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• W2091983485 abstract "Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β1A and β1D isoforms, although the interaction is stronger with β1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3. Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β1A and β1D isoforms, although the interaction is stronger with β1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3. Integrin-mediated cell adhesion connects the cell surface to the extracellular matrix in higher eukaryotes. It is a critical and common process in a plethora of physiological phenomena, including tissue integrity, embryogenesis, bone metabolism, hemostasis, and immunity. Integrin heterodimers form one of the most important cell-extracellular matrix receptors, and tight regulation of integrin ligand affinity is crucial for cell survival. The bi-directional signaling properties of integrins give them added interest (1Hynes R.O. Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6870) Google Scholar); key steps in their intracellular activation involve binding of Talin and Kindlin to the cytoplasmic tails of the integrin β-subunits (2Anthis N.J. Campbell I.D. The tail of integrin activation.Trends Biochem. Sci. 2011; 36: 191-198Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Kim C. Ye F. Ginsberg M.H. Regulation of integrin activation.Annu. Rev. Cell Dev. Biol. 2011; 27: 321-345Crossref PubMed Scopus (321) Google Scholar). The Kindlin family of proteins has recently emerged as a crucial component of focal adhesion assembly. There is evidence that, alongside Talin, they are co-activators of integrins (4Moser M. Legate K.R. Zent R. Fässler R. The tail of integrins, Talin, and Kindlins.Science. 2009; 324: 895-899Crossref PubMed Scopus (580) Google Scholar). In mammals there are three Kindlin isoforms, Kindlins 1, 2, and 3, all multidomain cytoplasmic proteins of ∼75 kDa. The domain structure of Kindlins and their homology with the Talin head domain are illustrated in Fig. 1. Like Talin they have F0, F1, F2, and F3 subdomains but in addition have a pleckstrin homology (PH) 5The abbreviations used are: PHpleckstrin homologySAXSsmall-angle x-ray scatteringAUCanalytical ultracentrifugationSECsize-exclusion chromatographyMWCOmolecular weight cut-offbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolDLSdynamic light scatteringMDmembrane-distal. domain inserted in the F2 domain. The F1, F2, and F3 domains have homology with FERM (4.1 band, ezrin, radixin, moesin) domains (5Elliott P.R. Goult B.T. Kopp P.M. Bate N. Grossmann J.G. Roberts G.C. Critchley D.R. Barsukov I.L. The Structure of the Talin head reveals a novel extended conformation of the FERM domain.Structure. 2010; 18: 1289-1299Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 6Meves A. Stremmel C. Gottschalk K. Fässler R. The Kindlin protein family: new members to the club of focal adhesion proteins.Trends Cell Biol. 2009; 19: 504-513Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 7Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. South A.P. Smith F.J. Prescott A.R. Wessagowit V. Oyama N. Akiyama M. Al Aboud D. Al Aboud K. Al Githami A. Al Hawsawi K. Al Ismaily A. Al-Suwaid R. Atherton D.J. Caputo R. Fine J.D. Frieden I.J. Fuchs E. Haber R.M. Harada T. Kitajima Y. Mallory S.B. Ogawa H. Sahin S. Shimizu H. Suga Y. Tadini G. Tsuchiya K. Wiebe C.B. Wojnarowska F. Zaghloul A.B. Hamada T. Mallipeddi R. Eady R.A. McLean W.H. McGrath J.A. Epstein E.H. Loss of Kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular matrix linker protein UNC-112, causes Kindler syndrome.Am. J. Hum. Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). There is also a long loop inserted into the F1 domain of Kindlins that is predicted to be unfolded (8Goult B.T. Bouaouina M. Harburger D.S. Bate N. Patel B. Anthis N.J. Campbell I.D. Calderwood D.A. Barsukov I.L. Roberts G.C. Critchley D.R. The structure of the N terminus of Kindlin-1: a domain important αiibβ3 integrin activation.J. Mol. Biol. 2009; 394: 944-956Crossref PubMed Scopus (69) Google Scholar) and which, in Kindlin-3, contains 109 residues. pleckstrin homology small-angle x-ray scattering analytical ultracentrifugation size-exclusion chromatography molecular weight cut-off 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol dynamic light scattering membrane-distal. The three mammalian Kindlins display tissue-specific expression patterns. Kindlin-1 is found primarily in the epidermis and, to a lesser extent, the colon, stomach, and kidneys (9Ussar S. Wang H.V. Linder S. Fässler R. Moser M. The Kindlins: subcellular localization and expression during murine development.Exp. Cell Res. 2006; 312: 3142-3151Crossref PubMed Scopus (199) Google Scholar). Kindlin-2 is ubiquitously expressed but is concentrated in striated and smooth muscle (9Ussar S. Wang H.V. Linder S. Fässler R. Moser M. The Kindlins: subcellular localization and expression during murine development.Exp. Cell Res. 2006; 312: 3142-3151Crossref PubMed Scopus (199) Google Scholar). Kindlin-3 was considered to be exclusively expressed in hematopoietic tissues (9Ussar S. Wang H.V. Linder S. Fässler R. Moser M. The Kindlins: subcellular localization and expression during murine development.Exp. Cell Res. 2006; 312: 3142-3151Crossref PubMed Scopus (199) Google Scholar) but it has also been found recently in endothelial tissues (10Bialkowska K. Ma Y.Q. Bledzka K. Sossey-Alaoui K. Izem L. Zhang X. Malinin N. Qin J. Byzova T. Plow E.F. The integrin co-activator Kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell.J. Biol. Chem. 2010; 285: 18640-18649Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Kindlin-3 was first identified during screening for B-cell-specific plasma membrane proteins in chronic lymphocytic leukemia (11Boyd R.S. Adam P.J. Patel S. Loader J.A. Berry J. Redpath N.T. Poyser H.R. Fletcher G.C. Burgess N.A. Stamps A.C. Hudson L. Smith P. Griffiths M. Willis T.G. Karran E.L. Oscier D.G. Catovsky D. Terrett J.A. Dyer M.J. Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B.Leukemia. 2003; 17: 1605-1612Crossref PubMed Scopus (66) Google Scholar). It has significant homology with Kindlin-1, which is mutated in Kindler syndrome (7Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. South A.P. Smith F.J. Prescott A.R. Wessagowit V. Oyama N. Akiyama M. Al Aboud D. Al Aboud K. Al Githami A. Al Hawsawi K. Al Ismaily A. Al-Suwaid R. Atherton D.J. Caputo R. Fine J.D. Frieden I.J. Fuchs E. Haber R.M. Harada T. Kitajima Y. Mallory S.B. Ogawa H. Sahin S. Shimizu H. Suga Y. Tadini G. Tsuchiya K. Wiebe C.B. Wojnarowska F. Zaghloul A.B. Hamada T. Mallipeddi R. Eady R.A. McLean W.H. McGrath J.A. Epstein E.H. Loss of Kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular matrix linker protein UNC-112, causes Kindler syndrome.Am. J. Hum. Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Kindlin-3 knock-out mice suffer from severe bleeding because of inactive platelet integrins and die of anemia (12Moser M. Bauer M. Schmid S. Ruppert R. Schmidt S. Sixt M. Wang H.V. Sperandio M. Fässler R. Kindlin-3 is required for β2 integrin-mediated leukocyte adhesion to endothelial cells.Nat. Med. 2009; 15: 300-305Crossref PubMed Scopus (288) Google Scholar, 13Moser M. Nieswandt B. Ussar S. Pozgajova M. Fässler R. Kindlin-3 is essential for integrin activation and platelet aggregation.Nat. Med. 2008; 14: 325-330Crossref PubMed Scopus (537) Google Scholar). Kindlin-3 is involved in podocyte formation in osteoclasts (14Schmidt S. Nakchbandi I. Ruppert R. Kawelke N. Hess M.W. Pfaller K. Jurdic P. Fässler R. Moser M. Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption.J. Cell Biol. 2011; 192: 883-897Crossref PubMed Scopus (134) Google Scholar) an observation that explains, in part, the observed osteopetrosis found in Kindlin-3-deficient mice and in human LAD-III sufferers (12Moser M. Bauer M. Schmid S. Ruppert R. Schmidt S. Sixt M. Wang H.V. Sperandio M. Fässler R. Kindlin-3 is required for β2 integrin-mediated leukocyte adhesion to endothelial cells.Nat. Med. 2009; 15: 300-305Crossref PubMed Scopus (288) Google Scholar, 14Schmidt S. Nakchbandi I. Ruppert R. Kawelke N. Hess M.W. Pfaller K. Jurdic P. Fässler R. Moser M. Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption.J. Cell Biol. 2011; 192: 883-897Crossref PubMed Scopus (134) Google Scholar, 15Malinin N.L. Zhang L. Choi J. Ciocea A. Razorenova O. Ma Y.Q. Podrez E.A. Tosi M. Lennon D.P. Caplan A.I. Shurin S.B. Plow E.F. Byzova T.V. A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans.Nat. Med. 2009; 15: 313-318Crossref PubMed Scopus (282) Google Scholar). Kindlin-3 also appears to have a role in red blood cell function (16Krüger M. Moser M. Ussar S. Thievessen I. Luber C.A. Forner F. Schmidt S. Zanivan S. Fässler R. Mann M. SILAC mouse for quantitative proteomics uncovers Kindlin-3 as an essential factor for red blood cell function.Cell. 2008; 134: 353-364Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). The phosphotyrosine-binding F3 subdomain of the Talin FERM domain has been shown to bind to an NPXY motif in the cytoplasmic tails of the integrin β-subunit, eliciting the activation of integrins to a high affinity state (17Anthis N.J. Wegener K.L. Critchley D.R. Campbell I.D. Structural diversity in integrin/Talin interactions.Structure. 2010; 18: 1654-1666Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Structural basis of integrin activation by Talin.Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Pulldown assays have demonstrated that Kindlins are also capable of binding to integrin β-tails (13Moser M. Nieswandt B. Ussar S. Pozgajova M. Fässler R. Kindlin-3 is essential for integrin activation and platelet aggregation.Nat. Med. 2008; 14: 325-330Crossref PubMed Scopus (537) Google Scholar, 19Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β-integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284: 11485-11497Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 20Has C. Herz C. Zimina E. Qu H.Y. He Y. Zhang Z.G. Wen T.T. Gache Y. Aumailley M. Bruckner-Tuderman L. Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes.Am. J. Pathol. 2009; 175: 1442-1452Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. The Kindler syndrome protein is regulated by transforming growth factor-β and involved in integrin-mediated adhesion.J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar); this binding is also involved in integrin activation in vivo (13Moser M. Nieswandt B. Ussar S. Pozgajova M. Fässler R. Kindlin-3 is essential for integrin activation and platelet aggregation.Nat. Med. 2008; 14: 325-330Crossref PubMed Scopus (537) Google Scholar, 22Ma Y.Q. Qin J. Wu C. Plow E.F. Kindlin-2 (Mig-2): a co-activator of β3 integrins.J. Cell Biol. 2008; 181: 439-446Crossref PubMed Scopus (272) Google Scholar, 23Montanez E. Ussar S. Schifferer M. Bösl M. Zent R. Moser M. Fässler R. Kindlin-2 controls bidirectional signaling of integrins.Genes Dev. 2008; 22: 1325-1330Crossref PubMed Scopus (332) Google Scholar). Thus, as currently understood, intracellular integrin activation involves both Talin and one of the Kindlins. Whether Talin and Kindlin bind together or separately to the β-tails had been unclear, but evidence that Kindlin-2 forms a ternary complex with β3 integrin tails and the Talin head domain has been found recently (24Bledzka K. Liu J. Xu Z. Perera H.D. Yadav S.P. Bialkowska K. Qin J. Ma Y.Q. Plow E.F. Spatial coordination of Kindlin-2 with Talin head domain in interaction with integrin β cytoplasmic tails.J. Biol. Chem. 2012; 287: 24585-24594Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). New data on the distinct roles of Kindlin and Talin binding in integrin function have also appeared (25Lefort C.T. Rossaint J. Moser M. Petrich B.G. Zarbock A. Monkley S.J. Critchley D.R. Ginsberg M.H. Fässler R. Ley K. Distinct roles for Talin-1 and Kindlin-3 in LFA-1 extension and affinity regulation.Blood. 2012; 119: 4275-4282Crossref PubMed Scopus (176) Google Scholar, 26Margadant C. Kreft M. de Groot D.J. Norman J.C. Sonnenberg A. Distinct roles of Talin and Kindlin in regulating integrin α5β1 function and trafficking.Curr. Biol. 2012; 22: 1554-1563Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). There are good structural data showing that Talin recognizes a conserved (“membrane proximal”) NPXY motif close to the transmembrane region of the integrin cytoplasmic tail (17Anthis N.J. Wegener K.L. Critchley D.R. Campbell I.D. Structural diversity in integrin/Talin interactions.Structure. 2010; 18: 1654-1666Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Structural basis of integrin activation by Talin.Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). There is no structural information about Kindlin-integrin interactions, but mutational and functional studies indicate that Kindlins bind to a second (“membrane distal”) conserved NPXY motif (19Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β-integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284: 11485-11497Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Mutational and biochemical analyses have also suggested that a Ser/Thr-rich region between the two NPXY motifs is critical for Kindlin binding to the β-integrin cytoplasmic tails; this is exemplified by a specific β3 integrin S732P mutation that causes Glanzmann thrombasthenia in humans (13Moser M. Nieswandt B. Ussar S. Pozgajova M. Fässler R. Kindlin-3 is essential for integrin activation and platelet aggregation.Nat. Med. 2008; 14: 325-330Crossref PubMed Scopus (537) Google Scholar, 19Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β-integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284: 11485-11497Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Here, we describe the expression, using a baculovirus-driven system, of recombinant Kindlin-3 in sufficient quantities to perform a biophysical characterization of the full-length protein. The three-dimensional arrangement of the domains within Kindlin-3 was investigated at low resolution using small-angle x-ray scattering in solution. Ab initio envelopes from small-angle x-ray scattering (SAXS) together with other shape estimates in solution reveal that Kindlin-3 is elongated and conformationally similar to Talin but with the prominent addition of the PH domain. Kindlin-3 is also shown to form a ternary complex with the Talin head region and integrin β-tails. Induced changes in NMR spectra show that Kindlin-3 binds directly to the membrane-distal NPXY motif and that the preceding Ser/Thr contributes to this binding. KINLDIN3 cDNA (a kind gift from R. Fässler, Max Planck Institute for Biochemistry, Martinsried, Germany) was amplified using primers 5′-aggagatataccatgATGGCGGGTATGAAGACAGC-3′ and 5′-gtgatggtgatgtttGAAGGCCTCATGGCCTCC-3′ and subsequently cloned into the pOPINE vector (27Berrow N.S. Alderton D. Sainsbury S. Nettleship J. Assenberg R. Rahman N. Stuart D.I. Owens R.J. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.Nucleic Acids Res. 2007; 35: e45Crossref PubMed Scopus (391) Google Scholar) encoding a C-terminal hexahistidine tag using the In-fusion enzyme system (Clontech). Plasmid DNA was sequence-verified (Geneservice, Ltd., Oxford, United Kingdom) and purified using standard methods. Baculovirus generation and insect cell culture maintenance were carried out using standard protocols (28Merrington C.L. Bailey M.J. Possee R.D. Manipulation of baculovirus vectors.Mol. Biotechnol. 1997; 8: 283-297Crossref PubMed Scopus (21) Google Scholar). Briefly, insect cells (Spodoptera frugiperda) were co-transfected with recombinant transfer vector, and linearized viral DNA was cultured in Sf900II serum-free media (Invitrogen) supplemented with 100 μg/ml penicillin and 100 μg/ml streptomycin. Virus-containing supernatant was harvested between 6 and 7 days after transfection. Virus was amplified prior to expression using standard methods with a multiplicity of infection of ∼0.1. Kindlin-3 expression was achieved by infecting suspension cultures of Sf9 cells in Sf900II supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, and 1% FCS with amplified recombinant virus at a multiplicity of infection of 1. Infected cells were harvested 72 h post-infection by centrifugation (1000 × g) and stored at −20 °C (or −80 °C for long-term storage). Frozen baculovirus-infected insect cell pellets expressing recombinant Kindlin-3 were thawed and resuspended with 50 mm NaH2PO4, pH 7.4, 500 mm NaCl, 10 mm imidazole, and 1% (v/v) Tween-20 supplemented with EDTA-free protease inhibitor mixture (Roche Applied Science) on ice prior to purification. Complete cell lysis was achieved by incubating the resuspension with the detergent. The recombinant protein was purified using three chromatographic steps after the lysate was clarified by centrifugation at 48,000 × g for 1 h at 4 °C. The supernatant was incubated with precharged and equilibrated nickel-Sepharose for 1–2 h at 4 °C. The beads were collected and washed using the batch method; 10 bed volumes of 50 mm NaH2PO4, pH 7.4, 500 mm NaCl, and 10 mm imidazole buffer were used to wash the beads. The protein was eluted and collected using 1–3 bed volumes of 50 mm NaH2PO4, pH 7.45, and 500 mm NaCl, and 500 mm imidazole. The protein composition of the eluant was assessed by SDS-PAGE and, in the first instance, Western blotting using an anti-His5 antibody. The eluant containing Kindlin-3 was pooled and buffer-exchanged into 20 mm Tris-HCl, pH 7.5, 200 mm NaCl via a series of dilutions into buffer, and the sample was concentrated using a centrifuge protein concentrator (Millipore) with a 50-kDa molecular weight cut-off (MWCO). The buffer-exchanged protein solution was applied onto a pre-equilibrated HiTrap heparin HP column (5 ml column volume, GE Healthcare). The protein that bound to the column was eluted using a linear NaCl gradient in the same buffer that increased from 200 mm to 1 m NaCl at a rate of 10 mm/ml. The protein composition of the fractionated eluant was assessed by SDS-PAGE and Western blotting, and those containing Kindlin-3 were pooled and concentrated using a centrifuge protein concentrator (Millipore) with a 50-kDa MWCO to 0.5 ml-2 ml sample size. Finally, the concentrated protein was polished and buffer-exchanged using size-exclusion chromatography (SEC). The protein was injected onto a pre-equilibrated Superdex S200 (16/60) or (10/30) (GE Healthcare) in 20 mm Tris-HCl, pH 7.5, 250 mm NaCl, and 1 mm DTT at a rate of 1 ml/min. The eluant from the column was fractionated into 1-ml samples, and the protein elution was monitored using absorbance at 280 nm. The fractions corresponding to a single absorbance peak that resulted from SEC were assessed by SDS-PAGE to determine homogeneity. The purified Kindlin-3 was assessed as >95% pure after this step. The protein was concentrated using a centrifuge protein concentrator (Millipore) with a 50-kDa MWCO to ∼15 mg/ml in the gel filtration buffer, flash-frozen in liquid nitrogen, and stored at −80 °C until used. The protein concentration was assessed spectroscopically using a calculated extinction coefficient (ϵ) of 109,320 m−1 cm−1 (assuming all Cys residues were reduced). Full-length Kindlin-3 was expressed and purified as described above. The C-terminal His6 tag was removed prior to size-exclusion chromatography by incubating the purified Kindlin-3 with carboxypeptidaseA-agarose (Sigma) at room temperature for 4 h. Kindlin-3 samples were prepared in buffer at pH 6.1 (10 mm bis-Tris, pH 6.1, 150 mm NaCl, and 2 mm DTT) or at pH 7.0 (20 mm HEPES, pH 7.0, 250 mm NaCl, and 2 mm TCEP). Full-length β-integrin tails were prepared as reported earlier for the β3 tail (29Oxley C.L. Anthis N.J. Lowe E.D. Vakonakis I. Campbell I.D. Wegener K.L. An integrin phosphorylation switch: the effect of β3 integrin tail phosphorylation on Dok1 and Talin binding.J. Biol. Chem. 2008; 283: 5420-5426Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 15N-Labeled tails were expressed in Escherichia coli grown in M9 minimal medium supplemented with 15NH4Cl. The tails were purified under denaturing conditions (50 mm sodium phosphate, 300 mm NaCl, 8 m urea, and 0.035% β-mercaptoethanol, pH 7.0) by Talon immobilized metal affinity chromatography (Clontech), eluting the polyhistidine-tagged integrin tail in 200 mm imidazole. Further purification was performed by C18 reverse phase HPLC using a 5–70% acetonitrile gradient. The polyhistidine tag was removed by overnight cleavage at 4 °C with 3C protease, and the cleaved tails were then purified again by C18 reverse phase HPLC. AUC was carried out using a Beckman (Palo Alto, CA) Optima XL-I AUC operating in velocity mode, initially with 12-mm sector-shaped centerpieces. Data were collected using 280 nm absorbance optics at a speed of 40,000 rpm with one scan taken every 6 min for a total of 60 scans. The sixth to 30th scans were determined as providing the best resolution of the sedimenting species using initial analyses in SedFit software (30Brown P.H. Schuck P. Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation.Biophys. J. 2006; 90: 4651-4661Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), and the data were analyzed using the c(s,f/f0) method whereby the distribution of apparent sedimentation coefficients shown by the sample is plotted over a third dimension, the population of species with diffusion coefficients of specific magnitudes as influenced by the frictional ratio of the species. For the initial study of isolated Kindlin-3, a resolution in s of 100 was used and a resolution in f/f0 of 10. For subsequent studies of mixtures of Kindlin-3, Talin head, and integrin β1A tails, 3-mm path-length cells, the 11th to 60th data scans, and a resolution of s = 50 were used. SedFit was also used to compute the hydrodynamic radius, frictional coefficient, and axial ellipsoidal ratio from the AUC data. The frictional coefficient is a unitless quantity that is the ratio between the theoretical behavior of a species, given its weight, partial specific volume, and the experimental conditions, and its actual sedimentation behavior. Plots presented as generated using ProFit software (Uetikon am See, Switzerland) were fitted with a Gaussian function to determine peak centers (which are the species sedimentation coefficients) and peak variances (which are proportional to the diffusion coefficient of the species). All samples were in 20 mm Tris, pH 7.5, 200 mm NaCl. Dynamic light scattering measurements were conducted on a Protein Solutions DynaPro instrument at 22 °C. Approximately 1 μg of protein was diluted in 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 1 mm DTT and centrifuged at 13,000 rpm for 5 min before being placed in the cuvette. SAXS data were collected at EMBL beamline X33 at the DORIS III storage ring, DESY (Hamburg, Germany) (31Roessle M.W. Klaering R. Ristau U. Robrahn B. Jahn D. Gehrmann T. Konarev P. Round A. Fiedler S. Hermes C. Svergun D.I. Upgrade of the small-angle X-ray scattering beamline X33 at the European Molecular Biology Laboratory, Hamburg.J. Appl. Crystallogr. 2007; 40: S190-S194Crossref Scopus (220) Google Scholar). The measurements were carried out at 288 K in 20 mm HEPES, pH 7.5, 300 mm NaCl, and 10 mm DTT. A MAR345 image plate at a sample detector distance of 2.43 m and wavelength λ of 0.93 Å, covering the momentum transfer range 0.04 < Q < 5.1 nm-1 (Q = 4π sin(θ)/λ where 2θ is the scattering angle), was used. The data were processed using standard procedures by the program package PRIMUS (32Konarev P. Volkov V.V. Sokolova A.V. Koch M.H. Svergun D.I. A Windows PC-based system for small-angle scattering data analysis.J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2351) Google Scholar). SAXS data sets for Kindlin-3 were collected at the following concentrations: 8.7, 4.35, 2.18, and 1.09 mg/ml. The reduced, rotationally averaged SAXS profiles were analyzed using the program GNOM (33Svergun D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria.J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2961) Google Scholar). A series of alternative possible maximum dimensions for Kindlin-3 were used to locate a Dmax defined by the data, which also showed a good fit to the experimental scattering curve. Distance distribution functions p(r) were then used to calculate reconstituted models ab initio with the program GASBOR (34Svergun D.I. Petoukhov M.V. Koch M.H. Determination of domain structure of proteins from X-ray solution scattering.Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). Models calculated starting from the premise of an unknown overall shape were consistently elongated, prolate structures, and therefore a prolate assumption was used in calculating the structures presented. For those samples showing no or limited intermolecular scattering (the two lowest concentrations), 20 models were calculated ab initio each time, and then they were sorted in pairwise groups of 10, aligned, averaged, and trimmed to a consensus core structure using the DAMAVER package (35Volkov V.V. Svergun D.I. Uniqueness of ab initio shape determination in small-angle scattering.J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1614) Google Scholar) running its constituent programs DAMSEL, DAMSUP, DAMAVER, and DAMFILT in turn and making use of the route supcomb20 for the alignment procedure, which allows enantiomer testing. This produced two independent scattering bead models from each data set, which were then aligned to each other in CHIMERA (36Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF chimera: a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (28178) Google Scholar), converted to CCP4 envelopes using the program GAP (37Grimes J.M. Burroughs J.N. Gouet P. Diprose J.M. Malby R. Ziéntara S. Mertens P.P. Stuart D.I. The atomic structure of the bluetongue virus core.Nature. 1998; 395: 470-478Crossref PubMed Scopus (489) Google Scholar), and correlated in Fourier space using the program WellMAP. 6J. F. Flanagan and R. J. C. Gilbert, unpublished data. The resolution achieved was estimated based on the 0.5 Fourier shell correlation criterion commonly used in electron microscopy (38Grigorieff N. Resolution measurement in structures derived from single particles.Acta C" @default.
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• W2091983485 title "Biophysical Analysis of Kindlin-3 Reveals an Elongated Conformation and Maps Integrin Binding to the Membrane-distal β-Subunit NPXY Motif" @default.
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