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• W2914284294 abstract "Focal adhesions (FAs) are specialized sites where intracellular cytoskeleton elements connect to the extracellular matrix and thereby control cell motility. FA assembly depends on various scaffold proteins, including the G protein–coupled receptor kinase-interacting protein 1 (GIT1), paxillin, and liprin-α. Although liprin-α and paxillin are known to competitively interact with GIT1, the molecular basis governing these interactions remains elusive. To uncover the underlying mechanisms of how GIT1 is involved in FA assembly by alternatively binding to liprin-α and paxillin, here we solved the crystal structures of GIT1 in complex with liprin-α and paxillin at 1.8 and 2.6 Å resolutions, respectively. These structures revealed that the paxillin-binding domain (PBD) of GIT1 employs distinct binding modes to recognize a single α-helix of liprin-α and the LD4 motif of paxillin. Structure-based design of protein variants produced two binding-deficient GIT1 variants; specifically, these variants lost the ability to interact with liprin-α only or with both liprin-α and paxillin. Expressing the GIT1 variants in COS7 cells, we discovered that the two PBD-meditated interactions play different roles in either recruiting GIT1 to FA or facilitating FA assembly. Additionally, we demonstrate that, unlike for the known binding mode of the FAT domain to LD motifs, the PBD of GIT1 uses different surface patches to achieve high selectivity in LD motif recognition. In summary, our results have uncovered the mechanisms by which GIT1's PBD recognizes cognate paxillin and liprin-α structures, information we anticipate will be useful for future investigations of GIT1–protein interactions in cells. Focal adhesions (FAs) are specialized sites where intracellular cytoskeleton elements connect to the extracellular matrix and thereby control cell motility. FA assembly depends on various scaffold proteins, including the G protein–coupled receptor kinase-interacting protein 1 (GIT1), paxillin, and liprin-α. Although liprin-α and paxillin are known to competitively interact with GIT1, the molecular basis governing these interactions remains elusive. To uncover the underlying mechanisms of how GIT1 is involved in FA assembly by alternatively binding to liprin-α and paxillin, here we solved the crystal structures of GIT1 in complex with liprin-α and paxillin at 1.8 and 2.6 Å resolutions, respectively. These structures revealed that the paxillin-binding domain (PBD) of GIT1 employs distinct binding modes to recognize a single α-helix of liprin-α and the LD4 motif of paxillin. Structure-based design of protein variants produced two binding-deficient GIT1 variants; specifically, these variants lost the ability to interact with liprin-α only or with both liprin-α and paxillin. Expressing the GIT1 variants in COS7 cells, we discovered that the two PBD-meditated interactions play different roles in either recruiting GIT1 to FA or facilitating FA assembly. Additionally, we demonstrate that, unlike for the known binding mode of the FAT domain to LD motifs, the PBD of GIT1 uses different surface patches to achieve high selectivity in LD motif recognition. In summary, our results have uncovered the mechanisms by which GIT1's PBD recognizes cognate paxillin and liprin-α structures, information we anticipate will be useful for future investigations of GIT1–protein interactions in cells. Focal adhesions (FAs) 4The abbreviations used are: FAfocal adhesionAMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidArfADP-ribosylation factorFAKfocal adhesion kinaseFATfocal adhesion kinase targetingGAPGTPase-activating proteinGIT1G-protein-coupled receptor kinase-interacting protein 1ITCisothermal titration calorimetryLDleucine-aspartic acid motifPAKp21-activated kinasePBDpaxillin-binding domainPDBProtein Data BankSAHsingle α-helixTrxthioredoxin. are protein-rich, highly specified subcellular structures in mediating the connection between cell and extracellular matrix, which are essential in various cellular processes, including cell spreading, migration, cancer invasion, and neuronal growth (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Transmembrane crosstalk between the extracellular matrix–cytoskeleton crosstalk.Nat. Rev. Mol. Cell Biol. 2001; 2 (11715046): 793-80510.1038/35099066Crossref PubMed Scopus (1841) Google Scholar2Eva R. Fawcett J. Integrin signalling and traffic during axon growth and regeneration.Curr. Opin. Neurobiol. 2014; 27 (24793179): 179-18510.1016/j.conb.2014.03.018Crossref PubMed Scopus (50) Google Scholar, 3Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.Annu. Rev. Cell Biol. 1988; 4 (3058164): 487-52510.1146/annurev.cb.04.110188.002415Crossref PubMed Scopus (1700) Google Scholar4Parsons J.T. Horwitz A.R. Schwartz M.A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension.Nat. Rev. Mol. Cell Biol. 2010; 11 (20729930): 633-64310.1038/nrm2957Crossref PubMed Scopus (1362) Google Scholar). These processes are closely associated with FA dynamics, which are regulated by FA-associated adaptor and scaffold proteins (5Burridge K. Focal adhesions: a personal perspective on a half century of progress.FEBS J. 2017; 284 (28796323): 3355-336110.1111/febs.14195Crossref PubMed Scopus (123) Google Scholar6Mitra S.K. Hanson D.A. Schlaepfer D.D. Focal adhesion kinase: in command and control of cell motility.Nat. Rev. Mol. Cell Biol. 2005; 6 (15688067): 56-6810.1038/nrm1549Crossref PubMed Scopus (1960) Google Scholar, 7Wehrle-Haller B. Structure and function of focal adhesions.Curr. Opin. Cell Biol. 2012; 24 (22138388): 116-12410.1016/j.ceb.2011.11.001Crossref PubMed Scopus (160) Google Scholar8Haase K. Al-Rekabi Z. Pelling A.E. Mechanical cues direct focal adhesion dynamics.Prog. Mol. Biol. Transl. Sci. 2014; 126 (25081616): 103-13410.1016/B978-0-12-394624-9.00005-1Crossref PubMed Scopus (18) Google Scholar). Among these FA-associated proteins, G protein–coupled receptor kinase–interacting proteins, GIT1 and GIT2 serve as GTPase-activating proteins (GAPs) for ADP-ribosylation factors (Arfs) to affect the assembly of integrin adhesion complexes to FAs (9Vitali T. Girald-Berlingeri S. Randazzo P.A. Chen P.W. Arf GAPs: a family of proteins with disparate functions that converge on a common structure, the integrin adhesion complex.Small GTPases. 2017; (28362242): 1-910.1080/21541248.2017.1299271Crossref PubMed Scopus (22) Google Scholar, 10Frank S.R. Adelstein M.R. Hansen S.H. GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover.EMBO J. 2006; 25 (16628223): 1848-185910.1038/sj.emboj.7601092Crossref PubMed Scopus (56) Google Scholar). focal adhesion α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ADP-ribosylation factor focal adhesion kinase focal adhesion kinase targeting GTPase-activating protein G-protein-coupled receptor kinase-interacting protein 1 isothermal titration calorimetry leucine-aspartic acid motif p21-activated kinase paxillin-binding domain Protein Data Bank single α-helix thioredoxin. The GIT proteins share a conserved domain architecture, which from N terminus to C terminus includes an Arf-GAP domain, three ankyrin repeats, a Spa homology domain, a coiled-coil region, and a paxillin-binding domain (PBD) (Fig. 1A). GITs are involved in regulating FA dynamics by interacting with many FA proteins (11Zhou W. Li X. Premont R.T. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.J. Cell Sci. 2016; 129 (27182061): 1963-197410.1242/jcs.179465Crossref PubMed Scopus (64) Google Scholar, 12Yoo S.M. Cerione R.A. Antonyak M.A. The Arf-GAP and protein scaffold Cat1/Git1 as a multifaceted regulator of cancer progression.Small GTPases. 2017; (28981399): 1-910.1080/21541248.2017.1362496PubMed Google Scholar). Among these interactions, the bindings of liprin-α, paxillin, and HIC5, a paxillin-like protein, to GIT1 are mediated by the PBD, folded as a four-helix bundle (13Asperti C. Astro V. Pettinato E. Paris S. Bachi A. de Curtis I. Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.PLoS One. 2011; 6 (21695141): e2075710.1371/journal.pone.0020757Crossref PubMed Scopus (9) Google Scholar14Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. Interaction between liprin-α and GIT1 is required for AMPA receptor targeting.J. Neurosci. 2003; 23 (12629171): 1667-167710.1523/JNEUROSCI.23-05-01667.2003Crossref PubMed Google Scholar, 15Zhang Z.M. Simmerman J.A. Guibao C.D. Zheng J.J. GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs.J. Biol. Chem. 2008; 283 (18448431): 18685-1869310.1074/jbc.M801274200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Schmalzigaug R. Garron M.L. Roseman J.T. Xing Y. Davidson C.E. Arold S.T. Premont R.T. GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.Cell. Signal. 2007; 19 (17467235): 1733-174410.1016/j.cellsig.2007.03.010Crossref PubMed Scopus (50) Google Scholar17Nishiya N. Shirai T. Suzuki W. Nose K. Hic-5 interacts with GIT1 with a different binding mode from paxillin.J. Biochem. 2002; 132 (12153727): 279-28910.1093/oxfordjournals.jbchem.a003222Crossref PubMed Scopus (36) Google Scholar). A similar fold is also found in focal adhesion kinase (FAK), termed the FAK-targeting (FAT) domain. Previous structural studies indicated that the FAT-like domains, including PBD, employ a similar way to recognize leucine-aspartic acid (LD) motifs, short helical interacting motifs (18Brown M.C. Curtis M.S. Turner C.E. Paxillin LD motifs may define a new family of protein recognition domains.Nat. Struct. Biol. 1998; 5 (9699628): 677-67810.1038/1370Crossref PubMed Scopus (99) Google Scholar, 19Hoellerer M.K. Noble M.E.M. Labesse G. Campbell I.D. Werner J.M. Arold S.T. Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.Structure. 2003; 11 (14527389): 1207-121710.1016/j.str.2003.08.010Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar20Alam T. Alazmi M. Gao X. Arold S.T. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs.Biochem. J. 2014; 460 (24870021): 317-32910.1042/BJ20140298Crossref PubMed Scopus (31) Google Scholar). The liprin-α family is composed of four highly conserved members (liprin-α1/2/3/4) in mammals; each contains the N-terminal coiled-coils and the three C-terminal sterile α motif repeats (21Serra-Pagès C. Medley Q.G. Tang M. Hart A. Streuli M. Liprins, a family of LAR transmembrane protein-tyrosine phosphatase-interacting proteins.J. Biol. Chem. 1998; 273 (9624153): 15611-1562010.1074/jbc.273.25.15611Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 22Zürner M. Schoch S. The mouse and human Liprin-α family of scaffolding proteins: genomic organization, expression profiling and regulation by alternative splicing.Genomics. 2009; 93 (19013515): 243-25310.1016/j.ygeno.2008.10.007Crossref PubMed Scopus (38) Google Scholar) (Fig. 1A). As the synaptic scaffold, liprin-α was identified as the GIT1-binding partner in neurons to mediate AMPA receptor targeting (14Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. Interaction between liprin-α and GIT1 is required for AMPA receptor targeting.J. Neurosci. 2003; 23 (12629171): 1667-167710.1523/JNEUROSCI.23-05-01667.2003Crossref PubMed Google Scholar). In addition to the well-known roles of liprin-α in neurons, emerging evidence has indicated that liprin-α plays an important role in nonneuronal cells by mediating the FA turnover during integrin-mediated migration (23Asperti C. Astro V. Totaro A. Paris S. de Curtis I. Liprin-α1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.J. Cell Sci. 2009; 122 (19690048): 3225-323210.1242/jcs.054155Crossref PubMed Scopus (49) Google Scholar, 24Astro V. Tonoli D. Chiaretti S. Badanai S. Sala K. Zerial M. de Curtis I. Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover.Sci. Rep. 2016; 6 (27659488): 3365310.1038/srep33653Crossref PubMed Scopus (28) Google Scholar). Paxillin acts as a scaffold to recruit different proteins in FAs via its five LD motifs and four LIM (Lin11, Isl-1, and Mec-3) domains (18Brown M.C. Curtis M.S. Turner C.E. Paxillin LD motifs may define a new family of protein recognition domains.Nat. Struct. Biol. 1998; 5 (9699628): 677-67810.1038/1370Crossref PubMed Scopus (99) Google Scholar) (Fig. 1A). By connecting integrin to F-actin and other FA-associated proteins, paxillin plays a critical role in assembly and disassembly of FAs (25Brown M.C. Turner C.E. Paxillin: adapting to change.Physiol. Rev. 2004; 84 (15383653): 1315-133910.1152/physrev.00002.2004Crossref PubMed Scopus (516) Google Scholar). Paxillin was suggested to recruit GIT1 to FA by the binding of the LD4 motif of paxillin to the PBD of GIT1, as the deletion of the LD4 motif or the PBD resulted in the disruption of localization of GIT1 to FA (16Schmalzigaug R. Garron M.L. Roseman J.T. Xing Y. Davidson C.E. Arold S.T. Premont R.T. GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.Cell. Signal. 2007; 19 (17467235): 1733-174410.1016/j.cellsig.2007.03.010Crossref PubMed Scopus (50) Google Scholar, 26West K.A. Zhang H.Y. Brown M.C. Nikolopoulos S.N. Riedy M.C. Horwitz A.F. Turner C.E. The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).J. Cell Biol. 2001; 154 (11448998): 161-17610.1083/jcb.200101039Crossref PubMed Scopus (146) Google Scholar, 27Manabe R. Kovalenko M. Webb D.J. Horwitz A.R. GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration.J. Cell Sci. 2002; 115 (11896197): 1497-1510Crossref PubMed Google Scholar). Although the PBD is crucial for the subcellular localization and function of GIT1, the molecular mechanisms underlying the PBD-mediated interactions remain elusive. A recent study indicated that the bindings of liprin-α and paxillin to GIT1 are mutually exclusive (13Asperti C. Astro V. Pettinato E. Paris S. Bachi A. de Curtis I. Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.PLoS One. 2011; 6 (21695141): e2075710.1371/journal.pone.0020757Crossref PubMed Scopus (9) Google Scholar). However, without detailed interaction information, it would be difficult to understand how these competitive interactions are coordinated and regulated in FA. Here, we determined crystal structures of GIT1_PBD in complex with a single α-helix (SAH) of liprin-α2 and the LD4 motif of paxillin. Although liprin-α2_SAH and paxillin_LD4 associate with the same binding groove on GIT1_PBD, surprisingly, these two helical peptides adopt binding orientations reversed to each other. Cellular analysis using the designed GIT1 mutations indicated that the GIT1/paxillin interaction controls the GIT1's localization to FA and the GIT1/liprin-α interaction promotes FA assembly. The structure of the GIT1_PBD/paxillin_LD4 complex reveals a novel LD recognition mode that involves a new surface patch on GIT1_PBD in addition to the canonical LD binding surface found in the FAT domain of FAK. To understand the assembly mechanism of GIT1, liprin-α, and paxillin, we investigated the binding of GIT1 to liprin-α and paxillin, which is mediated by the paxillin-binding domain of GIT1 (GIT1_PBD). To narrow down the minimal PBD-binding region in liprin-α2, various fragments were expressed in Escherichia coli and purified with high quality (Fig. 1A). As indicated by an isothermal titration calorimetry (ITC)-based assay, a small fragment containing residues 642–671 in liprin-α2 interacts with GIT1_PBD with a binding affinity of ∼3 μm (Fig. 1D). Neither an extension at the N-terminal nor at the C-terminal part of the fragment could significantly increase the binding affinity (Fig. S1A), suggesting that the 30-residue fragment contains essential and sufficient elements for the binding of liprin-α2 to GIT1_PBD. Although this fragment was predicted as a coiled-coil (14Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. Interaction between liprin-α and GIT1 is required for AMPA receptor targeting.J. Neurosci. 2003; 23 (12629171): 1667-167710.1523/JNEUROSCI.23-05-01667.2003Crossref PubMed Google Scholar), the molecular weight of the fragment measured by multiangle static light scattering matches the theoretical molecular weight of its monomeric state (Fig. 1C and Fig. S1B). Combining the CD spectrum of the fragment (Fig. S1C), we concluded that the fragment folds as a single α-helix. Therefore, the fragment in liprin-α2 was named hereafter as SAH. Similar to liprin-α2_SAH, the LD4 motif of paxillin (paxillin_LD4), also a single α-helix, interacts with GIT1_PBD with a binding affinity of ∼4 μm (Fig. 1E), consistent with the previous reports (15Zhang Z.M. Simmerman J.A. Guibao C.D. Zheng J.J. GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs.J. Biol. Chem. 2008; 283 (18448431): 18685-1869310.1074/jbc.M801274200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Schmalzigaug R. Garron M.L. Roseman J.T. Xing Y. Davidson C.E. Arold S.T. Premont R.T. GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.Cell. Signal. 2007; 19 (17467235): 1733-174410.1016/j.cellsig.2007.03.010Crossref PubMed Scopus (50) Google Scholar). As the bindings of liprin-α and paxillin to GIT1_PBD are mutually exclusive (13Asperti C. Astro V. Pettinato E. Paris S. Bachi A. de Curtis I. Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.PLoS One. 2011; 6 (21695141): e2075710.1371/journal.pone.0020757Crossref PubMed Scopus (9) Google Scholar) and liprin-α contains a LD-like motif in the SAH region (Fig. 1B), do liprin-α and paxillin share a similar LD-binding model, interacting with GIT1? To address this question, we aimed to solve the complex structures of GIT1_PBD/liprin-α2_SAH and GIT1_PBD/paxillin_LD4 by using X-ray crystallography. Gel filtration analysis showed that GIT1_PBD forms a stable complex with either liprin-α2_SAH or paxillin_LD4 in solution (Fig. S2, A and B). Crystallization trails for the two complex samples yielded high-quality crystals. The crystal structures of the two complexes, GIT1_PBD/liprin-α2_SAH and GIT1_PBD/paxillin_LD4, were successfully determined at 1.8 and 2.6 Å resolutions, respectively, by using the molecular replacement method (Table 1). Most residues of liprin-α2_SAH (29 of the 30 total residues) and paxillin_LD4 (21 of the 24 total residues) in these two structures were clearly assigned (Fig. 2, A and B). As a FAT-like domain, GIT1_PBD adopts the four-helix-bundle conformation. The overall structures of GIT1_PBD in the two complexes are essentially same as its apo-form structure (overall root mean square deviations of 0.9 and 1.0 Å, respectively), indicating that GIT1_PBD does not undergo conformational change upon target binding. Consistent with our biochemical analysis, both liprin-α2_SAH and paxillin_LD4 form single helices to interact with GIT1_PBD on the same surface composed of the first helix (H1) and the last helix (H4) (Fig. 2, C and D), well explaining the competitive binding of liprin-α and paxillin to GIT1_PBD (13Asperti C. Astro V. Pettinato E. Paris S. Bachi A. de Curtis I. Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.PLoS One. 2011; 6 (21695141): e2075710.1371/journal.pone.0020757Crossref PubMed Scopus (9) Google Scholar).Table 1Statistics of data collection and model refinementData collection Data setGIT1_PBD/liprin-α2_SAHGIT1_PBD/paxillin_LD4 Space groupP212121I4 Unit cell parameters a, b, c (Å)88.2, 38.6, 99.1128.8, 128.8, 47.6 α, β, γ (degrees)90, 90, 9090, 90, 90 Resolution range (Å)50–1.8 (1.83–1.8)50–2.6 (2.64–2.6) No. of unique reflections32,467 (1606)12,341 (621) Redundancy5.3 (5.6)6.7 (7.0) I/σ25.5 (2.0)35.1 (1.5) Completeness (%)99.2 (100)99.9 (100) Rmerge (%)aRmerge = Σ|Ii − Im|/ΣIi, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.9.8 (84.2)5.4 (98.6)Structure refinement Resolution (Å)50–1.8 (1.85–1.8)50–2.6 (2.86–2.6) Rcryst/Rfree (%)bR cryst = Σ‖Fo| − |Fc‖/Σ|Fo|, where Fo and Fc are observed and calculated structure factors. Rfree = ΣT‖Fo| − |Fc‖/ΣT|Fo|, where T is a test data set of about 5% of the total reflections randomly chosen and set aside prior to refinement.22.7 (32.5)/26.7 (33.4)19.4 (25.8)/22.6 (30.1) Root mean square deviations Bonds (Å)0.0030.002 Angles (degrees)0.60.5 Average B factor39.4131.0 No. of atoms Protein24382070 Ligand/ion30 Water molecules1410 Ramachandran plot Favored regions (%)99.798.9 Allowed regions (%)0.31.1 Outliner (%)00a Rmerge = Σ|Ii − Im|/ΣIi, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.b R cryst = Σ‖Fo| − |Fc‖/Σ|Fo|, where Fo and Fc are observed and calculated structure factors. Rfree = ΣT‖Fo| − |Fc‖/ΣT|Fo|, where T is a test data set of about 5% of the total reflections randomly chosen and set aside prior to refinement. Open table in a new tab Paxillin_LD4 interacts with GIT1_PBD using the similar LD-binding mode for FAT-like domains, in which the LD4 helix is parallel to the H1 helix (19Hoellerer M.K. Noble M.E.M. Labesse G. Campbell I.D. Werner J.M. Arold S.T. Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.Structure. 2003; 11 (14527389): 1207-121710.1016/j.str.2003.08.010Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 28Lulo J. Yuzawa S. Schlessinger J. Crystal structures of free and ligand-bound focal adhesion targeting domain of Pyk2.Biochem. Biophys. Res. Commun. 2009; 383 (19358827): 347-35210.1016/j.bbrc.2009.04.011Crossref PubMed Scopus (31) Google Scholar, 29Vanarotti M.S. Finkelstein D.B. Guibao C.D. Nourse A. Miller D.J. Zheng J.J. Structural basis for the interaction between Pyk2-FAT domain and leupaxin LD repeats.Biochemistry. 2016; 55 (26866573): 1332-134510.1021/acs.biochem.5b01274Crossref PubMed Scopus (13) Google Scholar30Vanarotti M.S. Miller D.J. Guibao C.D. Nourse A. Zheng J.J. Structural and mechanistic insights into the interaction between Pyk2 and paxillin LD motifs.J. Mol. Biol. 2014; 426 (25174335): 3985-400110.1016/j.jmb.2014.08.014Crossref PubMed Scopus (12) Google Scholar) (Fig. 2D and Fig. S3). However, to our surprise, despite containing an LD-like motif, liprin-α2_SAH binds to GIT1_PBD with the reverse orientation, in which the SAH helix is antiparallel to the H1 helix (Fig. 2C). Although the interacting orientations are different for the two PBD-binding peptides, both liprin-α2_SAH and paxillin_LD4 use their hydrophobic sides in their amphipathic helices to tightly pack with the H1/H4 groove of GIT1_PBD through hydrophobic interactions, which are further strengthened by hydrogen bondings (Fig. 2, E and F). In support of our findings, previous structural studies reported that hydrophobic interactions involving the GIT1_PBD/paxillin_LD4 interaction are critical for complex formation (15Zhang Z.M. Simmerman J.A. Guibao C.D. Zheng J.J. GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs.J. Biol. Chem. 2008; 283 (18448431): 18685-1869310.1074/jbc.M801274200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Schmalzigaug R. Garron M.L. Roseman J.T. Xing Y. Davidson C.E. Arold S.T. Premont R.T. GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.Cell. Signal. 2007; 19 (17467235): 1733-174410.1016/j.cellsig.2007.03.010Crossref PubMed Scopus (50) Google Scholar). Additionally, Asp-267 in paxillin_LD4 forms a salt bridge with Lys-758 in GIT1_PBD (Fig. 2F), which is a characteristic interaction in the LD-binding mode (15Zhang Z.M. Simmerman J.A. Guibao C.D. Zheng J.J. GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs.J. Biol. Chem. 2008; 283 (18448431): 18685-1869310.1074/jbc.M801274200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Schmalzigaug R. Garron M.L. Roseman J.T. Xing Y. Davidson C.E. Arold S.T. Premont R.T. GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.Cell. Signal. 2007; 19 (17467235): 1733-174410.1016/j.cellsig.2007.03.010Crossref PubMed Scopus (50) Google Scholar, 20Alam T. Alazmi M. Gao X. Arold S.T. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs.Biochem. J. 2014; 460 (24870021): 317-32910.1042/BJ20140298Crossref PubMed Scopus (31) Google Scholar). Sequence analysis further shows that the interface residues in GIT1, liprin-α2, and paxillin are highly conserved across species (Fig. 2, G–I), suggesting that the two different GIT1-binding modes observed for liprin-α2_SAH and paxillin_LD4 are likely to be shared by other members of the liprin-α family and HIC-5, respectively. As the bindings of liprin-α2_SAH and paxillin_LD4 to GIT1_PBD are mainly mediated by hydrophobic interactions (Fig. 2, E and F), we carefully analyzed the interfaces by comparing the buried hydrophobic residues of the two bound peptides. In the LD4 peptide, Leu-266LD4, Leu-269LD4, and Leu-273LD4, the conserved leucine residues in the LD core (Fig. 1B), interact with the hydrophobic groove formed by the H1 and H4 helices of GIT1_PBD, which is a general LD-binding feature identified in other FAT-like domains (Fig. S3). Interestingly, by aligning the two peptide-bound GIT1_PBD structures together, we found that the side chains of Ile-660SAH, Leu-657SAH, and Leu-653SAH in the SAH peptide could be overlapped precisely with those of the three leucine residues in the LD4 peptide (Fig. 3A), despite the different helical orientations between the two peptides. Likewise, the reversed amino acid sequence of the SAH peptide can be aligned to the LD4 sequence with the hydrophobic residues matched (Fig. 3B). This suggests the central roles of these hydrophobic residues in the binding of liprin-α2_SAH and paxillin_LD4 to GIT1_PBD. In addition to the three overlapped residues, a few C-terminal residues of both of the two peptides significantly contribute to the intermolecular hydrophobic interactions. As shown in Fig. 3A, Ile-664SAH and Ile-667SAH in the SAH peptide interact with the upper part of the H1/H4 groove, formed by the N terminus of H1 and the C terminus of H4, whereas the bulky side chain of Phe-276LD4 in the LD4 peptide occupies the H1/H4 groove in the lower part. In contrast, the corresponding positions of Ile-664SAH and Ile-667SAH in the LD4 peptide (Ala-262LD4 and Ala-259LD4) and of Phe-276LD4 in the SAH peptide (Ala-650SAH) are all alanine residues (Fig. 3, A and B), of which small side chains barely touch the H1/H4 groove, thereby contributing little to the bindings of either paxillin_LD4 or liprin-α2_SAH to GIT1_PBD. Thus, replacing Ala-650SAH with a phenylalanine in the SAH peptide may artificially create an additional PBD-binding site by mimicking the binding of Phe-276LD4 to GIT1_PBD. In line with this hypothesis, the A650F mutation of liprin-α2_SAH largely enhances the binding (Fig. S4). Based on the above analysis, we conclude that GIT1_PBD can specifically recognize liprin-α2_SAH and paxillin_LD4 by combinatorial usage of different surface patches in the H1/H4 groove, in which the upper and lower parts of the groove provide the binding specificities for the SAH and LD4 peptides, respectively, whereas the middle part of the groove creates the common binding surface for both of the two peptides (Fig. 3A). The binding mode differences between paxillin_LD4 and liprin-α2_SAH allow us to design GIT1 mutations, A754Q and I655Q, for disruption of specific target interactions with GIT1_PBD. The A754Q mutation largely decreases the hydrophobicity of the overlapped binding surface and imposes steric hindrance for interactions, thereby disrupting the bindings of GIT1_PBD to both liprin-α2_SAH and paxillin_LD4. By contrast, as Ile-655 locates on the SAH-binding surface on GIT1_PBD, the I655Q mutation presumably prevents GIT1_PBD from binding to liprin-α2_SAH while it retains the ability of GIT1_PBD to bind with paxillin_LD4. Fully consistent with our design, neither liprin-α2_SAH nor paxillin_LD4 shows detectable binding to the A754Q mutant, whereas the I655Q mutant interacts with paxillin_LD4 but not liprin-α2_SAH, as indicated by ITC-based measurements (Fig. 3C) and analytical gel filtration analysis (Fig. S2, C–F). As the core component of FA, paxillin is indispensable for FA assembly (25Brown M.C. Turner C.E. Paxillin: adapting to change.Physiol. Rev. 2004; 84 (15383653): 1315-133910.1152/physrev.00002.2004Crossref PubMed Scopus (516) Google Scholar, 31Kanteti R. Batra S.K. Lennon F.E. Salgia R. FAK and paxillin, two potential targets in pancreatic cancer.Oncotarget. 2016; 7 (26980710): 31586-31601Crossref PubMed Scopus (66) Google Scholar). Considering the localization of GIT1 and liprin-α to FA for controlling FA dynamics (23Asperti C. Astro V. Totaro A. Paris S. de Curtis I. Liprin-α1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.J. Cel" @default.
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• W2914284294 title "Structural basis of the target-binding mode of the G protein–coupled receptor kinase–interacting protein in the regulation of focal adhesion dynamics" @default.
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