suggesting that paxillin binding is a conserved function shared among the parvins
Summary
Protein Expression and Purification—Codon-optimized synthetic cDNA (GenScript; Piscataway, NJ) encoding human -parvin -Parvin proteins were expressed as N-terminal hexahistidine (His6)-tagged fusions in Escherichia coli and purified by nickel affinity (HisTrap), anion exchange (MonoQ), and/or size exclusion (Superdex) chromatography. When applicable, the His tag was removed with tobacco etch virus protease. For pulldown assays, purified -parvin and paxillin proteins were reapplied to beads. Pulldown Assays—GST-LD motif proteins on glutathione 4B beads were preblocked in 0.1% BSA and incubated with purified -parvin proteins at 0.1– 0.3 mg/ml in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 10% (v/v) glycerol). Beads were washed with binding buffer, and bound proteins were eluted and resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Equal amounts of -parvin proteins on beads were verified by Coomassie staining
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