The Structure of α-Parvin CH2-Paxillin LD1 Complex Reveals a Novel Modular Recognition for Focal Adhesion Assembly
2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês
10.1074/jbc.m801270200
ISSN1083-351X
AutoresXiaoxia Wang, Koichi Fukuda, In‐Ja L. Byeon, Algirdas Vėlyvis, Chuanyue Wu, Angela M. Gronenborn, Jun Qin,
Tópico(s)Glycosylation and Glycoproteins Research
Resumoα-Parvin is an essential component of focal adhesions (FAs), which are large multiprotein complexes that link the plasma membrane and actin cytoskeleton. α-Parvin contains two calponin homology (CH) domains and its C-terminal CH2 domain binds multiple targets including paxillin LD motifs for regulating the FA network and signaling. Here we describe the solution structure of α-parvin CH2 bound to paxillin LD1. We show that although CH2 contains the canonical CH-fold, a previously defined N-terminal linker forms an α-helix that packs unexpectedly with the C-terminal helix of CH2, resulting in a novel variant of the CH domain. Importantly, such packing generates a hydrophobic surface that recognizes the Leu-rich face of paxillin-LD1, and the binding pattern differs drastically from the classical paxillin-LD binding to four-helix bundle proteins such as focal adhesion kinase. These results define a novel modular recognition mode and reveal how α-parvin associates with paxillin to mediate the FA assembly and signaling. α-Parvin is an essential component of focal adhesions (FAs), which are large multiprotein complexes that link the plasma membrane and actin cytoskeleton. α-Parvin contains two calponin homology (CH) domains and its C-terminal CH2 domain binds multiple targets including paxillin LD motifs for regulating the FA network and signaling. Here we describe the solution structure of α-parvin CH2 bound to paxillin LD1. We show that although CH2 contains the canonical CH-fold, a previously defined N-terminal linker forms an α-helix that packs unexpectedly with the C-terminal helix of CH2, resulting in a novel variant of the CH domain. Importantly, such packing generates a hydrophobic surface that recognizes the Leu-rich face of paxillin-LD1, and the binding pattern differs drastically from the classical paxillin-LD binding to four-helix bundle proteins such as focal adhesion kinase. These results define a novel modular recognition mode and reveal how α-parvin associates with paxillin to mediate the FA assembly and signaling. The adhesion of cells to the extracellular matrix is mediated by focal adhesions (FAs), 2The abbreviations used are: FA, focal adhesion; CH, calponin homology; PKL, paxillin kinase linker; PBS, paxillin binding subdomain; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; MTSSL, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate. 2The abbreviations used are: FA, focal adhesion; CH, calponin homology; PKL, paxillin kinase linker; PBS, paxillin binding subdomain; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; MTSSL, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate. which are large, dynamic protein complexes that contain integrin transmembrane receptors and many other associated proteins (1Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Annu. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1697) Google Scholar, 2Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (492) Google Scholar, 3Lo S.H. Dev. Biol. 2006; 294: 280-291Crossref PubMed Scopus (132) Google Scholar). FAs function by mechanically linking the extracellular matrix to the actin cytoskeleton, thereby promoting strong attachment of cells to the extracellular matrix. They also act as a signaling machinery that transmits diverse signals between extracellular matrix and actin, regulating a variety of cellular processes such as cell migration, proliferation, and differentiation. Extensive genetic and cell biology studies have led to the identification of many FA proteins that have either scaffolding or signaling properties or both (4Martin K.H. Slack J.K. Boerner S.A. Martin C.C. Parsons J.T. Science. 2002; 296: 1652-1653Crossref PubMed Scopus (197) Google Scholar, 5Zaidel-Bar R. Itzkovitz S. Ma'ayan A. Iyengar R. Geiger B. Nat. Cell Biol. 2007; 9: 858-867Crossref PubMed Scopus (874) Google Scholar). Interestingly, all of these proteins are found almost exclusively as multidomain proteins that engage in multiple protein-protein interactions, leading to the formation of complex FA interaction network. The spatiotemporal assembly and disassembly of this network during various cell adhesive processes has been the subject of intense studies over the past decades (1Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Annu. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1697) Google Scholar, 2Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (492) Google Scholar, 5Zaidel-Bar R. Itzkovitz S. Ma'ayan A. Iyengar R. Geiger B. Nat. Cell Biol. 2007; 9: 858-867Crossref PubMed Scopus (874) Google Scholar), but the structural basis for how specific domains recognize each other to form the multiprotein FA complexes remain poorly understood. Parvins are a family (α,β,γ) of adaptor proteins (∼42 kDa) found in FAs (for review, see Refs. 6Sepulveda J.L. Wu C. Cell Mol. Life Sci. 2006; 63: 25-35Crossref PubMed Scopus (72) Google Scholar and 7Legate K.R. Montanez E. Kudlacek O. Fassler R. Nat. Rev. Mol. Cell Biol. 2006; 7: 20-31Crossref PubMed Scopus (543) Google Scholar). They contain two tandem copies of calponin homology (CH) domains and have been shown to play crucial roles in mediating the FA assembly and cell adhesion regulation via their CH domains, primarily the C-terminal CH2 (Fig. 1A). However, parvin CHs have very low sequence homology to conventional CH domains, which led to their classification as a distinct subfamily of CH-containing proteins (8Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (265) Google Scholar). Among parvins, α-parvin (also called actopaxin or CH-ILKBP) is the most extensively characterized (6Sepulveda J.L. Wu C. Cell Mol. Life Sci. 2006; 63: 25-35Crossref PubMed Scopus (72) Google Scholar). α-Parvin binds multiple proteins including paxillin, a well known FA regulator that contains five distinct N-terminal Leu-Asp (LD)-rich motifs (Fig. 1B) and four C-terminal LIM domains (9Schaller M.D. Oncogene. 2001; 20: 6459-6472Crossref PubMed Scopus (433) Google Scholar, 10Brown M.C. Turner C.E. Physiol. Rev. 2004; 84: 1315-1339Crossref PubMed Scopus (515) Google Scholar). The paxillin LD1 and LD4 motifs have been shown to trigger the early formation of focal adhesions (focal complexes) by specifically interacting with α-parvin CH2 (11Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar). On the other hand, the paxillin LD motifs are known to interact with a class of four helix bundle-containing FA proteins including FAK, vinculin, and paxillin kinase linker (PKL) (10Brown M.C. Turner C.E. Physiol. Rev. 2004; 84: 1315-1339Crossref PubMed Scopus (515) Google Scholar). The paxillin LD binding to FAK FAT has been extensively characterized at the structural level (12Liu G. Guibao C.D. Zheng J. Mol. Cell. Biol. 2006; 22: 2751-2760Crossref Scopus (87) Google Scholar, 13Hayashi I. Vuori K. Liddington R.C. Nat. Struct. Biol. 2002; 9: 101-106Crossref PubMed Scopus (169) Google Scholar, 14Hoellerer M.K. Noble M.E. Labesse G. Campbell I.D. Werner J.M. Arold S.T. Structure. 2003; 11: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Gao G. Prutzman K.C. King M.L. Scheswohl D.M. DeRose E.F. London R.E. Schaller M.D. Campbell S.L. J. Biol. Chem. 2004; 279: 8441-8451Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Bertolucci C.M. Guibao C.D. Zheng J. Protein Sci. 2005; 14: 644-652Crossref PubMed Scopus (48) Google Scholar). A universally conserved paxillin binding subdomain (termed PBS) for the LD binding has been thought to exist in all paxillin LD-binding proteins (17Brown M.C. Curtis M.S. Turner C.E. Nat. Struct. Biol. 1998; 5: 677-678Crossref PubMed Scopus (99) Google Scholar) including α-parvin CH2 (11Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar). However, the conventional CH domain is not a four-helix bundle (18Djinovic Carugo K. Bañuelos S. Saraste M. Nat. Struct. Biol. 1997; 4: 175-179Crossref PubMed Scopus (108) Google Scholar, 19Bañuelos S. Saraste M. Djinović Carugo K. Structure. 1998; 6: 1419-1431Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and the recognition of α-parvin CH2 by paxillin LDs to mediate specific focal complex formation remains unclear. As part of our effort to dissect the mechanism of the supramolecular FA assembly involving diverse protein-protein recognitions, we set out to investigate the molecular details of the α-parvin CH2/paxillin LD interaction. We show here that although α-parvin CH2 largely exhibits the canonical CH-fold, its N-terminal linker forms an α-helix that packs unexpectedly with the C-terminal helix of CH2, resulting in a novel variant of the CH domain. Importantly, such packing generates a hydrophobic surface that is highly conserved in the parvin family for recognizing the paxillin LD motifs. The LD binding pattern shown here is distinct from previously reported paxillin LD binding patterns to the four-helix bundle protein FAK, and the LD binding site also differs from the previously defined PBS. Thus, our results define a novel mode of modular recognition and reveal how α-parvin spatially associates with paxillin to mediate the specific multiprotein complex for FA assembly and signaling. Sample Preparation—DNA encoding for human α-parvin amino acid residues 222-372 and 244-372 were inserted into PET3a vector and PET15b (Novagen), respectively, transformed into Escherichia coli BL21(DE3), grown at 37 °C in minimal media to an A600 of 0.6-0.8, induced by 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and then left to grow for 3 h. 15N-Labeled and 15N/13C-labeled proteins were obtained by growing cultures in minimal media with 1.1 g of 15NH4Cl and/or 3.0 g of [13C]glucose as nitrogen and carbon sources, respectively. Cells were harvested by centrifugation, resuspended in 100 ml of lysis buffer (50 mm Tris-Cl, 50 mm NaCl, 1 mm EDTA, and 25 mg lysozyme, pH 8.0), then lysed by French Press twice. α-Parvin-(222-372) was purified with a DEAE column (GE Healthcare) using 50 mm Tris-Cl, pH 8.0, and a 0-1 m NaCl gradient. Fractions containing the target protein were concentrated and further purified by Superdex 75 (Amersham Biosciences) using 100 mm NaCl, 50 mm sodium phosphate buffer, pH 6.8. α-Parvin-(244-372) (α-parvin-C, see the text) with an N-terminal His tag was purified by nickel resin followed by the cleavage of the His tag and gel-filtration using Superdex G-75. Note that there is a natural Factor Xa cleavage site on 243-244 in the α-parvin-(222-372) construct so α-parvin-C-(244-372) can be also purified by Factor Xa cleavage on α-parvin-(222-372) followed by gel filtration. Peptides corresponding to LD1, N-terminal Cys-attached LD1, LD4, and mutant LD1 were synthesized and purified by the Lerner Research Institute (LRI) biotechnology core. The sample for NMR structure determination contained 0.4 mm [15N,13C]α-parvin-C plus 1 mm LD1 (natural abundance) in 50 mm sodium phosphate buffer and 100 mm NaCl, pH 6.8. α-Parvin-C at higher protein concentrations (>0.5 mm) and lower salt concentrations (<50 mm) tends to oligomerize as judged by NMR line broadening. For heteronuclear single quantrum coherence experiments, 0.25 mm 15N-labeled α-parvin-C was mixed with LD1, LD4, and all LD1 mutants in a 1:3 ratio. For chemical shift mapping analysis, 15N-labeled α-parvin-C was mixed with LD peptides in 1:1, 1:2, 1:3, and 1:4 ratios in a dose-dependent manner so that the peak movement can be traced. Limited Protease Digestion—α-Parvin-(222-372) (∼5 mg/ml) in 20 mm Tris, pH 7.5, 150 mm NaCl, 0.2 mm TCEP was treated with trypsin (Sigma) at a 1:200 (w/w) ratio of trypsin to α-parvin-(222-372), and the solution mixture was incubated at room temperature for 24 h. The proteolysis reaction was terminated by the addition of 1 mm phenylmethylsulfonyl fluoride and the solution was loaded onto Resource-Q column (GE Healthcare) equilibrated in 20 mm Tris, pH 7.5, 0.2 mm TCEP. α-Parvin-(222-372) was eluted with a linear gradient method of 0-1 m NaCl in 20 mm Tris, pH 7.5, 0.2 mm TCEP. Trypsin-cleaved α-parvin-(222-372) was separated by SDS-PAGE in 10% acrylamide gels, and transferred to Immobilon-P Transfer Membrane (Millipore) by a Trans-Blot SD Semi-dry Electrophoretic Transfer Cell (Bio-Rad). The membrane was excised with a razor blade, as guided by acrylamide gels stained with Coomassie Brilliant Blue, and washed extensively in Milli-Q water. N-terminal sequencing was performed using an Applied Biosystems Procise Sequencing System, model 492, attached to a model 140C Microgradient System and a 610A Data Analysis System (LRI Biotechnology Core). NMR Spectroscopy—All two- and three-dimensional heteronuclear NMR experiments were performed as previously described (20Clore G.M. Gronenborn A.M. Curr. Opin. Chem. Biol. 1998; 2: 564-570Crossref PubMed Scopus (103) Google Scholar). To maintain protein stability, all three-dimensional experiments were performed at 15 °C. NMR data were acquired using Bruker 500, 600, and 800 MHz spectrometers equipped with cryogenic probes. For backbone assignment, three-dimensional HNCACB and CBCA(CO)NH, HNCO, and HNCA were used. Side chain carbon and proton chemical shifts were obtained from C(CO)NH, HC(CO)NH, and HCCH-TOCSY experiments. Because of the interference of water signal, HCCH-TOCSY in 2H2O was used. Three-dimensional 13C/15N-edited NOESY (mixing time = 150 ms) was used to obtain NOE restraints. Three-dimensional 15N/13C-filtered NOESY (mixing time 150 ms) in 1H2 O or three-dimensional 13 C-filtered NOESY in 2H2O were performed to retrieve intermolecular NOE constraints between 15N/13C-labeled α-parvin-C in complex with unlabeled LD1. 1H assignment of unlabeled LD1 in the complex was made using two-dimensional 15N/13C-filtered NOESY and TOCSY. NOE constraints for the bound LD1 were obtained from two-dimensional 15N/13C-filtered NOESY as well as the transferred NOE experiment where 1 mm LD1 was mixed with 10% α-parvin-C fused to glutathione S-transferase (mixing time 400 ms). Note that because of its small size (10 residues, ∼1.0 kDa), the free LD1 peptide is like a small molecule with a fast tumbling rate, exhibiting almost no NOEs in our two-dimensional NOESY experiment (data not shown). However, when bound to CH2, we were able to see NOEs for the peptide in our transferred NOE experiment and two-dimensional-filtered NOESY experiment. These NOEs, which include sequential NHi - NHi+1 connectivity characteristic of α-helix, must be from the bound peptide, were then used for structure calculations. All NMR data were processed using NMRPipe (21Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11450) Google Scholar) and analyzed by PIPP (22Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (800) Google Scholar). For chemical shift mapping, weighted chemical shift changes were calculated using the equation: Δδobs[HN,N] = ([ΔδHN WHN]2 + [ΔδN WN]2)½, where WHN = 1 and WN = 0.154 are weighting factors based on the gyromagnetic ratios of 1H and 15N. KD was calculated using the approach as previously described (23Velyvis A. Vaynberg J. Yang Y. Vinogradova O. Zhang Y. Wu C. Qin J. Nat. Struct. Biol. 2003; 10: 558-564Crossref PubMed Scopus (55) Google Scholar). Paramagnetic Spin-labeling Experiment—The cysteine-specific spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate, MTSSL) was purchased from Sigma, Inc. MTSSL was attached to the synthetic N-ter-Cys-LD1 peptide using the following procedure: 1.0 mm LD1 peptide and a 10-fold excess of MTSSL were mixed and stirred for 12 h in a 4:1 (v/v) solution of 130 mm NaCl, 20 mm sodium phosphate buffer (pH 7.2), and acetonitrile. Spin-labeled LD1 was then purified by reverse-phase high-performance liquid chromatography and confirmed by mass spectroscopy. Structure Calculations—Structure calculation for the α-parvin-C·LD1 complex was performed using a combination of HADDOCK (23Velyvis A. Vaynberg J. Yang Y. Vinogradova O. Zhang Y. Wu C. Qin J. Nat. Struct. Biol. 2003; 10: 558-564Crossref PubMed Scopus (55) Google Scholar) and XPLOR-NIH (24Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2137) Google Scholar). First, the structures of α-parvin-C and LD1 were calculated separately using Xplor-NIH. The constraints included NOE-derived distances that had upper bounds of 2.5, 3.5, and 5.0 Å and the backbone φ,ψ angles obtained from program TALOS. Although the intermolecular NOEs from the α-parvin-C side can be unambiguously assigned, the majority of intermolecular NOEs involving LD1 Leu methyls are quite degenerate. However, we know that every Leu is involved in binding based on our mutagenesis data. Thus, we decided to first use the HADDOCK program to dock LD1 onto α-parvin-C. HADDOCK represents high ambiguity driven protein-protein docking, which is based on experimental data such as chemical shift perturbation data from NMR titration experiments or mutagenesis data (25Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1854) Google Scholar). Based on the chemical shift mapping data and intermolecular NOEs, we defined 10 active residues on α-parvin-C: Ala249, Thr252, Leu253, His256, Asp259, Lys260, Val264, Lys266, Ile269, and Glu287, and four passive residues: Asp251, Asp255, Phe271, and Val272. Four leucines in LD1 (Leu4, Leu7, Leu8, and Leu11) were defined as active residues, whereas the other six residues were passive residues. The side chains of all LD1 residues and α-parvin-C residues 244-272 were allowed for rearrangement. 1000 structures were calculate after rigid-body docking, followed by 200 structures after semiflexible simulated annealing, and 200 structures after water refinement. The final 200 structures by HADDOCK were found to converge quite well except that the bound LD1 was found in two opposite orientations (both orientations are roughly 50%). This was probably due to the ambiguous Leu constraints from LD1, which do not differentiate the two LD1 orientations. However, our spin labeling data readily eliminated one orientation because the N-terminal end of LD1 is spatially close to the linker especially Ala249 and Phe250 and the surrounding residues (supplemental Fig. S1). Using this HADDOCK-based structure, we were able to distinguish between previously ambiguous NOEs derived from the Leu side chains and further refine the structure. Hydrogen-bond restraints were incorporated during final stage of calculations based on hydrogen-exchange data and secondary-structure elements identified from previous rounds of structure calculations. Table 1 lists the detailed structural statistics of 20 final structures with the lowest energies. The PDB code for the final 20 structures and the NMR constraints is 2K2R. The BMRB accession code for the chemical shift assignments is 15760. Molecular images were drawn using PyMOL from The PyMOL Molecular Graphics System.TABLE 1Structural statistics of α-parvin-C·LD1 complexParametersNOE distance constraintsAll1422Sequential (|i − j| = 1)327 (15)aConstraints in the parentheses are for LD1.Medium (1 < |i − j| ≤ 5)193Long range (|i − j| > 5)172Intraresidue427 (37)aConstraints in the parentheses are for LD1.Intermolecular27H-bond256 (24)aConstraints in the parentheses are for LD1.Root mean square deviation from idealized covalent geometryBonds (Å)0.0079 ± 0.0008Angles (degree)0.873 ± 0.028Impropers (degree)0.628 ± 0.057ELJ (kcal mol−1)−509.1 ± 11.8Ramachandran plotMost favored regions (%)74.8 ± 3.6Additionally allowed regions (%)19.9 ± 4.3Generally allowed regions (%)4.4 ± 2.0Disallowed regions (%)0.8 ± 0.8Average root mean square deviation to the mean structurebα-Parvin-C-(249-372) and LD1-(3-12).Backbone (Å)0.61 ± 0.12Heavy atoms (Å)1.15 ± 0.21a Constraints in the parentheses are for LD1.b α-Parvin-C-(249-372) and LD1-(3-12). Open table in a new tab Structural Characterization of the α-Parvin/Paxillin Interaction—Previous biochemical and cell biology studies have shown that the C-terminal fragment of α-parvin-(222-372) binds not only to paxillin LD1, but also to LD4 (11Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar). Structure-based sequence analysis designated residues 222-263 to be part of a 60-residue linker between CH1 and CH2, with residues 264-372 forming CH2 (26Olski T.M. Noegel A.A. Korenbaum E. J. Cell Sci. 2001; 114: 525-538Crossref PubMed Google Scholar). However, although α-parvin CH2 is highly conserved in the parvin family, it has very low sequence homology to other actin-binding CH2 domains ( 75% identity, Fig. 1A), this spatial arrangement is likely conserved in the parvin family. Thus we have identified a novel variant of the CH-fold that may be specific for the parvin-mediated protein-protein interactions. LD1/α-Parvin-C Interface—LD1 in the bound state exhibits an amphipathic helical conformation that interacts primarily with the N-terminal αL-turn-αA segment, but also to the C-terminal αFof α-parvin-C (Fig. 3B). Importantly, packing of the linker helix αL with αA and αFin α-parvin-C generates a continuous hydrophobic surface that recognizes the Leu-rich face of the LD1 helix (Fig. 4). This interface involves all of the Leu residues from LD1 and the hydrophobic side chains of Ala249, Thr252, Leu253, Ala257, Lys260, Val264, and Thr267 from α-parvin-C (Fig. 4). The importance of the LD1 Leu residues is underscored by the aforementioned mutagenesis data in which every Leu/Ala mutation was found to disrupt the LD1 binding to α-parvin-C (supplemental Fig. S3). The previously defined linker region 249-260 makes significant contact with LD1 consistent with the chemical shift mapping and spin-labeling experiments. To further substantiate our observation, we mutated two interface residues, Thr-252 and Leu-253 in αL, which engage in multiple hydrophobic contacts with LD1 in our structure. The T252A/L253A mutant failed to bind to LD1 (supplemental Fig. S4), thereby demonstrating that αL is crucial for the α-parvin/paxillin interaction. LD1/α-Parvin Binding Topology Is Distinct from LD/FAK Complex—As mentioned above, paxillin LD motifs have been known to bind to a class of four-helix bundle proteins including the FAK FAT domain, vinculin, and PKL. Structural studies of the representative complex FAT/LD2 or LD4 have indicated that the LD binding mode is conserved in which the four-helix bundle interacts with two LD motifs (e.g. LD2 and LD4) at opposite faces (13Hayashi I. Vuori K. Liddington R.C. Nat. Struct. Biol. 2002; 9: 101-106Crossref PubMed Scopus (169) Google Scholar, 14Hoellerer M.K. Noble M.E. Labesse G. Campbell I.D. Werner J.M. Arold S.T. Structure. 2003; 11: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Gao G. Prutzman K.C. King M.L. Scheswohl D.M. DeRose E.F. London R.E. Schaller M.D. Campbell S.L. J. Biol. Chem. 2004; 279: 8441-8451Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Bertolucci C.M. Guibao C.D. Zheng J. Protein Sci. 2005; 14: 644-652Crossref PubMed Scopus (48) Google Scholar) (Fig. 5A). In contrast, we found that α-parvin-C has only one unique LD binding site. Furthermore, the folding of α-parvin-C and its binding topology to LD motif are drastically different from the arrangement seen for FAT (Fig. 5, A versus B). The LD target sequence in α-parvin-C versus those for the LD-FAK complex exhibit little similarity (Fig. 5c) despite the presence of hydrophobic binding features that appear to be important for both types of interactions. The main goal of this study was to understand the structural basis of α-parvin/paxillin recognition mediated by the C-terminal CH-containing fragment of the α-parvin and paxillin LD motif. Discovered nearly 18 years ago (27de Arruda M.V. Watson S. Lin C.S. Leavitt J. Matsudaira P. J. Cell Biol. 1990; 111: 1069-1079Crossref PubMed Scopus (157) Google Scholar), the CH domain has been widely known to function as a major actin binding domain. It is present in a variety of actin-binding proteins such as α-actinin, filamin, dystrophin, and spectrin. However, a growing quantity of data shows that the CH domain also plays important roles in binding to non-actin proteins, mediating diverse signaling and regulatory processes (e.g. Refs. 11Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar and 28Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (191) Google Scholar, 29LaLonde D.P. Brown M.C. Bouverat B.P. Turner C.E. J. Biol. Chem. 2005; 280: 21680-21688Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30Wei R.R. Al-Bassam J. Harrison S.C. Nat. Struct. Mol. Biol. 2007; 14: 54-59Crossref PubMed Scopus (254) Google Scholar). Numerous CH domain structures have been determined, including those of actin-binding proteins (for review, see Ref. 8Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (265) Google Scholar). However, no atomic structure of any CH domain-target complex has been reported to date. Our complex structure therefore provides the first atomic view of a CH domain-target complex. More importantly, it has resulted in several significant novel findings: (i) α-parvin-C was found to exhibit a distinct fold in which the N-terminal linker (based on sequence considerations alone) packs unexpectedly against the C-terminal helix of the canonical CH-fold. Importantly, this packing generates a hydrophobic surface that is crucial for recognizing the Leu-rich face of the paxillin LD motif. Our complex structure therefore defines a novel structural variant of the CH domain for the parvin family that is distinct from the CH domains of actin-binding proteins. The unique recognition surface in the present CH domain suggests that such a structural variant may have evolved for specifically interacting with non-actin targets, such as paxillin in focal adhesion assembly. (ii) The LD binding topology is distinctly different from that for the classical four-helix bundle proteins such as FAK FAT, vinculin, and PKL, suggesting that the paxillin LD motifs may engage in variable binding modes with different targets, triggering distinct signaling events. (iii) The LD1 binding site in our structure is quite different from the previously suggested LD binding site (PBS) (Lys277-Leu285, see Fig. 1A). We note that the PBS was identified based on sequence homology by which Lys277-Leu285 was found to be similar to the PBS in other paxillin LD-binding proteins including FAK, vinculin, and PKL (11Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1448Crossref PubMed Scopus (170) Google Scholar). Although point mutations of the PBS residues, V282G/L285R, abolished α-parvin binding to the paxillin LD motif, we note that Val282 and Leu285 in our structure are deeply buried in a hydrophobic core of α-parvin-C and thus their mutations into Gly or highly charged Arg likely disrupt the hydrophobic core and the structural integrity of the α-parvin-C-fold, which in turn impaired the α-parvin binding to paxillin. Recent structural studies have shown that LD binding sites on FAK also do not conform well the PBS rule (14Hoellerer M.K. Noble M.E. Labesse G. Campbell I.D. Werner J.M. Arold S.T. Structure. 2003; 11: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Gao G. Prutzman K.C. King M.L. Scheswohl D.M. DeRose E.F. London R.E. Schaller M.D. Campbell S.L. J. Biol. Chem. 2004; 279: 8441-8451Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Bertolucci C.M. Guibao C.D. Zheng J. Protein Sci. 2005; 14: 644-652Crossref PubMed Scopus (48) Google Scholar). In addition to binding to paxillin, α-parvin-C has also been shown to bind to ILK (28Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (191) Google Scholar) and TESK1 (31Straube A. Merdes A. Curr. Biol. 2007; 17: 1318-1325Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It remains to be determined how these proteins recognize α-parvin-C. These proteins may share an overlapping binding site with paxillin or bind to completely different regions in α-parvin-C, which may allow synergistic or competition-based switch mechanisms for regulating the supramolecular FA assembly. For example, deletion of the linker helix α-L in α-parvin was shown to have little effect on ILK binding (28Tu Y. Huang Y. Zhang Z. Hua Y. Wu C. J. Cell Biol. 2001; 153: 585-598Crossref PubMed Scopus (191) Google Scholar). In contrast, our results showed that mutation in this region (supplemental Fig. S5) abolished α-parvin-C binding to paxillin, suggesting that ILK and paxillin may bind to distinct regions in α-parvin. Paxillin LD1 has also been indicated to bind to the ILK kinase domain (32Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Thus, although LD1 binds weakly to α-parvin-C (Fig. S2), it is possible that LD1 binds simultaneously to ILK and α-parvin forming a three-way ternary complex. Interestingly, our gel filtration data revealed that α-parvin CH1 is a dimer (data not shown) suggesting that α-parvin may be a dimer. Because paxillin LD4 also binds to α-parvin-C, it is possible that one paxillin molecule may contact both CH2 subunits in the dimeric α-parvin via its LD1 and LD4, respectively. Thus whereas the binary interaction of LD/CH2 is weak as indicated in Fig. 2 and supplemental Fig. S2, it may become significant as part of the supramolecular focal adhesion assembly. Similar results have been found in the case of weak interaction between FA proteins PINCH and Nck2 (33Vaynberg J. Fukuda T. Chen K. Vinogradova O. Velyvis A. Tu Y. Ng L. Wu C. Qin J. Mol. Cell. 2005; 17: 513-523Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In summary, we have determined the structural basis of how paxillin and α-parvin specifically assemble as a critical component within FAs. Our structure provides the first atomic view of a novel type of modular interaction involving an LD motif and a distinct CH domain, providing the specificity for the paxillin/parvin interaction. The nature of the LD binding site suggests that a significantly revised assembly model has to be considered for α-parvin binding to paxillin and the regulation of higher order complex assembly and the formation of FAs. Because dysfunctions of paxillin or α-parvin have been implicated in numerous human disease developments (6Sepulveda J.L. Wu C. Cell Mol. Life Sci. 2006; 63: 25-35Crossref PubMed Scopus (72) Google Scholar, 10Brown M.C. Turner C.E. Physiol. Rev. 2004; 84: 1315-1339Crossref PubMed Scopus (515) Google Scholar), specific structure-based manipulation of the paxillin/α-parvin interaction may be useful not only for understanding the disease pathogenesis but also for developing new ways to treat these diseases. We thank Xiaolun Zhang, Xiangming Kong, Keyang Ding, Xian Mao, Rick Page, and Yanwu Yang for useful discussions and technical assistance. Download .pdf (.27 MB) Help with pdf files
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