Báo cáo khoa học: Actin-binding domain of mouse plectin Crystal structure and binding to vimentin pot

Eur J Biochem 271, 1873–1884 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04095.x Actin-binding domain of mouse plectin Crystal structure and binding to vimentin ˇ ˇ´ ´ ´ ´ ˇ ´ Jozef Sevcık1, L’ubica Urbanikova1, Julius Kost’an1,2, Lubomır Janda2 and Gerhard Wiche2 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic; 2Institute of Biochemistry and Molecular Cell Biology, Vienna BioCenter, University of Vienna, Austria Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2a) ˚ from mouse, to a resolution of 1.95 and 2.0 A Comparison of pleABD/2a with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity In fact, pleABD/2a has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD We show that vimentin binds to this site via the amino-terminal part of its rod domain This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks Plectin is a versatile cytoskeletal linker protein of very large size that is abundantly expressed in a wide variety of mammalian tissues and cell types As a cytolinker par excellence it has been found in association with various cytoskeletal structures and it has been shown to interact with a variety of distinct proteins on the molecular level (reviewed in [1]) Plectin’s putative role as a mechanical stabilizing element of cells was fully confirmed when EBS-MD, a severe skin blistering disease combined with muscular dystrophy, was traced to defects in the plectin gene [2], with plectindeficient mice showing a similar phenotype [3] Similar to other members of an emerging family of structurally related cytolinker proteins, referred to as the plakins [4,5], plectin displays a multidomain structure composed of a central  200 nm long rod segment flanked by globular domains [1] The interaction sites of various cytoskeletal proteins have been mapped to opposite ends of the molecule optimizing its potential as a cytoskeletal linker protein One of the better characterized interaction domains of plectin is its N-terminal actin-binding domain (ABD) which is of the canonical type, comprising two tandemly arranged calponin homology (CH) domains, CH1 and CH2 This relatively small domain ( 30 kDa) is found in many actin-binding and cytolinker proteins, such as a-actinin, dystonin, fimbrin, spectrin/fodrin, dystrophin and utrophin, to name a few However, in certain aspects the ABD of plectin seems to be unique Analysis of the plectin gene from mouse revealed the unusual high number of 14 alternatively spliced first exons, 11 of which are directly spliced into the first (exon 2) of seven exons encoding the ABD of plectin [6] In addition, two short exons (2a, 3a), optionally spliced into the ABD sequence (encoded by exons 2–8), lead to insertions of five or 12 amino acid-long segments between the regions encoded by exons and 3, and and 4, respectively Thus, not only three different isoforms of the plectin ABD itself exist but additionally there is the intriguing possibility that the various first exon-encoded sequences preceding the ABD differentially affect its functionality [7] So far, such variability of splicing variants has been described neither for other ABDs nor for the ABD of human plectin Interestingly, the ABD isoform most prominently expressed in muscle (containing the exon 2a-encoded sequence) has been shown to bind to actin more efficiently than other isoforms [6] Recent evidence suggests that the ABD, in particular individual CH subdomains, have functions other than binding to F-actin (reviewed in [8]) While the CH1 subdomain definitely interacts with F-actin, the CH2 subdomain seems to lack such intrinsic activity, but affects binding properties of the whole domain [9,10] In addition, calmodulin has been shown to regulate the interaction of Correspondence to G Wiche, Institute of Biochemistry and Molecular Cell Biology, Vienna BioCenter, University of Vienna, Dr Bohr Gasse 9, A-1030 Vienna, Austria Fax: + 43 4277 52854, Tel.: + 43 4277 52851, E-mail: gerhard.wiche@univie.ac.at Abbreviations: ABD, actin-binding domain; CH, calponin homology; IF, intermediate filament; TPCK, L-chloro-3-[4-tosylamido]4-phenyl-2-butanone Note: The atomic coordinates and structure factors (code 1SH5 and 1SH6) have been deposited in the Protein Data Bank, Reasearch Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) (Received 21 December 2003, revised 26 February 2004, accepted 18 March 2004) ˇ ˇ 1874 J Sevcı´ k et al (Eur J Biochem 271) dystrophin and utrophin with F-actin through direct binding to CH domains, although the physiological relevance of this is not clear [11,12] Furthermore, CH domains contain specific binding sites for phosphoinositides and PIP2 has been shown to modulate the actin-binding activity of a-actinin [13] and plectin [14] Another intriguing feature, so far characterized only for plectin ABD, is the direct interaction with the cytoplasmic tail domain of the integrin b4 subunit [15,16] For a number of proteins with either a single CH subdomain or tandemly arranged CH subdomains (ABD), interactions with intermediate filament (IF) proteins have been reported Calponin has been shown to interact with desmin, the major IF protein of smooth and striated muscle [17–19], and for fimbrin a colocalization with vimentin was observed in cultured macrophages [20] In both cases it was suggested that interactions were mediated by CH domains Based on this, the idea arose that the interaction of CH domains with IF subunit proteins may represent a highly conserved function common to CH protein family members As of recently, crystal structures of ABDs have been reported for fimbrin, utrophin, dystrophin and human plectin [21–24] Dystrophin and utrophin ABDs both crystallized as antiparallel dimers with an open conformation, whereas the ABD of fimbrin and human plectin were described as monomers with a closed conformation Unlike utrophin and dystrophin, fimbrin appears to associate with F-actin in the closed conformation [25] This could be due to lengths and conformation variability of linkers connecting individual CH subdomains in these proteins [21–24] To study the structural and functional relationships with other CH protein family members we have prepared two crystalline forms of the ABD of plectin and analyzed the atomic structures We show here that this ABD bears a close structural resemblance to the ABD of fimbrin and human plectin Extending this to the functional level, we further show that plectin, like fimbrin, interacts via its CH1 domain with the IF protein vimentin Furthermore, in affinitybinding assays using chymotryptic fragments of vimentin we identified the N-terminal region of vimentin’s central rod as ABD-docking site Materials and methods cDNA constructs A cDNA fragment corresponding to exons 2–8 including alternative exon 2a (pGR75; pleABD/2a) was prepared by PCR as described previously [6], except that the fragment was subcloned into a unique EcoRI site of expression vector pBN120 [26], a pET-15b derivate (Novagen, Madison, WI, USA) The same strategy was used for the cloning of ple1cABD/2a cDNA, corresponding to the plectin ABD preceded by 66 exon 1c-encoded amino acid residues (pGR147); ple1aABD starting with exon 1a-encoded sequences, but without exon 2a sequences; ple6–9 cDNA (pGR103) starting at the last codon of exon and extending to half of exon (ATC CGG); and ple4–8 (pDS19) cDNA starting with the first in-frame ATG in exon and extending close to the end of exon (GCA CAG) The differences were that ple4–8 cDNA was subcloned into pBN120 Ó FEBS 2004 through EcoRI and NdeI sites and that ple1aABD cDNA was subcloned into expression vector pGR66, a modified derivate of pBN120 missing a His-tag A fragment corresponding to repeat of mouse plectin (1039 bp) was generated by PCR (forward primer, 5¢-GGAATTCCGCGGTCTCCGCAAGC-3¢; reverse primer 5¢-GGAATTCAAGCGTACCAGCGCGGTAC-3¢), using mouse plectin cDNA (rat accession number X59601) as a template This fragment was subcloned into expression vector pBN120 resulting in plasmid pKAB1 Plasmid pFS129 encoding full length mouse vimentin (accession number M26251) without tag has been described previously [27] Plasmids pFS2 and pFS3, encoding the N-terminus of vimentin (Met1–Glu94) and the rod domain (Phe95–Ile411) of vimentin, respectively, were prepared similar to pKAB1, using primer pairs pFS2 forward (5¢-CCGGAATTCATGTGGACCAGGTCTGTG-3¢), and pFS2 reverse (5¢-CCGGAATTCCTCAGTGTTGATGG CGTC-3¢), and pFS3 forward (5¢-CCGGAATTCTTCAA GAACACCCGC-3¢), and pFS3 reverse (5’-CCGGAATT CAATCCTGCTCTCCTC-3’), and mouse vimentin cDNA as a template Plasmid pGP1 encoding the rod and C-terminus of vimentin (Phe95–Glu466) was obtained by subcloning a SacI/BamHI excision fragment of pFS129 in pFS3 pBN128, encoding the N-terminal and rod domain of vimentin (Met1–Ile411) was obtained by SalI/SmaI subcloning of mouse vimentin full-length cDNA, contained in pMC-V21 [27], into a modified pET-15b expression vector (pBN121) and deletion of the nucleotide sequence encoding the last 55 amino acids by PCR cloning All plasmids were verified by sequencing Protein expression and purification The protein used for crystallization (pleABD/2a) as well as ple1cABD/2a were isolated in the form of soluble Histagged fusion proteins and purified as described previously [28] To remove the His-tag prior to crystallization, pleABD/2a was treated with thrombin Ple1aABD without tag was purified from bacterial lysates in soluble form using several matrices (Phenyl-Sepharose fast flow, DEAE Sepharose CL-6B, Superdex 75TM) Recombinant fulllength vimentin encoded by pFS129 was isolated from lysed bacterial pellets following the inclusion body preparation procedure [29] Final pellets were dissolved in mM Tris/HCl, pH 7.5, M urea, mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM phenylmethanesulfonyl fluoride (solution A) and after centrifugation (40 000 g, 15 min, °C) supernatants were directly applied to a 10 mL DEAE Sepharose CL-6B column equilibrated with solution A Bound protein was eluted with a 60 mL gradient of NaCl (0–0.3 M) in solution A, and aliquots of fractions (2 mL) were analyzed by SDS/PAGE Vimentin-containing fractions were pooled, diluted : (v/v) with 50 mM sodium formate, pH 4.0, M urea, 10 mM 2-mercaptoethanol, 0.4 mM phenylmethanesulfonyl fluoride (solution B) and applied to a 10 mL CM Sepharose CL-6B column in solution B After washing, bound vimentin was eluted with a linear gradient of KCl (0–0.3 M) in solution B, and aliquots were stored at )20 °C His-tagged, truncated versions of vimentin and plectin ABD were purified by affinity chromatography as described [15] Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur J Biochem 271) 1875 Blot overlay assays Purified samples of proteins were subjected to SDS/PAGE using loading buffer with or without dithiothreitol Proteins were transferred to nitrocellulose membranes, blocked with 5% (w/v) nonfat milk powder in 1.5 mM KH2PO4, mM NaH2PO4, 137 mM NaCl, 2.6 mM KCl, pH 7.4 (solution C) Blots were overlaid with full-length mouse vimentin or ple1cABD/2a (both at concentrations of lgỈmL)1) in 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, mM EGTA, mM MgCl2, 0.1% (v/v) Tween 20, mM dithiothreitol, pH 7.5 After h of incubation, membranes were washed thoroughly with solution C supplemented with 0.1% (v/v) Tween 20 For detection of bound proteins we used affinity-purified goat anti-(mouse vimentin) IgG [30], diluted : 5000 (v/v), or isoform-specific affinity-purified rabbit antibodies to plectin 1c [31], diluted : 1000 (v/v), in combination with secondary HRP-coupled antisera and the SuperSignalÒ kit (Pierce, Rockford, IL, USA) Affinity chromatography PleABD/2a was coupled to CNBr-activated Sepharose 4B following the procedure outlined by Amersham Biosciences (Little Chalfont, UK) Vimentin purified in urea was dialyzed step by step against 6, 4, and M urea in solution D (10 mM Tris/acetate, pH 8.3, 0.1 mM EDTA, mM 2-mercaptoethanol) Each dialysis step was performed for 30 at room temperature, followed by dialysis against solution D overnight at °C Urea-free vimentin, precentrifuged at 100 000 g for 30 min, was digested with chymotrypsin (1 : 400, w/w) for 30 at 25 °C The reaction was stopped by the addition of L-chloro-3-[4-tosylamido]4-phenyl-2-butanone (TPCK) (final concentration of 100 lgỈmL)1) The digest was then immediately loaded onto a pleABD/2a Sepharose column equilibrated with 10 mM Tris/HCl, pH 7.5, 0.5 mM MgCl2, 0.2 mM dithiothreitol, 25 mM NaCl, 50 lgỈmL)1 TPCK (solution E) Bound protein was eluted with a linear gradient of 25–400 mM NaCl in solution E (10 000 g, min) The supernatants were used to form new drops, in which microcrystals had grown To obtain better crystals the procedure was repeated using solutions with lower concentrations of protein (10 mgỈmL)1) and precipitant [8% (w/v) PEG 8000] The precipitant solution was enriched with dioxane (2%, v/v) to reduce twinning tendency of crystals The first microcrystals were used as seeds Crystals reached dimensions of up to 0.8 mm after 1–2 days Data collection and processing The collection and processing of X-ray data from crystal form I (structure I) were described previously [28] Data ˚ from crystal form II (structure II) were collected to 2.0 A resolution at 100 K on EMBL X-11 beamline at the DORIS storage ring, DESY, Hamburg Crystals were soaked stepwise in cryoprotectant prepared from precipitant solution enriched with glucose (6, 12, 18 and 24%, w/v) before flash-freezing Conditions for data collection were optimized using the program BEST [32] Data collection statistics are summarized in Table (data from crystal form I are included for comparison) Dimensions of the unit cell, crystal symmetry and molecular mass of the protein gave a ˚ crystal packing density VM of 2.5 A3ỈDa)1, with a solvent content of 50% for one protein molecule in the asymmetric unit [33] Structure determination and refinement At the time when we solved the structure of pleABD/2a there were only three members of the actin-binding protein family for which the tertiary structure of their ABD was known, namely utrophin (Protein Data Bank code 1QAG), dystrophin (1DXX) and fimbrin (1AOA) (The structure of the human plectin ABD was reported later [24], and therefore could not be used in our structure determination) Table Data collection statistics Values in parentheses refer to the last resolution shell Parameter Crystallization Two crystalline forms (I and II) were prepared by the hanging-drop vapor diffusion method Monoclinic crystals (form I), belonging to the P21 space group with two molecules in the asymmetric unit, were grown from lL drops containing equivalent amounts of protein and precipitant solutions The protein solution was prepared by dissolving lyophilized samples in 0.05 M Tris/HCl, pH 9.0, to a concentration of 20 mgỈmL)1 The precipitant solution contained 10% (w/v) PEG 8000, 2% (v/v) dioxane, and 0.1 M Tris/HCl, pH 8.5–9.0 [28] Orthorhombic crystals in the P212121 space group with only one molecule in the asymmetric unit (form II) were prepared by the same method combined with seeding Drops (6 lL) were prepared by mixing equal volumes of protein solution (see above) and precipitant solution [10% (w/v) PEG 8000, 0.1 M cacodylate buffer, pH 6.5, 0.2 M calcium acetate] Drops were collected after 24 h of equilibration and the precipitate was removed by centrifugation Crystal form I Crystal form II ˚ Wavelength (A) Beamline Temperature (K) ˚ Resolution range (A) 1.100 X31 193 20.0–1.95 (1.97–1.95) P21 0.812 X11 100 50.0–2.00 (2.02–2.00) P212121 55.31 108.92 63.75 115.25 0.3 60 96.2 (71.7) 6.0 (40.0) 22.2 (2.1) 32.52 51.23 144.72 90 0.4 50 97.6 (86.3) 3.8 (31.0) 33.9 (3.5) Space group Unit cell parameters ˚ a (A) ˚ b (A) ˚) c (A b (°) Mosaicity Solvent content (%) Completeness (%) R(I)mergea (%) I/r(I) R(I)merge ¼ S/I )/SI, where I is an individual intensity measurement and is the average intensity for this reflection with summation over all data a ˇ ˇ 1876 J Sevcı´ k et al (Eur J Biochem 271) Amino acid sequence identities of the ABDs of utrophin, dystrophin and fimbrin with pleABD/2a, determined by the program FASTA [34], are 48, 47 and 23%, respectively Because of differences in the relative orientation of CH1 and CH2 subdomains in the structures of utrophin, dystrophin and fimbrin, not the whole ABD, but the CH1 and CH2 subdomains of utrophin were used as two model structures PleABD/2a structure I was determined by molecular replacement using the program MOLREP [35] four times, twice with CH1 and twice with CH2 After each MOLREP session the solution was subjected to five cycles of refinement with the program REFMAC5 [36] to improve the model, and the resulting PDB file was fixed in the next MOLREP session Using this procedure, R factors in the four MOLREP sessions were 58, 52, 45 and 38%, and correlation coefficients 25, 39, 54 and 68% MOLREP unambiguously showed that there were two protein molecules in the asymmetric unit (hereafter referred to as molecules A and B) For building the model, the program ARP/WARP in the mode WARPNTRACE [37] was used The program automatically built a model consisting of 378 out of 490 residues (molecules A and B) with a connectivity index of 0.92 The remaining residues, including those forming the loop connecting the CH1 and CH2 subdomains, were built manually using the program O [38] running on a Silicon Graphics Station Structure I was refined with the program REFMAC5 Sparse matrix was used as the method of minimization Refinement of the structure was altered with correcting the amino acid sequence and building the parts which were different from those of utrophin and which were not built by WARPNTRACE The structure was refined against 95% of the data, the remaining 5% (randomly excluded from the full data set) were used for crossvalidation in which Rfree was calculated to follow the progress of refinement [39] After each refinement cycle, ARP, an automated refinement procedure [40], was applied for modeling and updating the solvent structure After the R factor had fallen to about 20%, refinement continued with anisotropic temperature factors and hydrogen atoms generated in standard geometries Structure II was determined by molecular replacement using molecule A from structure I as a model Solution was straightforward giving an R factor and correlation coefficient of 39 and 63%, respectively Refinement was performed in the same way as that of structure I Refinement statistics of both structures, average temperature factors, and target deviations against stereochemical restraints are given in Table For visualization and rebuilding of the structures O and XTALVIEW programs were used [38,41] Other methods MALDI-TOF MS as well as ESI MS were performed at the mass spectrometry unit, Vienna Biocenter, Vienna, Austria Results Description of the structures Like other ABDs of the canonical CH1-CH2 type, the ABD of plectin is encoded by seven exons, the first four encoding Ó FEBS 2004 Table Refinement statistics Parameter structure I structure II ˚ Resolution limits (A) 30.0–2.0 50.0–2.0 42 811/2286 15 889/848 No of reflections all/Rfree set Rwork (%) 15.1 19.7 Rfree (%) 19.4 29.9 Rall (%) 15.3 20.2 ˚ ESU based on Rfree (A) 0.12 0.21 Protein atoms, molecule A/B 1919/1919 1884 Water molecules 188 58 Wilson plot B factor 32.6 33.8 ˚ Average B (A2) main-chain A molecule 42.2 36.3 B molecule 39.3 – side-chain A molecule 46.6 39.8 B molecule 44.2 – Waters 46.2 38.9 Rms deviation from ideal geometry (target values are given in parentheses) ˚ Bond distances (A) 0.03 (0.02) 0.04 (0.02) Bond angles (°) 2.02 (1.94) 2.59 (1.94) ˚ Chiral centers (A3) 0.22 (0.20) 0.21 (0.20) ˚ Planar groups (A) 0.01 (0.02) 0.02 (0.02) ˚ Main-chain bond B-values (A2) 2.16 (1.50) 2.38 (1.50) ˚ Main-chain angle B-values (A2) 3.44 (2.00) 3.57 (2.00) ˚ Side-chain bond B-values (A2) 4.70 (3.00) 5.27 (3.00) ˚ Side-chain angle B-values (A2) 7.20 (4.50) 7.26 (4.50) the CH1 subdomain and the remaining three the CH2 subdomain The mouse plectin ABD isoform analyzed here, pleABD/2a, is unique, as it contains a five amino acid-long sequence (Fig 1) inserted by differential splicing of a short exon (exon 2a) between the first two exons of the ABD [6] Comprising 245 residues, the analyzed recombinant protein contained pleABD/2a as a 237 residues-long fragment (amino acids 181–417; EMBL accession number AF 188008) flanked by six amino-terminal (GSHMEF) and two carboxy-terminal (EF) residues (added as cloning requirement) The amino acid residues in the structures are numbered from to 245 according to the sequence of the recombinant protein, i.e amino acids with numbers 7–243 in the structure correspond to 181–417 in the AF 188008 sequence Two crystal structures of pleABD/2a were determined One of them, structure I was derived from a monoclinic crystal containing two protein molecules (A and B) in the asymmetric unit, the other, structure II, from an orthorhombic crystal, containing one molecule in the asymmetric unit (for details see Materials and methods) As found in structure I, molecule A, pleABD/2a is an a-protein consisting of 11 helices: a1 (residues 8–25), a2 (48–58), a3 (70–86), a4 (96– 100), a5 (104–118), a6 (134–145), a7 (165–172), a8 (181–186), a9 (189–204), a10 (212–215), and a11 (222–235) (Figs and 2A) Helices a1–a5 form the CH1 and helices a6–a11 the CH2 subdomain The subdomains are connected by a flexible 15 residues-long loop (119–133) For five N-terminal (GSHME) and eight C-terminal residues (RVPGAQEF) of both molecules in structure I there was no electron density observed In structure II there was no electron density for the first seven (N-terminal) nor for the last eight (C-terminal) Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur J Biochem 271) 1877 Fig Amino acid sequence alignment and structural features of ABDs from mouse and human plectin, utrophin, dystrophin and fimbrin Boxes indicate helices (a1–a11) as given by the program PROCHECK [44] Connecting segments between the CH1 and CH2 subdomains are in red The three amino acid residues differing in the human and mouse plectin sequence are in gray italics (*) Note that in the PDB structure 1MB8 only two differing amino acid residues were reported The sequence encoded by exon 2a is double underlined Amino acid residues of the identified actinbinding sites of fimbrin and dystrophin are shaded Numbers on the right correspond to amino acid positions Dashes in sequences indicate gaps introduced to allow maximum alignment PLM, mouse plectin ABD (EMBL accession no AF188008); PLH, human plectin ABD (EMBL accession no U53204); UTR, utrophin ABD (PDB code 1QAG); FIM, L-fimbrin ABD1 (PDB code 1AOA); DYS, dystrophin ABD (PDB code 1DXX) residues It is reasonable to conclude that N- and C-terminal residues formed highly disordered tails The molecular mass of the recombinant protein was 28 631 Da, as determined by mass spectrometry This was in a good agreement with its theoretical mass of 28 656 Da, indicating that the structure indeed contained all of the 245 encoded residues ABDs can adopt open (e.g utrophin) or closed (e.g fimbrin) conformations PleABD/2a was in a closed conformation in which the CH1 and CH2 subdomains were facing each other through helices a1 and a5, and helices a10 (including the subsequent loop) and a11 Molecules A and B in the asymmetric unit of the pleABD/2a structure I formed a crystallographic dimer (Fig 2B) In the contact area of the two molecules there were two hydrogen bonds formed between side chains of Glu125(A)/Asn88(B) and Lys130(A)/ Glu131(B) and one bond mediated by a water molecule Asn151(A)/W66/Asp150(B) The solvent-accessible surface ˚ buried at the plectin dimer interface was 760 A2, corres˚ ponding to  3% (380 A2) of the surface of each isolated ˚ molecule (11 800 A2) This was far below the minimum of 9% required for classification of a dimer as a protein complex [42], suggesting that the crystallographic dimer hardly could exist in solution Moreover, structure II has confirmed that pleABD/2a can exist as a monomer in solution The contacts between molecules in the crystal lattice apparently did not change the orientation of the CH1 with respect to the CH2 subdomain in spite of the fact that the loop connecting the two subdomains theoretically could allow various orientations including those found in utrophin It can be concluded that the conformation of pleABD/2a as found in structures I and II is stable and not subject to conformational changes due to different crystal packing Quality of protein structure models The final R and Rfree factors for structure I were 15.3 and 19.4% (Table 2) There were two protein molecules (A and B) and 188 solvent molecules in the asymmetric unit The Ramachandran plot [43] calculated by the program PROCHECK [44] showed that 92.1 and 94.4% of the residues of molecules A and B, respectively, were in the most favored regions The remainder was in additionally allowed regions except for Thr158, which in both molecules had the same dihedral angles being just at the outer border of the additionally allowed region Thr158 was localized in a surface loop and there was no doubt about its conformation, as the electron density in this region was clear ˇ ˇ 1878 J Sevcı´ k et al (Eur J Biochem 271) Ó FEBS 2004 Fig Ribbon representation of the pleABD/ 2a structure I and comparison with utrophin and fimbrin ABDs (A) Stereo view of pleABD/2a Individual helices are numbered (B) Crystallographic dimer as seen in the asymmetric unit The views are related by 90° rotation around a horizontal axis Molecules A and B are shown in red and blue, respectively (C) Overlap (stereo view) of CH1 and CH2 subdomains of pleABD/2a molecule A with the CH1 subdomain of utrophin molecule A and the CH2 subdomain of utrophin molecule B Utrophin molecules are colored in light (molecule A) and dark blue (molecule B) The plectin molecule is in red (D) Overlap (stereo view) of pleABD/2a (red) with fimbrin ABD (green) Figures were generated using the program MOLSCRIPT [52] A diagram of the average main-chain temperature factors of molecules A and B as a function of residue number (Fig 3A) revealed an amazing similarity of their temperature factor profiles, confirming the conformational stability of the pleABD/2a molecule; temperature factors were highest in the loop connecting the two subdomains For structure II, the final R and Rfree factors were 20.2 and 29.9% (Table 2) The structure contained one protein Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur J Biochem 271) 1879 Fig Comparisons of mouse and human ABDs Average main chain B factor values of mouse ABDs (A), and differences between Ca atoms in superpositioned structures (B) are plotted as a function of residue numbers IA, structure I molecule A; IB structure I molecule B; II, structure II; HP, structure of human plectin and 58 solvent molecules in the asymmetric unit and in the Ramachandran plot there were 88.2% of the residues in the most favored region and 11.8% in additionally allowed regions Fluctuation of B-values of structure II was similar to that of structure I, with average B-values of structure II being slightly lower (Fig 3A), which could be due to the fact that structure II data were collected at cryogenic temperature Refinement parameters of structure II were slightly worse compared to those of structure I, which was unexpected considering that the crystal data seemed to be better for structure II Comparison of ABDs from mouse plectin, human plectin, fimbrin, utrophin and dystrophin The major difference in the tertiary structures of human and mouse plectin was found at their N termini, where the first a-helical segment (a1) of human plectin exceeded that from mouse by nine amino acid residues However, since six of these additional residues were encoded by one of the first exons (1c) preceding the actual ABD-encoding exons 2–8, this does not reflect a structural difference of mouse and human ABDs per se To document minor structural differences between human and mouse plectin ADBs, structure I molecule B (IB), structure II (II), and the structure of human plectin (HP) were superimposed on structure I molecule A (IA) Figure 3B shows the deviations of Ca atoms observed in these superpositions as a function of residue number (with the exception of the first N- and the last C-terminal residues, ˚ which differed by more than A) Excluding the residues for which distances between corresponding Ca atoms ˚ exceeded 1.75 A (Fig 3B, dashed line), the rms displacement values were 0.25 (IB/IA, 232 Ca atoms), 0.48 (II/IA, ˚ 225 atoms), and 0.62 A (HP/IA, 224 atoms) The largest ˚ differences in Ca positions (up to 2.5 A) were found in the surface loop region, which was not unexpected considering that each molecule has a different environment in the crystal Least squares superpositions of mouse plectin with the corresponding subdomains of human plectin, utrophin, dystrophin and fimbrin are summarized in Table In these superpositions only the CH1 (8–119) and CH2 (134–236) subdomains from structure I molecule A were used; the connecting segment was excluded as its conformation differs substantially among the various proteins compared (helix in utrophin and dystrophin, helix–loop in fimbrin, and loop in plectin) Furthermore, as the ABDs of utrophin and dystrophin adopt an open conformation (contrary to the plectin ABD), the CH1 domain from utrophin molecule A and the CH2 domain from molecule B were used in the overlap The different numbers of Ca atoms involved in Table Superposition of ABD domains (without segment connecting subdomains) IA(B), structure I, molecule A(B); II, structure II; HP, human plectin ABD No of Ca atomsa ˚ rmsd (A) IA/IB IA/II IB/II IA/HP IA/utrophinb IA/dystrophinb IA/fimbrin 218 211 212 203 192 176 78 0.23 0.44 0.48 0.55 0.73 0.82 1.12 ˚ Ca atoms separated by more than 1.75 A were omitted b CH1 is from utrophin (dystrophin) molecule A, CH2 from utrophin (dystrophin) molecule B (see Figs 2C,D, and relevant text) a ˇ ˇ 1880 J Sevcı´ k et al (Eur J Biochem 271) Ó FEBS 2004 superpositions (reflecting the degree of structural similarities) clearly showed highest similarity of mouse plectin with human plectin and lowest with fimbrin (Table 3) The nearly identical conformations of the pleABD/2a structure and the corresponding subdomains of the A and B molecules of utrophin and dystrophin probably have not arisen by chance and may have significance for functional properties of these ABDs The ABD of plectin binds to vimentin Fig Overlay of various plectin ABD versions with full-length vimentin Recombinant versions of the plectin ABD starting with exon 1aencoded sequences (ple1aABD), or starting with exon 2-encoded sequences and containing 2a-encoded sequences (pleADB/2a), or lacking part of (ple4–8), or the whole CH1 domain (ple6–9), as well as a fragment corresponding to the repeat domain of plectin (positive control), and BSA (negative control) were subjected, in duplicate, to 12.5% SDS/PAGE Proteins on one gel were blotted onto a nitrocellulose membrane and overlaid with recombinant full-length mouse vimentin (B), proteins on a second gel were stained with Coomassie Blue (A) All proteins, except for ple6–9 and BSA, showed significant binding to vimentin The IF protein vimentin has been reported to specifically interact with the CH1 subdomain of the first (N-terminal) of fimbrin’s two ABDs [20] In light of the extensive structural resemblance of fimbrin and plectin ABDs it was therefore of interest to assess IF-binding activity of the plectin ABD Moreover, a second IF protein interaction domain at the N terminus in addition to its C-terminal IF-binding site [26] would raise plectin’s functional versatility, particularly as a cytoskeletal crosslinking element To assess the plectin ABD–vimentin interaction, in vitro overlay assays were performed Ple1aABD, an ABD version of plectin preceded by a sequence encoded by exon 1a[6], pleABD/2a and truncated versions of the plectin ABD missing half of the CH1 domain (ple4–8), or the complete CH1 domain (ple6– 9) were immobilized on nitrocellulose membranes and overlaid with full-length vimentin In agreement with previously reported findings [20] all proteins, except the one missing the entire CH1 domain (ple6–9) and the negative control (BSA), showed binding to vimentin, similar to the positive control (recombinant plectin repeat 5) (Fig 4) Thus only the CH1, but not the CH2, domain of plectin’s ABD was found capable of binding to vimentin To specify the subdomain of vimentin that bound to plectin’s ABD, several truncated versions of vimentin were expressed in Escherichia coli and subjected to overlay assays The ABD version used in these experiments contained the preceding 66 amino acid residues-long sequence specific for plectin isoform 1c (ple1cABD/2a), enabling detection of bound protein via isoform 1c-specific antibodies [31] This fragment showed strong binding to full-length vimentin and to VimNR, a truncated version of vimentin containing its N-terminal domain and central rod domain, but lacking the C-terminal domain (Fig 5) Only very weak interactions were observed with vimentin fragments corresponding to the N-terminal (VimN), or rod domains alone (VimR), or to the rod in conjunction with the C-terminal domain (VimRC) These data suggested that the plectin ABD-binding site of vimentin was contained in the N-terminal part of the molecule comprising the head domain and possibly parts of the rod The head domain of vimentin had previously been shown to harbor the binding site(s) for the CH1 subdomain of fimbrin’s N-terminal ABD [20], whereas desmin, an IF protein of similar type, was found to interact with the corresponding domain of calponin via the N-terminal part of its rod domain [19] Interestingly, fimbrin and calponin have been found to interact with tetrameric forms of soluble vimentin and desmin As we were unable to cosediment pleABD/2a with filamentous vimentin in sedimentation assays (data not shown) it seems that the plectin ABD interacts with vimentin in the same way Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur J Biochem 271) 1881 Elution and SDS/PAGE of bound proteins revealed that a single major fragment of  18 kDa was retained on the column (Fig 6) This fragment could be mapped to ÔCoil 1Õ, an N-terminal segment of the vimentin rod domain (amino acid residue positions 124–276 of mouse vimentin, EMBL accession no M26251), using MALDI-TOF mass spectrometric sequence analysis Discussion Fig Overlay of recombinant vimentin fragments with plectin ABD Recombinant versions of vimentin subdomains were immobilized on nitrocellulose membranes as described in Fig 4, and overlaid with the ple1cABD/2a Vim, full-length vimentin; VimN, N-terminal domain; VimR, rod domain; VimRC, vimentin without N-terminal domain; VimNR, vimentin without C-terminal domain To detect vimentinbound plectin ABD, plectin isoform 1c-specific antibodies [31] were used Note the strong binding of plectin ABD to full-length vimentin and VimNR, but only weak or no binding to other vimentin fragments To confirm the specificity of the plectin ABD–vimentin interaction and to more precisely map vimentin’s plectin ABD-binding site, vimentin purified in urea was kept in its soluble (tetrameric) form by dialysis into solution D (see Material and methods) The protein was than subjected to limited chymotryptic digestion and fragments generated were applied to a pleABD/2a-Sepharose affinity column Because of its expression in the form of several distinct isoforms, the ABD of plectin is unique among those of other actin-binding protein family members [6] The isoform crystallized and analyzed here contains an extra five amino acids (HWRAE; positions 28–32) encoded by exon 2a, a differentially spliced exon inserted between the common exons and This insertion makes the connecting loop of helices a1 and a2 longer Amino acid residues contained in this loop are located closely to one (the first) of three segments identified as direct docking sites for actin within dystrophin’s ABD [22] (Fig 1) This insertion in pleABD/ 2a may result in higher flexibility of this region and the surface exposure and amino acid composition of this segment (facilitating charge–charge as well as hydrophobic interactions) may improve binding properties of the ABD In fact, we have previously shown that this isoform exhibits a higher affinity to actin than several other isoforms without the 2a sequence [6] Our analysis revealed that both, mouse and human pleABD/2a are structurally similar to the fimbrin ABD, although these domains share only little sequence identity The fimbrin and plectin ABDs have a similar closed conformation, differing from the open conformation described for the ABDs of dystrophin and utrophin [21–24] It is interesting to note that until now, sequences corresponding to exons 2a and 3a of mouse plectin have been identified neither in other family members, nor in human plectin In this view, insertion of exon 2a into the sequence of human pleABD/2a as reported [24] seems without rational explanation Although the ABDs of fimbrin, utrophin and a-actinin are related, they appear to have different effects on F-actin conformation upon binding and thus may use different mechanisms of association [25,45,46] Up to now a consen- Fig Affinity-binding of proteolytically derived fragments of vimentin to pleABD/2a Partial chymotryptic digestion of vimentin and affinitychromatography of fragments on a pleABD2a-Sepharose column was carried out as described in the text SDS/PAGE of eluted fractions is shown Lanes 1–11, wash fractions; 12–27, salt-gradient elution of bound proteins Co, sample loaded onto column The molecular mass of size markers run in left-most lane is indicated The18 kDa fragment of vimentin binding to pleABD/2a mapped to the N-terminal part of the vimentin rod domain, as determined by MALDI-TOF mass spectrometry ˇ ˇ 1882 J Sevcı´ k et al (Eur J Biochem 271) sus on the mode of binding (open or closed conformation of ABD) of utrophin and dystrophin to actin filaments has not been reached [47,48] It has been reported that fimbrin [25] and human plectin ABDs [24] bind to actin filaments in their closed conformation In view of these findings it is expected that pleABD/2a could adopt a closed as well as an open conformation in binding to actin, as the connecting segment between the CH1 and CH2 domains is flexible and long enough to allow both conformations (as proposed also for human plectin [24]) The notion of pleABD/2a adopting an open conformation is supported by similar characteristics of contact areas in the CH1 and CH2 structures of pleABD/ 2a, utrophin, and dystrophin ABD of fimbrin is expected to bind only in its closed conformation, in agreement with the finding that 72% of its contact area (excluding the connecting segment) is hydrophobic and there is no ˚ hydrogen bond shorter than 3.25 A On the other hand, only 59% of the corresponding contact areas of pleABD/2a and utrophin are hydrophobic and there are five and six hydrogen bonds, respectively Moreover, the secondary structure prediction of pleABD/2a suggested a helix to exist between the CH1 and CH2 subdomains The sites of pleABD/2a responsible for binding to actin have not been identified so far Assuming that they are similar to those reported for dystrophin [22], the amino acid sequences 13–22, 88–116 and 131–146 might be involved, slightly differing from the binding segments predicted for fimbrin [25] (Fig 1) The ABDs of dystrophin and utrophin crystallized as antiparallel dimers [21,22], whereas fimbrin and human plectin ABDs crystallized in monomeric form [23,24] When pleABD/2a was crystallized at pH 9, we also found two molecules in the asymmetric unit [28] However, conditions at pH 6.5 led to the formation of crystals with only one pleABD/2a molecule in the asymmetric unit The assessment of the contact area between molecules A and B in pleABD/2a crystals obtained at pH (crystal form I) showed that these molecules not interact strongly enough to form dimers which could exist also in solution Therefore, the crystallographic dimer observed in the asymmetric unit was probably an artefact of crystallization rather than a dimerization product Although CH subdomains forming a canonical ABD structurally seem to be highly conserved, data are accumulating that show functional diversity of CH1 and CH2 subdomains [8] It was reported that proteins containing either single CH subdomains (calponin), or more complex ABDs (fimbrin) can interact with IF proteins Calponin binds to desmin, the major IF protein in smooth and skeletal muscle [17–19] and fimbrin interacts with and colocalizes with vimentin in filopodia, retraction fibres, and podosomes at the ventral surface of cultured macrophages [20] In both cases there is evidence that binding occurred to IF subunit proteins in their nonfilamentous state [17–20] Fimbrin was unable to bind to polymerized vimentin in cosedimentation assays and binding occurred at a stoichiometry of : 4, suggesting that the IF protein was in its tetrameric form [20] As one may expect on the basis of their structural similarity, the ABDs of fimbrin and plectin apparently also have a number of functions in common, including binding to vimentin Similar to fimbrin [20] the ABD of plectin failed Ó FEBS 2004 to cosediment with vimentin filaments (data not shown), suggesting that it, too, interacted with soluble IF protein subunits rather than with their filamentous polymers Binding assays revealed that the major vimentin-binding interface was localized within the CH1 subdomain of plectin Since ple4–8 (pleABD/2a lacking half of the CH1 domain) bound to vimentin, sequences preceding those encoded by exon apparently did not contribute to this interaction However, as fragments corresponding to the first half of plectin’s CH1 domain (exons 2–4) have not been examined in our studies, a role of this part in vimentinbinding can not be fully excluded Using an overlay assay we found strong binding of plectin’s ABD to a fragment of vimentin comprising the rod and its N-terminal domains, but when examined individually, both of these domains showed only weak binding Using a similar method, it had been shown that a vimentin fragment lacking the C terminus (N410/VimNR) was capable of binding to fimbrin, contrary to a fragment lacking the initial 102 amino acid residues (102C/VimRC), suggesting that the fimbrin-binding site was located in the N-terminal head domain of vimentin [20] The N-terminal vimentin fragment (VimN) and the rod fragment (VimR) used in our experiments ended and started, respectively, at Phe95 Consequently, the rod-containing fragment used in our experiments and the N-terminal fragment N410/VimNR used in [20] overlapped by a few amino acid residues With this consideration, the results of our overlay assay were consistent with the findings reported in [20] Using as an alternative method affinity-binding of proteolytic fragments of vimentin in combination with mass spectrometry, we found that plectin bound to a  18 kDa fragment corresponding to ÔCoil 1Õ [49], the N-terminal part of the ahelical rod domain of vimentin This is in agreement with similar experiments in which the calponin-binding site was restricted to the N-terminal part of the desmin rod domain [19] However, in the overlay assay, the rod domain of vimentin (VimR) alone showed little binding to plectin As fragments obtained by partial proteolytic digestion of properly folded full-length vimentin are more likely to preserve the structure of the native protein than recombinantly prepared fragments, we assume the true plectin ABD-binding site of vimentin to be localized in the Nterminal part of its rod domain Binding to the evolutionary highly conserved CH domains could be a common feature of IF proteins in general Likewise, considering that the ahelical coiled-coil structure of vimentin’s rod domain is highly conserved in all IF protein family members, plectin’s ABD may bind also to other IF proteins, such as desmin The functional significance of the plectin ABD–vimentin interaction remains elusive The primary activity of the ABD supposedly is actin-binding, a function demonstrated for both fimbrin and plectin [14,50] The CH domain of calponin, on the other hand, doesn’t seem to be required for actin-binding as calponin interacts with actin via its C-terminal domain [51] Thus, with an IF protein-binding site residing in their N-terminal CH domains in addition to their genuine actin-binding activities, fimbrin, plectin and calponin, might influence the assembly and organization of both actin and IF cytoskeletal networks The interaction of fimbrin’s ABD with vimentin has been shown to be adhesion dependent and it has been suggested that this complex is a Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur J Biochem 271) 1883 transient structure involved in early cell adhesion [20] In smooth muscle cells calponin may be an integral component of desmin IFs in the vicinity of dense bodies [18] The interaction between IF proteins, such as desmin or vimentin, and proteins containing one or more CH domains could represent a highly conserved function related to the establishment of cell adhesion structures By sequestering soluble vimentin at certain sites, such as focal adhesion contacts, plectin may favor and/or initiate IF network formation at these sites by locally increasing IF protein concentrations Once filament assembly has been initiated, plectin may stabilize and anchor the filaments at these sites via its alternative C-terminal IF-binding site Future experiments should show whether this model can be verified Acknowledgements We thank our colleagues Kamaran Abdoulrahman, Branislav Nikolic, Gernot Putz, Gunther Rezniczek, Daniel Spazierer and Ferdinand ă Steinbock, for providing various reagents and for valuable discussions ă We are grateful to the EMBL Hamburg team for providing us with synchrotron facilities and for help in data collection We would also like to thank the European Community for supporting J.S and L.U through the Access to Research Infrastructure Action of the Improving Human Potential Programme to the EMBL Hamburg Outstation (contract HPRI-CT-1999–00017) This work was supported by the Slovak Academy of Sciences Grant 2/1018/21 (J.S and L.U.), Austrian Science Research Fund Grant P14520 (G.W.), and an Austrian Federal Ministry of Education, Science, and Culture Research Contract (G.W.) 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Kaulfus, P., Matsudaira, P & Weber, K (1981) F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments J Biol Chem 256, 9283–9288 51 Gimona, M & Mital, R (1998) The single CH domain of calponin is neither sufficient nor necessary for F-actin binding J Cell Sci 111, 1813–1821 52 Kraulis, P.J (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystalogr 24, 946–950  ... strong binding of plectin ABD to full-length vimentin and VimNR, but only weak or no binding to other vimentin fragments To confirm the specificity of the plectin ABD? ?vimentin interaction and to more... difference of mouse and human ABDs per se To document minor structural differences between human and mouse plectin ADBs, structure I molecule B (IB), structure II (II), and the structure of human plectin. .. protein Ó FEBS 2004 Structure and vimentin -binding of plectin ABD (Eur J Biochem 271) 1879 Fig Comparisons of mouse and human ABDs Average main chain B factor values of mouse ABDs (A), and differences