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• W2019078052 abstract "Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces. Here, we investigated if there are similar inter-domain interactions around domain 4 in the N-terminal rod 1 region of FLN. Protein crystal structures revealed a new type of domain organization between domains 3, 4, and 5. In this module, domains 4 and 5 interact rather tightly, whereas domain 3 has a partially flexible interface with domain 4. NMR peptide titration experiments showed that within the three-domain module, domain 4 is capable for interaction with a peptide derived from platelet glycoprotein Ib. Crystal structures of FLN domains 4 and 5 in complex with the peptide revealed a typical β sheet augmentation interaction observed for many FLN ligands. Domain 5 was found to stabilize domain 4, and this could provide a mechanism for the regulation of domain 4 interactions. Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces. Here, we investigated if there are similar inter-domain interactions around domain 4 in the N-terminal rod 1 region of FLN. Protein crystal structures revealed a new type of domain organization between domains 3, 4, and 5. In this module, domains 4 and 5 interact rather tightly, whereas domain 3 has a partially flexible interface with domain 4. NMR peptide titration experiments showed that within the three-domain module, domain 4 is capable for interaction with a peptide derived from platelet glycoprotein Ib. Crystal structures of FLN domains 4 and 5 in complex with the peptide revealed a typical β sheet augmentation interaction observed for many FLN ligands. Domain 5 was found to stabilize domain 4, and this could provide a mechanism for the regulation of domain 4 interactions. Filamins (FLNs) 3The abbreviations used are: FLNfilaminGPIbglycoprotein IbEOMensemble optimization methodMDmolecular dynamicsSAXSsmall angle x-ray scatteringHSQCheteronuclear single quantum coherencePDBProtein Data Bank. are homodimeric actin cross-linking proteins that are required for multicellular tissue differentiation. All three FLN genes (FLNA, FLNB, and FLNC) are essential in mouse and truncation or substitution mutations cause developmental defects in humans (1.Fürst D.O. Goldfarb L.G. Kley R.A. Vorgerd M. Olivé M. van der Ven P.F. Filamin C-related myopathies. Pathology and mechanisms.Acta Neuropathol. 2013; 125: 33-46Crossref PubMed Scopus (80) Google Scholar, 2.Kley R.A. Serdaroglu-Oflazer P. Leber Y. Odgerel Z. van der Ven P.F. Olivé M. Ferrer I. Onipe A. Mihaylov M. Bilbao J.M. Lee H.S. Höhfeld J. Djinović-Carugo K. Kong K. Tegenthoff M. Peters S.A. Stenzel W. Vorgerd M. Goldfarb L.G. Fürst D.O. Pathophysiology of protein aggregation and extended phenotyping in filaminopathy.Brain. 2012; 135: 2642-2660Crossref PubMed Scopus (58) Google Scholar) (Fig. 1). The diversity of phenotypes caused by FLN mutations can be explained by at least 90 proteins that interact with FLNs (3.Nakamura F. Stossel T.P. Hartwig J.H. The filamins. Organizers of cell structure and function.Cell Adh. Migr. 2011; 5: 160-169Crossref PubMed Scopus (323) Google Scholar). The interaction partners can be classified to at least three different categories: transmembrane proteins, cytoskeletal proteins, and intracellular signaling proteins. Thus, FLNs are involved in stabilization and regulation of plasma membrane, regulation of actin cytoskeleton, and intracellular signaling (3.Nakamura F. Stossel T.P. Hartwig J.H. The filamins. Organizers of cell structure and function.Cell Adh. Migr. 2011; 5: 160-169Crossref PubMed Scopus (323) Google Scholar, 4.Nakamura F. Heikkinen O. Pentikäinen O.T. Osborn T.M. Kasza K.E. Weitz D.A. Kupiainen O. Permi P. Kilpeläinen I. Ylänne J. Hartwig J.H. Stossel T.P. Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.PloS One. 2009; 4: e4928Crossref PubMed Scopus (54) Google Scholar, 5.Razinia Z. Mäkelä T. Ylänne J. Calderwood D.A. Filamins in mechanosensing and signaling.Annu. Rev. Biophys. 2012; 41: 227-246Crossref PubMed Scopus (164) Google Scholar, 6.Zhou A.-X. Hartwig J.H. Akyürek L.M. Filamins in cell signaling, transcription and organ development.Trends Cell Biol. 2010; 20: 113-123Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 7.van der Flier A. Sonnenberg A. Structural and functional aspects of filamins.Biochim. Biophys. Acta. 2001; 1538: 99-117Crossref PubMed Scopus (329) Google Scholar, 8.Stossel T.P. Condeelis J. Cooley L. Hartwig J.H. Noegel A. Schleicher M. Shapiro S.S. Filamins as integrators of cell mechanics and signalling.Nat. Rev. Mol. Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (820) Google Scholar). filamin glycoprotein Ib ensemble optimization method molecular dynamics small angle x-ray scattering heteronuclear single quantum coherence Protein Data Bank. Structurally, vertebrate FLNs consist of an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains (the dimer is shown in Fig. 1). Domains 1–15 are referred to as rod 1 and 16–24 as rod 2. The structure of the actin-binding domain is similar to that of α-actinin and is composed of two calponin homology domains (9.Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin). A molecular leaf spring.J. Cell Biol. 1990; 111: 1089-1105Crossref PubMed Scopus (429) Google Scholar, 10.Ruskamo S. Ylänne J. Structure of the human filamin A actin-binding domain.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 1217-1221Crossref PubMed Scopus (15) Google Scholar). The FLN Ig domains have a characteristic structure and they can be regarded as protein interaction modules. They often interact with other proteins by a β sheet augmentation mechanism. In this mechanism, the interaction partner forms an additional β-strand next to the strand C of the FLN domain and simultaneously interacts with the hydrophobic groove between the strands C and D, called the CD face (11.Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. The structure of the GPIb-filamin A complex.Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (134) Google Scholar, 12.Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wegener K.L. Campbell I.D. Ylänne J. Calderwood D.A. The molecular basis of filamin binding to integrins and competition with talin.Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 13.Lad Y. Jiang P. Ruskamo S. Harburger D.S. Ylänne J. Campbell I.D. Calderwood D.A. Structural basis of the migfilin-filamin interaction and competition with integrin tails.J. Biol. Chem. 2008; 283: 35154-35163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 14.Ithychanda S.S. Das M. Ma Y.-Q. Ding K. Wang X. Gupta S. Wu C. Plow E.F. Qin J. Migfilin, a molecular switch in regulation of integrin activation.J. Biol. Chem. 2009; 284: 4713-4722Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It is using this mode that ligands like transmembrane receptors of the integrin family (12.Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wegener K.L. Campbell I.D. Ylänne J. Calderwood D.A. The molecular basis of filamin binding to integrins and competition with talin.Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 15.Heikkinen O.K. Ruskamo S. Konarev P.V. Svergun D.I. Iivanainen T. Heikkinen S.M. Permi P. Koskela H. Kilpeläinen I. Ylänne J. Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin.J. Biol. Chem. 2009; 284: 25450-25458Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16.Takala H. Nurminen E. Nurmi S.M. Aatonen M. Strandin T. Takatalo M. Kiema T. Gahmberg C.G. Ylänne J. Fagerholm S.C. β2 Integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding.Blood. 2008; 112: 1853-1862Crossref PubMed Scopus (126) Google Scholar), the platelet von Willebrand factor receptor subunit glycoprotein Ib (GPIb) (11.Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. The structure of the GPIb-filamin A complex.Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (134) Google Scholar), and the cystic fibrosis transmembrane conductance regulator (17.Smith L. Page R.C. Xu Z. Kohli E. Litman P. Nix J.C. Ithychanda S.S. Liu J. Qin J. Misra S. Liedtke C.M. Biochemical basis of the interaction between cystic fibrosis transmembrane conductance regulator and immunoglobulin-like repeats of filamin.J. Biol. Chem. 2010; 285: 17166-17176Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 18.Playford M.P. Nurminen E. Pentikäinen O.T. Milgram S.L. Hartwig J.H. Stossel T.P. Nakamura F. Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.J. Biol. Chem. 2010; 285: 17156-17165Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) interact with FLNa domains 17, 19, 21, and 23. Also, signaling protein FilGAP uses the same mechanism for interacting with FLNa domain 23 (4.Nakamura F. Heikkinen O. Pentikäinen O.T. Osborn T.M. Kasza K.E. Weitz D.A. Kupiainen O. Permi P. Kilpeläinen I. Ylänne J. Hartwig J.H. Stossel T.P. Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.PloS One. 2009; 4: e4928Crossref PubMed Scopus (54) Google Scholar) and migfilin with FLNa domain 21 (13.Lad Y. Jiang P. Ruskamo S. Harburger D.S. Ylänne J. Campbell I.D. Calderwood D.A. Structural basis of the migfilin-filamin interaction and competition with integrin tails.J. Biol. Chem. 2008; 283: 35154-35163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 14.Ithychanda S.S. Das M. Ma Y.-Q. Ding K. Wang X. Gupta S. Wu C. Plow E.F. Qin J. Migfilin, a molecular switch in regulation of integrin activation.J. Biol. Chem. 2009; 284: 4713-4722Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Biochemical studies have also suggested that FLNa domain 4 interacts in a similar way with many of the ligands (19.Ithychanda S.S. Hsu D. Li H. Yan L. Liu D.D. Liu D. Das M. Plow E.F. Qin J. Identification and characterization of multiple similar ligand-binding repeats in filamin. Implication on filamin-mediated receptor clustering and cross-talk.J. Biol. Chem. 2009; 284: 35113-35121Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Interestingly, in the dimerization interface of FLNc and FLNa, domain 24 also utilizes the CD face, but the β sheet augmentation occurs through strand D (20.Pudas R. Kiema T.-R. Butler P.J. Stewart M. Ylänne J. Structural basis for vertebrate filamin dimerization.Structure. 2005; 13: 111-119Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21.Seo M.-D. Seok S.-H. Im H. Kwon A.-R. Lee S.J. Kim H.-R. Cho Y. Park D. Lee B.-J. Crystal structure of the dimerization domain of human filamin A.Proteins. 2009; 75: 258-263Crossref PubMed Scopus (17) Google Scholar). There are three structurally characterized, closely interacting domain pairs in the C-terminal rod 2 region of FLNa: domains 16–17, 18–19, and 20–21 (15.Heikkinen O.K. Ruskamo S. Konarev P.V. Svergun D.I. Iivanainen T. Heikkinen S.M. Permi P. Koskela H. Kilpeläinen I. Ylänne J. Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin.J. Biol. Chem. 2009; 284: 25450-25458Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22.Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding.EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (116) Google Scholar). Interestingly, in FLNa domain pairs 18–19 and 20–21, the function of the even numbered domain is to mask the CD face of the odd domain (15.Heikkinen O.K. Ruskamo S. Konarev P.V. Svergun D.I. Iivanainen T. Heikkinen S.M. Permi P. Koskela H. Kilpeläinen I. Ylänne J. Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin.J. Biol. Chem. 2009; 284: 25450-25458Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22.Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding.EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (116) Google Scholar) and this masking can be relieved by (a) low pico-Newton range mechanical forces that lead to a tighter interaction between FLN domain and ligand (23.Pentikäinen U. Ylänne J. The regulation mechanism for the auto-inhibition of binding of human filamin A to integrin.J. Mol. Biol. 2009; 393: 644-657Crossref PubMed Scopus (52) Google Scholar, 24.Ehrlicher A.J. Nakamura F. Hartwig J.H. Weitz D.A. Stossel T.P. Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A.Nature. 2011; 478: 260-263Crossref PubMed Scopus (263) Google Scholar, 25.Rognoni L. Stigler J. Pelz B. Ylänne J. Rief M. Dynamic force sensing of filamin revealed in single-molecule experiments.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 19679-19684Crossref PubMed Scopus (114) Google Scholar); or (b) displacement of the even numbered domain by the ligand itself, albeit with a lower affinity (26.Ithychanda S.S. Qin J. Evidence for multisite ligand binding and stretching of filamin by integrin and migfilin.Biochemistry. 2011; 50: 4229-4231Crossref PubMed Scopus (22) Google Scholar, 27.Ruskamo S. Gilbert R. Hofmann G. Jiang P. Campbell I.D. Ylänne J. Pentikäinen U. The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended.Biochem. J. 2012; 446: 261-269Crossref PubMed Scopus (22) Google Scholar). To find out if similar structural mechanisms could regulate the interactions of FLN domain 4, we solved the crystal structures of FLNa domains 3–5, FLNc domains 4–5, and FLNc domains 4–5 in complex with GPIb peptide. These structures disclosed a completely new type of interaction between three Ig domains. Fragments of the human FLNC and FLNA cDNA (GenBankTM AJ012737 and AB593010.1) were PCR amplified according to the predicted domain boundaries (7.van der Flier A. Sonnenberg A. Structural and functional aspects of filamins.Biochim. Biophys. Acta. 2001; 1538: 99-117Crossref PubMed Scopus (329) Google Scholar) and cloned to the GST fusion protein vector pGTVL1 (Structural Genomics Consortium, University of Oxford) according to the ligation-independent cloning method (28.Gileadi O. Burgess-Brown N.A. Colebrook S.M. Berridge G. Savitsky P. Smee C.E. Loppnau P. Johansson C. Salah E. Pantic N.H. High throughput production of recombinant human proteins for crystallography.Methods Mol. Biol. 2008; 426: 221-246Crossref PubMed Scopus (68) Google Scholar). The final products were verified by DNA sequencing. The proteins were expressed in Escherichia coli BL21 Gold cells (Agilent Technologies) at 37 °C for 4 h with 0.4 mm isopropyl β-d-1-thiogalactopyranoside. The bacterial pellets were lysed using French press. The proteins were captured with glutathione-agarose column (Protino glutathione-agarose 4B, Macherey-Nagel), released with Tobacco Etch virus protease (Invitrogen), and further purified by size exclusion chromatography with a HiLoad 26/60 Superdex 75 column (GE Healthcare) in 100 mm NaCl, 1 mm dithiothreitol (DTT), 20 mm Tris, pH 7.5 (FLNc fragments), and 100 mm NaCl, 1 mm DTT, 20 mm Tris, pH 8.0 (FLNa fragments). Finally, the proteins were concentrated using Centriprep YM-10000 (Millipore). Mutants were generated using the QuikChange Multisite-directed mutagenesis kit (Agilent Technologies). 13C/15N-Labeled FLNc4–5, FLNc5, and FLNa3–5 were expressed in E. coli in standard d-glucose/M9 minimal medium. These proteins were purified in 50 mm sodium phosphate, 100 mm NaCl, 1 mm DTT using the same protocol as described above for the unlabeled fragments. Crystallization trials with hanging drop vapor diffusion method were set up for the purified recombinant proteins at room temperature. First crystals for FLNc4–5 were obtained in 1.4 m sodium potassium phosphate. The condition was optimized using a gradient of salt concentration and the final crystals were mounted from 1.6 m sodium potassium phosphate. These were first cryo-protected by adding 30% glycerol (final concentration) to the mother liquor and then frozen in liquid nitrogen. The crystals for FLNa3–5 were obtained in 0.1 m sodium malonate, pH 4, 12% (w/v) polyethylene glycol (PEG) 3,350, and frozen in liquid nitrogen using 0.1 m sodium malonate, pH 4, 35% (w/v) polyethylene glycol 3,350. Equimolar mixture (1 mm each) of FLNc4–5 with GPIb peptide (residues 573–596, RGSLPTFRSSLFLWVRPNGRVGPL, numbering according to Uniprot ID P07359) was used to obtain co-crystals in 0.1 m HEPES, pH 7.5, 20% (w/v) PEG 8,000 using a 1:2 ratio of protein to mother liquor. The crystals were cryo-protected with 6.6% ethylene glycol and 16.6% glycerol. The diffraction data were collected at 100 K at the ESRF beamline ID23-1 (wavelength = 1.07227 Å) (FLNc4–5) and ID29 (wavelength = 0.976250 Å) (FLNa3–5 and FLNc4–5/GPIb). The data were processed with XDS (29.Kabsch W. XDS.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11211) Google Scholar) and the structures were solved using molecular replacement with the program Phaser (30.McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14421) Google Scholar) using FLNc23 (PDB code 2NQC) chain A as the search model for FLNc4–5, FLNc4–5 (PDB code 3V8O), and FLNc23 (2D7Q) as models for FLNa3–5 structure, and FLNc4–5 (PDB code 3V8O) for the FLNc4–5/GPIb structure. Refinement and model building were performed by programs Refmac5 (31.Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13850) Google Scholar, 32.Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. Read R.J. Vagin A. Wilson K.S. Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 235-242Crossref PubMed Scopus (9196) Google Scholar) and Coot (33.Emsley P. Cowtan K. Coot. Model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23210) Google Scholar) for FLNc4–5 and FLNc4–5/GPIb. The model for FLNa3–5 was built using ARP/wARP 7.3 (34.Langer G. Cohen S.X. Lamzin V.S. Perrakis A. Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7.Nat. Protoc. 2008; 3: 1171-1179Crossref PubMed Scopus (1316) Google Scholar) and Coot and refined using Refmac5. The structure of FLNc4–5 was refined using medium noncrystallographic symmetry restraints between chains A and B. Structure factors and coordinates were deposited in the PDB with codes 3V8O (FLNc4–5), 4M9P (FLNa3–5), and 4MGX (FLNc4–5/GPIb). For FLNc4–5, 89.2% of amino acids were in the most favored region, 9.8% in additionally allowed regions, and 1% in generously allowed region of the Ramachandran plot. The values for FLNa3–5 were 92.1, 7.5, and 0.4%, respectively, and for FLNc4–5/GPIb, 77.6, 19.0, and 3.4%, respectively. Crystal structure figures were generated using PyMOL (Schrödinger LLC, Portland, OR). The domain-domain interface was analyzed using the PISA server (35.Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6761) Google Scholar). Rendering was done in Chimera (36.Pettersen 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 (27891) Google Scholar) according to the sequence alignment made with Clustal Omega (37.Sievers F. Wilm A. Dineen D. Gibson T.J. Karplus K. Li W. Lopez R. McWilliam H. Remmert M. Söding J. Thompson J.D. Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.Mol. Syst. Biol. 2011; 7: 539Crossref PubMed Scopus (9081) Google Scholar) using FLNa/b/c3–5, FLNa3–5 of Mus musculus, Gallus gallus, Danio rerio, and Drosophila melanogaster. Small angle x-ray scattering (SAXS) data were collected on the EMBL (European Molecular Biology Laboratory) X33 beamline at the DESY, Hamburg (38.Roessle M.W. Klaering R. Ristau U. Robrahn B. Jahn D. Gehrmann T. Konarev P. Round A. Fiedler S. Hermes C. Svergun D. 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) (MAR345 image plate, sample-detector distance of 2.7 m and wavelength λ = 0.15 nm, covering the momentum transfer range 0.08 < s < 4.9 nm−1 (where s = 4π sin(θ)/λ, 2θ = scattering angle)); MAX IV Laboratory, beamline Cassiopeia I911-SAXS at the MAX II storage ring (Lund, Sweden) (39.Labrador A. Cerenius Y. Svensson C. Theodor K. Plivelic T. The yellow mini-hutch for SAXS experiments at MAX IV Laboratory.J. Phys. Conf. Ser. 2013; 425 (072019)Crossref Scopus (106) Google Scholar) (PILATUS 1M image plate, sample-detector distance of 2.2 m and wavelength 0.091 nm, covering the momentum transfer range 0.1 < s < 3 nm−1) and ESRF (European Synchrotron Radiation Facility), beamline BM29 (40.Pernot P. Round A. Barrett R. De Maria Antolinos A. Gobbo A. Gordon E. Huet J. Kieffer J. Lentini M. Mattenet M. Morawe C. Mueller-Dieckmann C. Ohlsson S. Schmid W. Surr J. Theveneau P. Zerrad L. McSweeney S. Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution.J. Synchrotron Radiat. 2013; 20: 660-664Crossref PubMed Scopus (288) Google Scholar) (PILATUS 1M image plate, sample-detector distance of 2.9 m and wavelength 0.10 nm, covering the momentum transfer range 0.01 < s < 5 nm−1). The protein concentrations were in the range of 1–10 mg/ml in purification buffer supplemented with 10 mm DTT. Buffer subtractions were conducted with either BioXTAS RAW software (41.Nielsen S.S. Noergaard Toft K. Snakenborg D. Jeppesen M.G. Jacobsen J.K. Vestergaard B. Kutter. J.P. Arleth L. BioXTAS RAW, a software program for highthroughput automated small-angle X-ray scattering data reduction and preliminary analysis.J. Appl. Crystallogr. 2009; 42: 959-964Crossref Scopus (180) Google Scholar) or ATSAS package program PRIMUS (42.Konarev P.V. Volkov V.V. Sokolova A.V. Koch M.H.J. Svergun D.I. PRIMUS. A Windows PC-based system for small-angle scattering data analysis.J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2341) Google Scholar) and the scattering intensity (I) was extrapolated to zero solute concentration. The forward scattering I(0) and the radius of gyration (Rg) were calculated using the program GUINIER (43.Guinier A. La diffraction des rayons X aux très petits angles. Application à l'étude de phénomènes ultramicroscopiques.Ann. Phys. 1939; 12: 161-237Crossref Google Scholar), where at very small angles (s × Rg < 1.3), the scattering intensity is the following. l(s)=l(0)exp(-1/3(Rgs)2)(Eq. 1) The distance distribution functions p(r) and the maximum particle dimensions Dmax were calculated for all fragments using the program GNOM (44.Svergun D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria.J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2945) Google Scholar). The molecular mass of the constructs were evaluated by comparing the forward scattering with that from reference solution of bovine serum albumin (BSA) with molecular mass 66 kDa using Equation 2. Mwsample=l(0)sample×Mwref/l(0)ref(Eq. 2) Assuming the samples are monodisperse, Porod's law was applied to find out the excluded volume of the hydrated particle as, V=2π2l(0)/∫0∞s2 lexp(s)ds(Eq. 3) and to check the S−4 decay in scattering intensity at higher angles (45.Rambo R.P. Tainer J.A Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law.Biopolymers. 2011; 95: 559-571Crossref PubMed Scopus (371) Google Scholar). Kratky plot (46.Kratky O. Glatter O. Small angle x-ray scattering. Academic Press, London1982Google Scholar) (I(s) × s2 versus s) was evaluated to check for the flexibility of the protein at higher scattering angles. The scattering patterns were further used to generate low resolution ab initio models of FLNc4–5 and FLNa3–5 by the programs DAMMIF (47.Franke D. Svergun D.I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering.J. Appl. Crystallogr. 2009; 42: 342-346Crossref PubMed Scopus (1181) Google Scholar) or GASBOR (48.Svergun 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 (1140) Google Scholar). Ten rounds of DAMMIF or GASBOR were done to generate models that were averaged using the program DAMAVER (49.Volkov V.V. Svergun D.I. Uniqueness of ab initio shape determination in small-angle scattering.J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1610) Google Scholar) to find the best model with common structural features. The scattering intensities of the crystal structures of FLNc4–5 (Protein Data Bank code 3V8O) and FLNa3–5 (PDB code 4M9P) were calculated using CRYSOL (50.Svergun D. Barberato C. Koch M.H.J. CRYSOL. A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates.J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2764) Google Scholar) and the superposition of the DAMAVER generated envelope with the respective crystal structures are done with the SUPCOMB program of the ATSAS package and the figures are made using PyMOL. To assess the flexibility of domain 3 in the FLNa and FLNc3–5 fragments, the ensemble optimization method (EOM) (51.Bernadó P. Mylonas E. Petoukhov M.V. Blackledge M. Svergun D.I. Structural characterization of flexible proteins using small-angle x-ray scattering.J. Am. Chem. Soc. 2007; 129: 5656-5664Crossref PubMed Scopus (926) Google Scholar) was used. Similar analysis was performed for FLNc4–5 as an internal control. First, 10,000 randomized models (pool) were generated for each fragment using the native chain option in the RanCH program of the EOM package. The scattering profiles of each of these models were compared with the experimental scattering of the respective fragments (FLNa3–5, FLNc3–5, and FLNc4–5). A genetic algorithm was used to select a set of representative models (FLNa3–5 = 24; FLNc3–5 = 18, and FLNc4–5 = 12) from the pool such that the average scattering from the selected models fits the experimental scattering. The results were represented as Rg and Dmax distribution profiles using GraphPad Prism version 4 for Windows (GraphPad Software Inc., La Jolla, CA). Pull-down assays were performed using GST-tagged FLNc4 bound to glutathione-agarose resin. 1–20 μm FLNc5 and FLNc5R755D was allowed to interact for 1 h at 23 °C after which the resin was washed three times with 500 μl of PBS, 1 mm DTT and eluted with 10 μl of the SDS-PAGE sample buffer. Proteins were fractionated by SDS-PAGE and visualized with Coomassie stain. Intensities of the protein bands were quantified by ImageJ (52.Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ. 25 years of image analysis.Nat. Methods. 2012; 9: 671-675Crossref PubMed Scopus (34883) Google Scholar). Data were plotted using GraphPad Prism version 5 for Windows to produce a non-linear regression curve for one site total and nonspecific binding (specific = Bmax × X/(X + Kd); nonspecific = NS × X + background, where X = ligand concentration). Thermal stability of FLNc4, FLNc5, and FLNc4–5 was determined using Bio-Rad C1000 thermal cycler, CFx96 Real-Time system. Unfolding of the proteins was monitored using the fluorescent dye SYPRO Orange (Invitrogen), which binds to the hydrophobic core of the protein as it unfolds. A temperature gradient was set up from 20 to 95 °C with 0.5 °C/30-s increments. Each sample contained 100 μm protein, except FLNc4 (150 μm), and 5× dye in 25 μl total volume. NMR samples were prepared in 50 mm NaH2PO4, 100 mm NaCl, 1 mm DTT buffer at pH 7.0. D2O was added to obtain ∼8% solutions. Protein concentrations were 0.4–1.2 mm. Measurement temperatures were 28 °C for FLNc5 and FLNc4–5, and 35 °C for FLNa3–5. For the chemical" @default.
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• W2019078052 date "2014-03-01" @default.
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• W2019078052 title "A Novel Structural Unit in the N-terminal Region of Filamins" @default.
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