Title: Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin
Abstract: Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism. Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NMR structures revealed that the domain packing in IgFLNa18–19 resembles the structure of IgFLNa20–21. In both domain pairs the integrin-binding site is masked, although the details of the domain-domain interaction are partly distinct. The structure of IgFLNa16–17 revealed a new domain packing mode where the adhesion receptor binding site of domain 17 is not masked. Sequence comparison suggests that similar packing of three tandem filamin domain pairs is present throughout the animal kingdom, and we propose that this packing is involved in the regulation of filamin interactions through a mechanosensor mechanism. Actin cytoskeleton is a dynamic network that is involved in many fundamental cellular processes such as cell differentiation, morphology, endocytosis, exocytosis, cytokinesis, and cell movement. These events are regulated by proteins that interact with monomeric and filamentous actin. Filamins are actin filament-binding and cross-linking proteins. Filamin A and filamin B are both ubiquitously expressed, and their mutations in human patients cause developmental abnormalities in brain, cartilage, bones, and epithelial tissues (1Feng Y. Walsh C.A. Nat. Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (424) Google Scholar). Filamin C is muscle-specific, and mutations thereof cause myofibrillar myopathy (2Vorgerd M. van der Ven P.F. Bruchertseifer V. Löwe T. Kley R.A. Schröder R. Lochmüller H. Himmel M. Koehler K. Fürst D.O. Huebner A. Am. J. Hum. Genet. 2005; 77: 297-304Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Mice with targeted deletion of any of the filamin genes die either during development or soon after birth (3Dalkilic I. Schienda J. Thompson T.G. Kunkel L.M. Mol. Cell. Biol. 2006; 26: 6522-6534Crossref PubMed Scopus (128) Google Scholar, 4Feng Y. Chen M.H. Moskowitz I.P. Mendonza A.M. Vidali L. Nakamura F. Kwiatkowski D.J. Walsh C.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19836-19841Crossref PubMed Scopus (243) Google Scholar, 5Zhou X. Tian F. Sandzén J. Cao R. Flaberg E. Szekely L. Cao Y. Ohlsson C. Bergo M.O. Borén J. Akyürek L.M. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3919-3924Crossref PubMed Scopus (98) Google Scholar, 6Farrington-Rock C. Kirilova V. Dillard-Telm L. Borowsky A.D. Chalk S. Rock M.J. Cohn D.H. Krakow D. Hum. Mol. Genet. 2008; 17: 631-641Crossref PubMed Scopus (42) Google Scholar). These phenotypes are thought to reflect the roles of filamins as scaffolds of signaling pathways required for cell differentiation, regulators of cell migration, and stabilizers of cytoskeleton and cell membranes (1Feng Y. Walsh C.A. Nat. Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (424) Google Scholar, 7Popowicz G.M. Schleicher M. Noegel A.A. Holak T.A. Trends Biochem. Sci. 2006; 31: 411-419Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Filamins bind to actin filaments mainly via their N-terminal actin-binding domains and interact with other proteins via the 24 filamin type immunoglobulin-like domains (IgFLN), 3The abbreviations used are:IgFLNfilamin immunoglobulin-like domainGPglycoproteinNOEnuclear Overhauser enhancementRMSDroot mean square deviationSAXSsmall angle x-ray scattering.also called filamin repeats (8Nakamura F. Osborn T.M. Hartemink C.A. Hartwig J.H. Stossel T.P. J. Cell Biol. 2007; 179: 1011-1025Crossref PubMed Scopus (216) Google Scholar). Especially the C-terminal IgFLNs 16–24 contain several protein-protein interaction sites (1Feng Y. Walsh C.A. Nat. Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (424) Google Scholar). Our previous structural studies have revealed that many proteins interact with filamins by forming an additional β-strand next to strand C of an individual IgFLN. The platelet von Willebrand factor receptor, glycoprotein (GP) Ibα, interacts in this way with IgFLNa17 (9Nakamura F. Pudas R. Heikkinen O. Permi P. Kilpeläinen I. Munday A.D. Hartwig J.H. Stossel T.P. Ylänne J. Blood. 2006; 107: 1925-1932Crossref PubMed Scopus (139) Google Scholar). The integrin family adhesion receptor β subunits interact with IgFLNa21 and to a lesser extent with IgFLNa19 (10Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wegener K.L. Campbell I.D. Ylänne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 11Takala H. Nurminen E. Nurmi S.M. Aatonen M. Strandin T. Takatalo M. Kiema T. Gahmberg C.G. Ylänne J. Fagerholm S.C. Blood. 2008; 112: 1853-1862Crossref PubMed Scopus (130) Google Scholar). Furthermore, some signaling proteins use a similar interaction mode: the adaptor protein migfilin interacts with IgFLNa21 (12Lad Y. Jiang P. Ruskamo S. Harburger D.S. Ylänne J. Campbell I.D. Calderwood D.A. J. Biol. Chem. 2008; 283: 35154-35163Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and the Rho family GTPase-activating protein FilGAP interacts with IgFLNa23 (13Ohta Y. Hartwig J.H. Stossel T.P. Nat. Cell Biol. 2006; 8: 803-814Crossref PubMed Scopus (303) Google Scholar, 14Nakamura 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. PLOS One. 2009; 4: e4928Crossref PubMed Scopus (57) Google Scholar). filamin immunoglobulin-like domain glycoprotein nuclear Overhauser enhancement root mean square deviation small angle x-ray scattering. Although structural details are known from many filamin interactions, it is not completely clear how these interactions are regulated. In some cases the regulation involves competition between multiple binding partners (10Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wegener K.L. Campbell I.D. Ylänne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 11Takala H. Nurminen E. Nurmi S.M. Aatonen M. Strandin T. Takatalo M. Kiema T. Gahmberg C.G. Ylänne J. Fagerholm S.C. Blood. 2008; 112: 1853-1862Crossref PubMed Scopus (130) Google Scholar). Alternative splicing (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar), proteolysis of filamin (16Azam M. Andrabi S.S. Sahr K.E. Kamath L. Kuliopulos A. Chishti A.H. Mol. Cell. Biol. 2001; 21: 2213-2220Crossref PubMed Scopus (217) Google Scholar, 17Raynaud F. Jond-Necand C. Marcilhac A. Fürst D. Benyamin Y. Int. J. Biochem. Cell Biol. 2006; 38: 404-413Crossref PubMed Scopus (2) Google Scholar, 18Heuzé M.L. Lamsoul I. Baldassarre M. Lad Y. Lévêque S. Razinia Z. Moog-Lutz C. Calderwood D.A. Lutz P.G. Blood. 2008; 112: 5130-5140Crossref PubMed Scopus (67) Google Scholar), and ligand phosphorylation (11Takala H. Nurminen E. Nurmi S.M. Aatonen M. Strandin T. Takatalo M. Kiema T. Gahmberg C.G. Ylänne J. Fagerholm S.C. Blood. 2008; 112: 1853-1862Crossref PubMed Scopus (130) Google Scholar) also contribute to the regulation. Recently, it has become apparent that conformational changes in filamins may also be involved. For instance, actomyosin contraction exposes hidden cysteine residues in filamins (19Johnson C.P. Tang H.Y. Carag C. Speicher D.W. Discher D.E. Science. 2007; 317: 663-666Crossref PubMed Scopus (306) Google Scholar). This opens the possibility that forces transmitted through actin filament may open up binding sites, and filamin may thus be involved in mechanosensor signaling. We have recently found a structural mechanism by which mechanical forces could regulate interactions at the C-terminal part of filamin. Our recent crystal structure revealed that IgFLNa20 forms a compact pair with IgFLNa21, and in this pair the N-terminal part of IgFLNa20 masks the integrin-binding site on IgFLNa21 (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar). It is possible that this masking could be released by mechanical forces. Four lines of evidence led us to hypothesize that in addition to the IgFLNa20–21 pair, other similar domain pairs could exist at the C terminus of filamin: (i) the overall structure of the C-terminal part (IgFLNs 16–24) of filamin is relatively more compact than the N-terminal part of the molecule (IgFLNs 1–15) (8Nakamura F. Osborn T.M. Hartemink C.A. Hartwig J.H. Stossel T.P. J. Cell Biol. 2007; 179: 1011-1025Crossref PubMed Scopus (216) Google Scholar); (ii) the N-terminal sequences of even-numbered domains 16, 18, and 20 differ from other IgFLNs (20Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1089-1105Crossref PubMed Scopus (435) Google Scholar) (sequence alignment is shown in supplemental Fig. S1); (iii) in single-domain solution NMR structures of IgFLNc16, IgFLNb16, 18, and 20, the N-terminal part is not folded with the rest of the domain; and (iv) according to biochemical experiments, IgFLNa18 masks integrin binding to IgFLNa19 (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar). We report here small angle x-ray scattering (SAXS) analysis showing that IgFLNa16–17 and 18–19 have overall dimensions very similar to those of the previously known domain pair IgFLNa20–21. The IgFLNa22–23 construct was much more elongated, which is indicative for two independently folded noninteracting domains. Further, the atomic structures solved with NMR spectroscopy show that IgFLNa18–19 forms a pair similar to IgFLNa20–21, but the details of the interaction and orientation of the domains differ. On the other hand, IgFLNa16–17 forms an entirely novel type of domain pair. Sequence comparisons predict that these three interdependent domain pairs are conserved from nematodes to vertebrates, suggesting that the arrangement has special regulatory functions. The IgFLNa12–13 (amino acids 1353–1542), IgFLNa16–17 (amino acids 1772–1956), IgFLNa18–19 (amino acids 1954–2141), IgFLNa20–21 (amino acids 2141–2329), and IgFLNa22–23 (amino acids 2330–2522) fragments were generated by polymerase chain reaction and cloned into the modified pGEX vector (GE Healthcare). The inserts were checked by DNA sequencing. Glutathione S-transferase fusion proteins then were produced in Escherichia coli BL21 Gold cells and purified with glutathione-Sepharose 4 Fast Flow (GE Healthcare) according to the manufacturer's instructions. Glutathione S-transferase was cleaved by TEV protease at 4 °C for 16 h. The buffer was changed in a HiPrep 26/10 desalting column (GE Healthcare) to 100 mm NaCl, 1 mm dithiothreitol, 20 mm Tris, pH 8, and glutathione S-transferase was removed in a glutathione-Sepharose 4 Fast Flow column. The further protein purifications were performed by gel filtration in a HiLoad 26/60 Superdex 75 column (GE Healthcare), and finally the proteins were concentrated with Centriprep YM-3000 or YM-10000 (Millipore). Synchrotron radiation x-ray scattering data were collected on the EMBL X33 beamline at the DORIS III storage ring, DESY, Hamburg (21Roessle M.W. Klaering R. Ristau U. Robrahn B. Jahn D. Gehrmann T. Konarev P. Round A. Fiedler S. Hermes C. Svergun D. J. Appl. Crystallogr. 2007; 40: S190-S194Crossref Scopus (220) Google Scholar). Solutions of FLNa fragments IgFLNa12–13, IgFLNa16–17, IgFLNa18–19, IgFLNa20–21, and IgFLNa22–23 in 100 mm NaCl, 10 mm dithiothreitol, 20 mm Tris, pH 8.0, were adjusted to concentrations of 2.8–9.6, 2.3–9.9, 1.9–7.8, 3.7–9.7, and 3.3–10.0 mg/ml, respectively. The MAR345 image plate at sample-detector distance 2.7 m and wavelength λ = 0.15 nm, covering the momentum transfer range 0.12 < s < 4.9 nm−1 (s = 4π sin(θ)/λ, where 2θ is the scattering angle) was used. The data were averaged after normalization to the intensity of the incident beam, the scattering of the buffer was subtracted, and the difference data were extrapolated to zero solute concentration following standard procedures. All of the data manipulations were performed using the program package PRIMUS (22Konarev P.V. Petoukhov M.V. Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2006; 39: 277-286Crossref Scopus (527) Google Scholar). The forward scattering I(0) and the radius of gyration (Rg) were evaluated using the Guinier approximation (23Guinier A. Ann. Physics. 1939; 12: 161-237Crossref Google Scholar), assuming that at very small angles (s < 1.3/Rg) the intensity is represented as I(s) = I(0) exp(−1/3(Rgs)2). These parameters were also computed from the entire scattering patterns using the program GNOM (24Svergun D. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (3006) Google Scholar), providing the distance distribution functions p(r) and the maximum particle dimensions Dmax. The molecular mass of the solute was evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (molecular mass, 66 kDa). The excluded volume of the hydrated particle (the Porod volume) was computed as follows (25Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 17-51Google Scholar).V=2π2I0/∫0∞s2IexpSds(Eq. 1) Prior to this analysis, an appropriate constant was subtracted from each data point to force the s−4 decay of the intensity at higher angles following Porod's law (25Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 17-51Google Scholar) for homogeneous particles. This “shape scattering” curve was further used to generate low resolution ab initio models of fragments IgFLNa12–13, 16–17, 18–19, 20–21, and 22–23 by the program DAMMIN (26Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1767) Google Scholar) and DAMMIF (27Franke D. Svergun D.I. J. Appl. Crystallogr. 2009; 42: 342-346Crossref PubMed Scopus (1209) Google Scholar), which represent the protein by an assembly of densely packed beads. Simulated annealing was employed to build a compact interconnected configuration of beads inside a sphere with the diameter Dmax that fits the experimental data Iexp(s) to minimize the discrepancy,χ2=1N−1∑jIexpsj−CIcalcSjσsj2(Eq. 2) where N is the number of experimental points, c is a scaling factor, and Icalc(sj) and σ(sj) are the calculated intensity and the experimental error at the momentum transfer sj, respectively. The program GASBOR (28Svergun D.I. Petoukhov M.V. Koch M.H. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar) was used to create ab initio models of proteins consisting of dummy residues instead of beads. In this program a simulated annealing protocol is employed to construct a model with a protein-like distribution of beads that provides the best fit to the experimental data. For the ab initio analyses, multiple runs were performed to verify the stability of the solution. The results from 10 separate runs of DAMMIN, DAMMIF, and GASBOR were averaged to determine common structural features using the program DAMAVER (29Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1634) Google Scholar). The calculated parameters for IgFLNa20–21 were estimated based on the crystal structure of IgFLNa19–21 (Protein Data Bank code 2j3s) (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar) and for IgFLNa16–17 and FLNa18–19 based on the NMR structures using the program CRYSOL (30Svergun D. Barberato C. Koch M.H.J. J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2812) Google Scholar). NMR sample preparation and conditions, spectroscopic details, and chemical shift assignment have been described earlier (31Heikkinen O.K. Permi P. Koskela H. Ylänne J. Kilpeläinen I. Biomol. NMR Assignments. 2009; 3: 53-56Crossref PubMed Scopus (6) Google Scholar). For structure determination three-dimensional 13C- and 15N-edited NOE spectroscopy-heteronuclear single-quantum coherence spectra were recorded on a Varian INOVA 800-MHz spectrometer equipped with 5-mm z-gradient triple resonance probehead at 30 °C. Spectrum acquisition and processing was done using VNMRJ 2.1 and VNMR 6.1C software (Varian Inc.). Sparky 3.110 was used for spectrum analysis (Goddard TD, Kneller DG. University of California, San Francisco, CA). Dihedral angle constraints for χ and ψ angles were extracted from chemical shift data using TALOS software (32Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2741) Google Scholar). The dihedral angle restraints were parameterized as (TALOS prediction ± 2 S.D.). Structure calculation was done using the automatic NOE assignment and torsion angle dynamics mode of CYANA 2.1 (33Herrmann T. Güntert P. Wüthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1338) Google Scholar). Based on the lowest target function, 10% of calculated structures were chosen for further refinement. Molecular dynamics refinement of the final structures was done using a generalized Born implicit solvent model in AMBER 8.0 (38Case D.A. Darden T.A. Cheatham III, T.E. Simmerling C.L. Wang J. Duke R.E. Luo R. Merz K.M. Wang B. Pearlman D.A. Crowley M. Brozell S. Tsui V. Gohlke H. Mongan J. Hornak V. Cui G. Beroza P. Schafmeister C. Caldwell J.W. Ross W.S. Kollman P.A. AMBER 8. University of California, San Francisco, CA2004Google Scholar). Quality control of the structure families was done with WHAT CHECK (34Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1823) Google Scholar) and PROCHECK-NMR (35Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4507) Google Scholar) programs. Domain-domain interaction interfaces were analyzed on a ProtorP server (36Reynolds C. Damerell D. Jones S. Bioinformatics. 2009; 25: 413-414Crossref PubMed Scopus (163) Google Scholar). Experimental procedures of relaxation rate and heteronuclear NOE determination and peptide titrations can be found in the supplemental materials. To compare the binding of IgFLNa16–17 and IgFLNa17 fragments to GPIbα, the synthetic GPIbα peptide containing residues 556–577 (EZBiolab Inc., Westfield, IN) was coupled to an N-hydroxysuccinimide activated Sepharose 4 Fast Flow (GE Healthcare) at 4 °C according to the manufacturer's instructions. Purified IgFLNa16–17 and IgFLNa17 were incubated for 1 h at 23 °C with 20 μl of the peptide-Sepharose in 1% Triton X-100, 150 mm NaCl, 20 mm Tris, pH 7.4. The Sepharose was centrifuged at 15,000 × g for 1 min and washed twice with 300 μl of the binding buffer. The proteins were eluted with 10 μl of SDS electrophoresis sample buffer and run on a SDS-polyacrylamide gel. To study the presence of compact domain pairs in the C-terminal part of filamin A, we expressed and purified two-domain fragments and analyzed them by SAXS. The location of the studied fragments in filamin is shown in Fig. 1. IgFLNa12–13 was used as a control because no domain pair formation was expected in this area. All of the constructs behaved well in SAXS, and neither aggregation nor dimerization was observed. The experimental SAXS curves from the constructs are shown in Fig. 2, and the overall parameters computed from the data are presented in Table 1. The experimental values for radius of gyration (Rg) and maximal dimension (Dmax) from IgFLNa16–17, 18–19, and 20–21 were similar to the theoretical values calculated from the crystallographic structure of the IgFLNa20–21 pair (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar) (Table 1). However, the values of Rg and Dmax for the IgFLNa12–13 and 22–23 were significantly higher, pointing to more extended structures. The long-tailed shape of distance distribution function p(r) for IgFLNa12–13 and 22–23 (Fig. 2, insets) was also consistent with elongated shapes.FIGURE 2.Experimental SAXS data of two-domain fragments and fits of ab initio models. The x-ray scattering data from IgFLNa16–17, 18–19 and 22–23 (A) and IgFLNa12–13 and 22–23 (B) are displayed as dots. The scattering from typical ab initio models computed by DAMMIN/GASBOR is displayed as full lines. The plots display the logarithm of the scattering intensity as a function of momentum transfers, and successive curves are displaced down by one logarithmic unit for clarity. The distance distribution functions are presented in the insets.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Summary of SAXS measurementsFLNa12–13FLNa16–17FLNa18–19FLNa20–21FLNa22–23ObservedObservedCalculatedObservedCalculatedObservedCalculatedObservedC (mg/ml)2.8/9.62.3/9.91.9/7.83.7/9.33.3/10.0Rg (nm)2.39 ± 0.011.93 ± 0.031.812.11 ± 0.042.111.91 ± 0.031.972.77 ± 0.05Dmax (nm)8.6 ± 0.56.0 ± 0.56.36.5 ± 0.57.66.1 ± 0.56.39.0 ± 0.5Vp (nm3)25 ± 433 ± 42734 ± 43331 ± 42932 ± 4MMexp (kDa)14 ± 422 ± 418.721 ± 320.416.8 ± 418.424 ± 4χab1.451.691.871.371.78 Open table in a new tab Typical low resolution shapes of IgFLNa16–17 and 18–19 reconstructed ab initio by DAMMIN (26Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1767) Google Scholar) and GASBOR (28Svergun D.I. Petoukhov M.V. Koch M.H. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar) (Fig. 3) provided good fits to the experimental data with the discrepancy factors of χ = 1.69–1.87 (curves are shown in Fig. 2) and displayed similar overall dimensions with IgFLNa20–21 (Fig. 3). The shape of IgFLNa18–19 had more pronounced features of two domains, whereas the envelope of IgFLNa16–17 was a bit more compact. Typical ab initio low resolution models of IgFLNa12–13 and 22–23 revealed that the shapes of these tandem domains significantly differ from those of IgFLNa16–17, 18–19, and 20–21. The shape of IgFLNa12–13 and 22–23 is elongated, suggesting a conventional head-to-tail arrangement of the domains (Fig. 3). In conclusion, our SAXS analysis suggests that IgFLNa16–17 and 18–19 form interacting domain pairs similar to those of IgFLNa20–21. IgFLNa22–23, on the other hand, does not appear to form such a pair. To study the atomic details of the IgFLNa18–19 and IgFLNa16–17 domain pairs, NMR spectroscopy was employed. The chemical shift assignments have been published elsewhere (31Heikkinen O.K. Permi P. Koskela H. Ylänne J. Kilpeläinen I. Biomol. NMR Assignments. 2009; 3: 53-56Crossref PubMed Scopus (6) Google Scholar). Chemical shift mapping between single domains IgFLNa18 and 19 and the two-domain construct IgFLN18–19 showed that the largest changes were located at β-strands C and D as well as at the EF loop of IgFLNa19 (supplemental Fig. S3). These changes confirmed that the two domains indeed interact with each other in solution and that the interaction resembles the one found previously for IgFLNa20–21 (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J. 2007; 26: 3993-4004Crossref PubMed Scopus (121) Google Scholar). The structure of IgFLNa18–19 was solved with solution state NMR spectroscopy. Details of the structure calculations are described in supplemental materials, and structure quality indicators are presented in Table 2. Altogether, 2930 NOE-derived distance restraints and 235 chemical shift-based dihedral angle restraints were used in the structure calculation. The mutual orientation of the domains was based on 76 interdomain distance restraints, of which 42 are located between β-strand A of domain 18 and the CD face of domain 19 (see supplemental Fig. S2B for graphical representation of the interdomain distance restraints). Average backbone RMSD from the mean structure for the double-domain (residues 1960–2135) was ∼1 Å. The coordinate precision of single domains was better; average backbone RMSD from the mean structure was 0.8 Å for IgFLNa18 (residues 1960–2045) and 0.3 Å for IgFLNa19 (residues 2046–2135). There was some fluctuation in the mutual orientation of the domains in the structure ensemble (Fig. 4A). However, relaxation properties of the two domains were similar (supplemental Fig. S7), suggesting that the domains tumble in solution as a single unit and do not substantially wobble relative to each other. The fluctuation in domain orientation in the structure ensemble is most probably due to the relatively low number of NOE restraints between the main bodies of domain 18 and domain 19. Over 90% of the residues reside in the most favored regions of the Ramachandran plot. The IgFLNa18–19 structure family conforms well to the SAXS data (Table 1).TABLE 2NMR structure statisticsFLNa16–17FLNa18–19Amino acids1772–19561954–2141Number of structures4020Structure restraintsTotal distance restraints34392930Short range |i − j| ≤ 116551483Medium range, 1 < |i − j| < 5317331Long range, |i − j| ≥ 514671116Interdomain9976Distance restraints/residue18.515.3ϕ and ψ dihedral angle restraints202235Violation statisticsMaximum NOE restraint violation (Å)0.220.22Number of NOE violations > 0.10 Å (n ± S.D.)8.2 ± 2.62.2 ± 1.1Maximum ϕ/ψ dihedral angle violation (°)7.614.2Number of ϕ/ψ dihedral angle violations > 5° (n ± S.D.)0.13 ± 0.330.15 ± 0.37EnergiesAverage restraint violation energy (kcal/mol ± S.D.)29.24 ± 1.3016.23 ± 1.74Average AMBER energy (kcal/mol ± S.D.)−4640.48 ± 13.01−5440.69 ± 16.61RMSD from ideal covalent geometryBond lengths (Å ± S.D.)0.0098 ± 0.00010.0097 ± 0.0002Bond angles (° ± S.D.)2.18 ± 0.012.22 ± 0.01Average coordinate RMSD from the mean structure (Å ± S.D.)Residues 1787–1954Residues 1960–2135Backbone atoms0.48 ± 0.081.01 ± 0.32Heavy atoms0.85 ± 0.071.31 ± 0.29Ramachandran map regions (%)Residues in most favored regions89.191.7Additionally allowed regions9.87.5Generously allowed regions0.90.4Disallowed regions0.20.4 Open table in a new tab The structure of the IgFLNa18-19 domain pair reveals that IgFLNa19 is folded as a conventional Ig domain, but IgFLNa18 does not constitute a complete Ig-fold (Fig. 4B). The first β-strand of IgFLNa18 is not folded as part of domain 18 but is instead bound to the CD face of IgFLNa19. In addition to the interaction between β-strand A of IgFLNa18 and β-strand C of IgFLNa19, also some hydrophobic contacts (particularly Leu1963 with Ile2092 and Thr2094) contribute to the interaction (Fig. 4C). A hydrogen bond is formed between the Ser2961 hydroxyl group and, depending on substructure of the structure family, either amide hydrogen or carbonyl oxygen of Val2090. The absence of β-strand A leaves the hydrophobic core of domain 18 partly exposed. This hydrophobic core anchors domain 18 orthogonally to the N-terminal end of IgFLNa19. A closer look at the structure shows that the side chain of Tyr2077 is pointing outwards from the BC loop of IgFLNa19 and sticks into the hydrophobic core of domain 18 (Fig. 4D). Many hydrophobic core residues of IgFLNa18 (Ile1971, Phe2011, Pro2013, Val2037, and Ile2039) interact with the aromatic side chain of Tyr2077. Also, residues of the AB loop of IgFLNa18 (e.g. Ala1969) and of the domain linker contribute to the domain interface. Total domain-domain interaction surface area between IgFLNa18 and 19 is 980 Å2. If the β-strand interaction is neglected, the interaction surface is considerably smaller: only 380 Å2. Overall structure of IgFLNa18–19 is similar to that of IgFLNa20–21 (15Lad Y. Kiema T. Jiang P. Pentikäinen O.T. Coles C.H. Campbell I.D. Calderwood D.A. Ylänne J. EMBO J.