Actin-binding domain of mouse plectin

Actin-binding domain of mouse plectinCrystal structure and binding to vimentin

Jozef Sevcık1, L’ubica Urbanikova1, Julius Kost’an1,2, Lubomır Janda2 and Gerhard Wiche2

1Institute 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, iscomposed of several subdomains that harbor binding sitesfor a variety of different interaction partners. A canonicalactin-binding domain (ABD) comprising two calponinhomology domains (CH1 and CH2) is located in proximityto its amino terminus. However, the ABD of plectin isunique among actin-binding proteins as it is expressed in theform of distinct, plectin isoform-specific versions. We havedetermined the three-dimensional structure of two distinctcrystalline forms of one of its ABD versions (pleABD/2a)from mouse, to a resolution of 1.95 and 2.0 A. Comparisonof pleABD/2a with the ABDs of fimbrin and utrophinrevealed 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 compactfold as the human plectin ABD and the fimbrin ABD, dif-fering from the open conformation described for the ABDsof utrophin and dystrophin. Plectin harbors a specificbinding site for intermediate filaments of various typeswithin its carboxy-terminal R5 repeat domain. Our experi-ments revealed an additional vimentin-binding site of plec-tin, residing within the CH1 subdomain of its ABD. Weshow that vimentin binds to this site via the amino-terminalpart of its rod domain. This additional amino-terminalintermediate filament protein binding site of plectin mayhave a function in intermediate filament dynamics andassembly, rather than in linking and stabilizing intermediatefilament networks.

Plectin is a versatile cytoskeletal linker protein of verylarge size that is abundantly expressed in a wide varietyof mammalian tissues and cell types. As a cytolinker parexcellence it has been found in association with variouscytoskeletal structures and it has been shown to interact witha variety of distinct proteins on the molecular level (reviewedin [1]). Plectin’s putative role as a mechanical stabilizingelement of cells was fully confirmed when EBS-MD, a severeskin blistering disease combined with muscular dystrophy,was traced to defects in the plectin gene [2], with plectin-deficient mice showing a similar phenotype [3].

Similar to other members of an emerging family ofstructurally related cytolinker proteins, referred to as theplakins [4,5], plectin displays a multidomain structurecomposed of a central � 200 nm long rod segment flankedby globular domains [1]. The interaction sites of variouscytoskeletal proteins have been mapped to opposite ends ofthe molecule optimizing its potential as a cytoskeletal linker

protein. One of the better characterized interaction domainsof plectin is its N-terminal actin-binding domain (ABD)which is of the canonical type, comprising two tandemlyarranged calponin homology (CH) domains, CH1 andCH2. This relatively small domain (� 30 kDa) is foundin many actin-binding and cytolinker proteins, such asa-actinin, dystonin, fimbrin, spectrin/fodrin, dystrophin andutrophin, to name a few.

However, in certain aspects the ABD of plectin seems tobe unique. Analysis of the plectin gene from mouse revealedthe unusual high number of 14 alternatively spliced firstexons, 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 ABDsequence (encoded by exons 2–8), lead to insertions offive or 12 amino acid-long segments between the regionsencoded by exons 2 and 3, and 3 and 4, respectively. Thus,not only do three different isoforms of the plectin ABD itselfexist but additionally there is the intriguing possibility thatthe various first exon-encoded sequences preceding theABD differentially affect its functionality [7]. So far, suchvariability of splicing variants has been described neither forother ABDs nor for the ABD of human plectin. Interest-ingly, the ABD isoform most prominently expressed inmuscle (containing the exon 2a-encoded sequence) has beenshown to bind to actin more efficiently than other isoforms[6]. Recent evidence suggests that the ABD, in particularindividual CH subdomains, have functions other thanbinding to F-actin (reviewed in [8]). While the CH1subdomain definitely interacts with F-actin, the CH2subdomain seems to lack such intrinsic activity, but affectsbinding properties of the whole domain [9,10]. In addition,calmodulin has been shown to regulate the interaction of

Correspondence toG. 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: [email protected]

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)

Eur. J. Biochem. 271, 1873–1884 (2004) � FEBS 2004 doi:10.1111/j.1432-1033.2004.04095.x

dystrophin and utrophin with F-actin through directbinding to CH domains, although the physiological rele-vance of this is not clear [11,12]. Furthermore, CH domainscontain specific binding sites for phosphoinositides and PIP2

has been shown to modulate the actin-binding activity ofa-actinin [13] and plectin [14]. Another intriguing feature,so far characterized only for plectin ABD, is the directinteraction with the cytoplasmic tail domain of the integrinb4 subunit [15,16].

For a number of proteins with either a single CHsubdomain or tandemly arranged CH subdomains (ABD),interactions with intermediate filament (IF) proteins havebeen reported. Calponin has been shown to interact withdesmin, the major IF protein of smooth and striated muscle[17–19], and for fimbrin a colocalization with vimentin wasobserved in cultured macrophages [20]. In both cases it wassuggested that interactions were mediated by CH domains.Based on this, the idea arose that the interaction ofCH domains with IF subunit proteins may represent ahighly conserved function common to CH protein familymembers.

As of recently, crystal structures of ABDs have beenreported for fimbrin, utrophin, dystrophin and humanplectin [21–24]. Dystrophin and utrophin ABDs bothcrystallized as antiparallel dimers with an open conforma-tion, whereas the ABD of fimbrin and human plectin weredescribed as monomers with a closed conformation. Unlikeutrophin and dystrophin, fimbrin appears to associate withF-actin in the closed conformation [25]. This could be due tolengths and conformation variability of linkers connectingindividual CH subdomains in these proteins [21–24]. Tostudy the structural and functional relationships with otherCH protein family members we have prepared two crystal-line forms of the ABD of plectin and analyzed the atomicstructures. We show here that this ABD bears a closestructural resemblance to the ABD of fimbrin and humanplectin. Extending this to the functional level, we furthershow that plectin, like fimbrin, interacts via its CH1 domainwith the IF protein vimentin. Furthermore, in affinity-binding assays using chymotryptic fragments of vimentinwe identified the N-terminal region of vimentin’s central rodas ABD-docking site.

Materials and methods

cDNA constructs

A cDNA fragment corresponding to exons 2–8 includingalternative exon 2a (pGR75; pleABD/2a) was prepared byPCR as described previously [6], except that the fragmentwas subcloned into a uniqueEcoRI site of expression vectorpBN120 [26], a pET-15b derivate (Novagen, Madison, WI,USA). The same strategy was used for the cloning ofple1cABD/2a cDNA, corresponding to the plectin ABDpreceded by 66 exon 1c-encoded amino acid residues(pGR147); ple1aABD starting with exon 1a-encodedsequences, but without exon 2a sequences; ple6–9 cDNA(pGR103) starting at the last codon of exon 5 and extendingto half of exon 9 (ATC CGG); and ple4–8 (pDS19) cDNAstarting with the first in-frame ATG in exon 4 and extendingclose to the end of exon 8 (GCA CAG). The differenceswere that ple4–8 cDNA was subcloned into pBN120

through EcoRI and NdeI sites and that ple1aABD cDNAwas subcloned into expression vector pGR66, a modifiedderivate of pBN120 missing a His-tag.

A fragment corresponding to repeat 5 of mouse plectin(1039 bp) was generated by PCR (forward primer,5¢-GGAATTCCGCGGTCTCCGCAAGC-3¢; reverseprimer5¢-GGAATTCAAGCGTACCAGCGCGGTAC-3¢), usingmouse plectin cDNA (rat accession number X59601) as atemplate. This fragment was subcloned into expressionvector pBN120 resulting in plasmid pKAB1.

Plasmid pFS129 encoding full length mouse vimentin(accession number M26251) without tag has been describedpreviously [27]. Plasmids pFS2 and pFS3, encoding theN-terminus of vimentin (Met1–Glu94) and the rod domain(Phe95–Ile411) of vimentin, respectively, were preparedsimilar to pKAB1, using primer pairs pFS2 forward(5¢-CCGGAATTCATGTGGACCAGGTCTGTG-3¢), andpFS2 reverse (5¢-CCGGAATTCCTCAGTGTTGATGGCGTC-3¢), and pFS3 forward (5¢-CCGGAATTCTTCAAGAACACCCGC-3¢), and pFS3 reverse (5’-CCGGAATTCAATCCTGCTCTCCTC-3’), and mouse vimentin cDNAas a template. Plasmid pGP1 encoding the rod andC-terminus of vimentin (Phe95–Glu466) was obtained bysubcloning a SacI/BamHI excision fragment of pFS129 inpFS3. pBN128, encoding the N-terminal and rod domain ofvimentin (Met1–Ile411) was obtained by SalI/SmaI sub-cloning of mouse vimentin full-length cDNA, contained inpMC-V21 [27], into a modified pET-15b expression vector(pBN121) and deletion of the nucleotide sequence encodingthe last 55 amino acids by PCR cloning. All plasmids wereverified by sequencing.

Protein expression and purification

The protein used for crystallization (pleABD/2a) as well asple1cABD/2a were isolated in the form of soluble His-tagged fusion proteins and purified as described previously[28]. To remove the His-tag prior to crystallization, pleA-BD/2a was treated with thrombin. Ple1aABD without tagwas purified from bacterial lysates in soluble form usingseveral matrices (Phenyl-Sepharose 6 fast flow, DEAESepharose CL-6B, Superdex 75TM). Recombinant full-length vimentin encoded by pFS129 was isolated fromlysed bacterial pellets following the inclusion body prepar-ation procedure [29]. Final pellets were dissolved in 5 mM

Tris/HCl, pH 7.5, 8 M urea, 1 mM EDTA, 10 mM 2-merca-ptoethanol, 0.4 mM phenylmethanesulfonyl fluoride (solu-tion A) and after centrifugation (40 000 g, 15 min, 4 �C)supernatants were directly applied to a 10 mL DEAESepharose 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 frac-tions were pooled, diluted 1 : 5 (v/v) with 50 mM sodiumformate, pH 4.0, 8 M urea, 10 mM 2-mercaptoethanol,0.4 mM phenylmethanesulfonyl fluoride (solution B) andapplied to a 10 mL CM Sepharose CL-6B column insolution B. After washing, bound vimentin was eluted witha linear gradient of KCl (0–0.3 M) in solution B, andaliquots were stored at )20 �C. His-tagged, truncatedversions of vimentin and plectin ABD were purified byaffinity chromatography as described [15].

1874 J. Sevcık et al. (Eur. J. Biochem. 271) � FEBS 2004

Blot overlay assays

Purified samples of proteins were subjected to SDS/PAGEusing loading buffer with or without dithiothreitol. Proteinswere transferred to nitrocellulose membranes, blockedwith 5% (w/v) nonfat milk powder in 1.5 mM KH2PO4,8 mM NaH2PO4, 137 mM NaCl, 2.6 mM KCl, pH 7.4(solution C). Blots were overlaid with full-length mousevimentin or ple1cABD/2a (both at concentrations of5 lgÆmL)1) in 4.3 mM Na2HPO4, 1.4 mM KH2PO4,137 mM NaCl, 2.7 mM KCl, 1 mM EGTA, 2 mM MgCl2,0.1% (v/v) Tween 20, 1 mM dithiothreitol, pH 7.5. After 1 hof incubation, membranes were washed thoroughly withsolution C supplemented with 0.1% (v/v) Tween 20. Fordetection of bound proteins we used affinity-purified goatanti-(mouse vimentin) IgG [30], diluted 1 : 5000 (v/v), orisoform-specific affinity-purified rabbit antibodies to plectin1c [31], diluted 1 : 1000 (v/v), in combination with secon-dary HRP-coupled antisera and the SuperSignal� kit(Pierce, Rockford, IL, USA).

Affinity chromatography

PleABD/2a was coupled to CNBr-activated Sepharose 4Bfollowing the procedure outlined by Amersham Biosciences(Little Chalfont, UK). Vimentin purified in urea wasdialyzed step by step against 6, 4, and 2 M urea in solutionD (10 mM Tris/acetate, pH 8.3, 0.1 mM EDTA, 5 mM

2-mercaptoethanol). Each dialysis step was performed for30 min at room temperature, followed by dialysis againstsolution D overnight at 4 �C. Urea-free vimentin, precen-trifuged at 100 000 g for 30 min, was digested with chymo-trypsin (1 : 400, w/w) for 30 min at 25 �C. The reaction wasstopped by the addition of L-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK) (final concentration of100 lgÆmL)1). The digest was then immediately loadedonto a pleABD/2a Sepharose column equilibrated with10 mM Tris/HCl, pH 7.5, 0.5 mM MgCl2, 0.2 mM dithio-threitol, 25 mM NaCl, 50 lgÆmL)1 TPCK (solution E).Bound protein was eluted with a linear gradient of25–400 mM NaCl in solution E.

Crystallization

Two crystalline forms (I and II) were prepared by thehanging-drop vapor diffusion method. Monoclinic crystals(form I), belonging to the P21 space group with twomolecules in the asymmetric unit, were grown from 4 lLdrops containing equivalent amounts of protein andprecipitant solutions. The protein solution was preparedby dissolving lyophilized samples in 0.05 M Tris/HCl,pH 9.0, to a concentration of 20 mgÆmL)1. The precipitantsolution 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 withonly one molecule in the asymmetric unit (form II) wereprepared by the same method combined with seeding.Drops (6 lL) were prepared by mixing equal volumes ofprotein 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 equili-bration and the precipitate was removed by centrifugation

(10 000 g, 5 min). The supernatants were used to form newdrops, in which microcrystals had grown. To obtain bettercrystals the procedure was repeated using solutions withlower concentrations of protein (10 mgÆmL)1) and precip-itant [8% (w/v) PEG 8000]. The precipitant solution wasenriched with dioxane (2%, v/v) to reduce twinningtendency of crystals. The first microcrystals were used asseeds. Crystals reached dimensions of up to 0.8 mm after1–2 days.

Data collection and processing

The collection and processing of X-ray data from crystalform I (structure I) were described previously [28]. Datafrom crystal form II (structure II) were collected to 2.0 Aresolution at 100 K on EMBL X-11 beamline at theDORIS storage ring, DESY, Hamburg. Crystals weresoaked stepwise in cryoprotectant prepared from precipi-tant solution enriched with glucose (6, 12, 18 and 24%, w/v)before flash-freezing. Conditions for data collection wereoptimized using the program BEST [32]. Data collectionstatistics are summarized in Table 1 (data from crystal formI are included for comparison). Dimensions of the unit cell,crystal symmetry and molecular mass of the protein gave acrystal packing density VM of 2.5 A3ÆDa)1, with a solventcontent of 50% for one protein molecule in the asymmetricunit [33].

Structure determination and refinement

At the time when we solved the structure of pleABD/2athere were only three members of the actin-binding proteinfamily for which the tertiary structure of their ABD wasknown, namely utrophin (Protein Data Bank code 1QAG),dystrophin (1DXX) and fimbrin (1AOA). (The structure ofthe human plectin ABD was reported later [24], andtherefore could not be used in our structure determination).

Table 1. Data collection statistics. Values in parentheses refer to the

last resolution shell.

Parameter Crystal form I Crystal form II

Wavelength (A) 1.100 0.812

Beamline X31 X11

Temperature (K) 193 100

Resolution range (A) 20.0–1.95

(1.97–1.95)

50.0–2.00

(2.02–2.00)

Space group P21 P212121

Unit cell parameters

a (A) 55.31 32.52

b (A) 108.92 51.23

c (A) 63.75 144.72

b (�) 115.25 90

Mosaicity 0.3 0.4

Solvent content (%) 60 50

Completeness (%) 96.2 (71.7) 97.6 (86.3)

R(I)mergea (%) 6.0 (40.0) 3.8 (31.0)

I/r(I) 22.2 (2.1) 33.9 (3.5)

a R(I)merge ¼ S/I)<I>/SI, where I is an individual intensity

measurement and <I> is the average intensity for this reflection

with summation over all data.

� FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1875

Amino acid sequence identities of the ABDs of utrophin,dystrophin and fimbrin with pleABD/2a, determined by theprogram FASTA [34], are 48, 47 and 23%, respectively.Because of differences in the relative orientation of CH1 andCH2 subdomains in the structures of utrophin, dystrophinand fimbrin, not the whole ABD, but the CH1 and CH2subdomains of utrophin were used as two model structures.PleABD/2a structure I was determined by molecularreplacement 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 refine-ment 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 coeffi-cients 25, 39, 54 and 68%. MOLREP unambiguously showedthat there were two protein molecules in the asymmetric unit(hereafter referred to as molecules A and B). For build-ing the model, the program ARP/WARP in the modeWARPNTRACE [37] was used. The program automaticallybuilt a model consisting of 378 out of 490 residues (moleculesA and B) with a connectivity index of 0.92. The remainingresidues, including those forming the loop connecting theCH1 and CH2 subdomains, were built manually using theprogram 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 correctingthe amino acid sequence and building the parts whichwere different from those of utrophin and which were notbuilt by WARPNTRACE. The structure was refined against95% of the data, the remaining 5% (randomly excludedfrom the full data set) were used for crossvalidation inwhich Rfree was calculated to follow the progress ofrefinement [39]. After each refinement cycle, ARP, anautomated refinement procedure [40], was applied formodeling and updating the solvent structure. After theR factor had fallen to about 20%, refinement continuedwith anisotropic temperature factors and hydrogen atomsgenerated in standard geometries.Structure II was determined by molecular replacement

using molecule A from structure I as a model. Solution wasstraightforward giving an R factor and correlation coeffi-cient of 39 and 63%, respectively. Refinement was per-formed in the same way as that of structure I. Refinementstatistics of both structures, average temperature factors,and target deviations against stereochemical restraints aregiven in Table 2. For visualization and rebuilding of thestructures O and XTALVIEW programs were used [38,41].

Other methods

MALDI-TOF MS as well as ESI MS were performedat the mass spectrometry unit, Vienna Biocenter, Vienna,Austria.

Results

Description of the structures

Like other ABDs of the canonical CH1-CH2 type, the ABDof plectin is encoded by seven exons, the first four encoding

the CH1 subdomain and the remaining three the CH2subdomain. The mouse plectin ABD isoform analyzed here,pleABD/2a, is unique, as it contains a five amino acid-longsequence (Fig. 1) inserted by differential splicing of a shortexon (exon 2a) between the first two exons of the ABD [6].Comprising 245 residues, the analyzed recombinant proteincontained pleABD/2a as a 237 residues-long fragment(amino acids 181–417; EMBL accession number AF188008) flanked by six amino-terminal (GSHMEF) andtwo carboxy-terminal (EF) residues (added as cloningrequirement). The amino acid residues in the structuresare numbered from 1 to 245 according to the sequence ofthe recombinant protein, i.e. amino acids with numbers7–243 in the structure correspond to 181–417 in the AF188008 sequence.

Two crystal structures of pleABD/2a were determined.One of them, structure I was derived from a monocliniccrystal containing two protein molecules (A and B) in theasymmetric unit, the other, structure II, from an orthorhom-bic crystal, containing one molecule in the asymmetric unit(for details seeMaterials andmethods).As found in structureI, molecule A, pleABD/2a is an a-protein consisting of 11helices: 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 1 and2A). Helices a1–a5 form the CH1 and helices a6–a11 theCH2 subdomain. The subdomains are connected by aflexible 15 residues-long loop (119–133). For five N-terminal(GSHME) and eight C-terminal residues (RVPGAQEF) ofboth molecules in structure I there was no electron densityobserved. In structure II there was no electron density for thefirst seven (N-terminal) nor for the last eight (C-terminal)

Table 2. Refinement statistics.

Parameter structure I structure II

Resolution limits (A) 30.0–2.0 50.0–2.0

No. of reflections all/Rfree set 42 811/2286 15 889/848

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)


residues. It is reasonable to conclude that N- and C-terminalresidues formed highly disordered tails. The molecular massof the recombinant proteinwas 28 631 Da, as determinedbymass spectrometry. This was in a good agreement with itstheoretical mass of 28 656 Da, indicating that the structureindeed 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 confor-mation in which the CH1 and CH2 subdomains were facingeach other through helices a1 and a5, and helices a10(including the subsequent loop) and a11. Molecules A and Bin the asymmetric unit of the pleABD/2a structure I formeda crystallographic dimer (Fig. 2B). In the contact area of thetwo molecules there were two hydrogen bonds formedbetween side chains of Glu125(A)/Asn88(B) and Lys130(A)/Glu131(B) and one bond mediated by a water moleculeAsn151(A)/W66/Asp150(B). The solvent-accessible surfaceburied at the plectin dimer interface was 760 A2, corres-ponding to � 3% (380 A2) of the surface of each isolatedmolecule (11 800 A2). This was far below the minimum of9%required forclassificationofadimerasaproteincomplex[42], suggesting that the crystallographic dimer hardly couldexist in solution. Moreover, structure II has confirmed thatpleABD/2a can exist as a monomer in solution.

The contacts between molecules in the crystal latticeapparently did not change the orientation of the CH1 withrespect to the CH2 subdomain in spite of the fact that theloop connecting the two subdomains theoretically couldallow various orientations including those found inutrophin. It can be concluded that the conformation ofpleABD/2a as found in structures I and II is stable and notsubject to conformational changes due to different crystalpacking.

Quality of protein structure models

The final R and Rfree factors for structure I were 15.3 and19.4% (Table 2). There were two protein molecules (A andB) and 188 solvent molecules in the asymmetric unit. TheRamachandran plot [43] calculated by the program PRO-

CHECK [44] showed that 92.1 and 94.4% of the residues ofmolecules A and B, respectively, were in the most favoredregions. The remainder was in additionally allowed regionsexcept for Thr158, which in both molecules had the samedihedral angles being just at the outer border of theadditionally allowed region. Thr158 was localized in asurface loop and there was no doubt about its conforma-tion, as the electron density in this region was clear.

Fig. 1. 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 actin-

binding 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).


A diagram of the average main-chain temperature factorsof molecules A and B as a function of residue number(Fig. 3A) revealed an amazing similarity of their tempera-ture factor profiles, confirming the conformational stability

of the pleABD/2a molecule; temperature factors werehighest in the loop connecting the two subdomains.

For structure II, the final R and Rfree factors were 20.2and 29.9% (Table 2). The structure contained one protein

Fig. 2. 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) Crystal-

lographic 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].


and 58 solvent molecules in the asymmetric unit and in theRamachandran plot there were 88.2% of the residues in themost favored region and 11.8% in additionally allowedregions. Fluctuation of B-values of structure II was similarto that of structure I, with average B-values of structure IIbeing slightly lower (Fig. 3A), which could be due to the factthat structure II data were collected at cryogenic tempera-ture. Refinement parameters of structure II were slightlyworse compared to those of structure I, which wasunexpected considering that the crystal data seemed to bebetter for structure II.

Comparison of ABDs from mouse plectin, human plectin,fimbrin, utrophin and dystrophin

The major difference in the tertiary structures of human andmouse plectin was found at their N termini, where the firsta-helical segment (a1) of human plectin exceeded that frommouse by nine amino acid residues. However, since six ofthese additional residues were encoded by one of the firstexons (1c) preceding the actual ABD-encoding exons 2–8,this does not reflect a structural difference of mouse andhuman ABDs per se.

To document minor structural differences betweenhuman 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 inthese superpositions as a function of residue number (withthe exception of the first N- and the last C-terminal residues,which differed by more than 5 A). Excluding the residuesfor which distances between corresponding Ca atomsexceeded 1.75 A (Fig. 3B, dashed line), the rms displace-ment 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 thesurface loop region, which was not unexpected consideringthat each molecule has a different environment in thecrystal.

Least squares superpositions of mouse plectin with thecorresponding subdomains of human plectin, utrophin,dystrophin and fimbrin are summarized in Table 3. In thesesuperpositions only the CH1 (8–119) and CH2 (134–236)subdomains from structure I molecule A were used; theconnecting segment was excluded as its conformation differssubstantially among the various proteins compared (helixin utrophin and dystrophin, helix–loop in fimbrin, and loopin plectin). Furthermore, as the ABDs of utrophin anddystrophin adopt an open conformation (contrary to theplectin ABD), the CH1 domain from utrophin molecule Aand the CH2 domain from molecule B were used in theoverlap. The different numbers of Ca atoms involved in

Fig. 3. Comparisons of mouse and human

ABDs. Average main chain B factor values of

mouse ABDs (A), and differences between Caatoms in superpositioned structures (B) are

plotted as a function of residue numbers. IA,

structure Imolecule A; IB structure Imolecule

B; II, structure II; HP, structure of human

plectin.

Table 3. 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 218 0.23

IA/II 211 0.44

IB/II 212 0.48

IA/HP 203 0.55

IA/utrophinb 192 0.73

IA/dystrophinb 176 0.82

IA/fimbrin 78 1.12

a 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).


superpositions (reflecting the degree of structural similarit-ies) clearly showed highest similarity of mouse plectin withhuman plectin and lowest with fimbrin (Table 3). Thenearly identical conformations of the pleABD/2a structureand the corresponding subdomains of the A and Bmolecules of utrophin and dystrophin probably have notarisen by chance and may have significance for functionalproperties of these ABDs.

The ABD of plectin binds to vimentin

The IF protein vimentin has been reported to specificallyinteract with the CH1 subdomain of the first (N-terminal) offimbrin’s two ABDs [20]. In light of the extensive structuralresemblance of fimbrin and plectin ABDs it was therefore ofinterest to assess IF-binding activity of the plectin ABD.Moreover, a second IF protein interaction domain at the Nterminus in addition to its C-terminal IF-binding site [26]would raise plectin’s functional versatility, particularly as acytoskeletal crosslinking element. To assess the plectinABD–vimentin interaction, in vitro overlay assays wereperformed. Ple1aABD, an ABD version of plectin precededby a sequence encoded by exon 1a[6], pleABD/2a andtruncated versions of the plectin ABD missing half of theCH1 domain (ple4–8), or the complete CH1 domain (ple6–9) were immobilized on nitrocellulose membranes andoverlaid with full-length vimentin. In agreement withpreviously reported findings [20] all proteins, except theone missing the entire CH1 domain (ple6–9) and thenegative control (BSA), showed binding to vimentin, similarto the positive control (recombinant plectin repeat 5)(Fig. 4). Thus only the CH1, but not the CH2, domain ofplectin’s ABD was found capable of binding to vimentin.

To specify the subdomain of vimentin that bound toplectin’s ABD, several truncated versions of vimentin wereexpressed inEscherichia coli and subjected to overlay assays.The ABD version used in these experiments contained thepreceding 66 amino acid residues-long sequence specificfor plectin isoform 1c (ple1cABD/2a), enabling detection ofbound protein via isoform 1c-specific antibodies [31]. Thisfragment showed strong binding to full-length vimentin andto VimNR, a truncated version of vimentin containing itsN-terminal domain and central rod domain, but lacking theC-terminal domain (Fig. 5). Only very weak interactionswere observed with vimentin fragments corresponding tothe N-terminal (VimN), or rod domains alone (VimR), or tothe rod in conjunction with the C-terminal domain (Vim-RC). These data suggested that the plectin ABD-bindingsite of vimentin was contained in the N-terminal part ofthe molecule comprising the head domain and possiblyparts of the rod.

The head domain of vimentin had previously been shownto harbor the binding site(s) for the CH1 subdomain offimbrin’s N-terminal ABD [20], whereas desmin, an IFprotein of similar type, was found to interact with thecorresponding domain of calponin via the N-terminal partof its rod domain [19]. Interestingly, fimbrin and calponinhave been found to interact with tetrameric forms of solublevimentin and desmin. As we were unable to cosedimentpleABD/2a with filamentous vimentin in sedimentationassays (data not shown) it seems that the plectin ABDinteracts with vimentin in the same way.

Fig. 4. Overlay of various plectin ABD versions with full-length vimen-

tin. Recombinant versions of the plectin ABD starting with exon 1a-

encoded 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 5 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 nitrocel-

lulose 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.


To confirm the specificity of the plectin ABD–vimentininteraction and to more precisely map vimentin’s plectinABD-binding site, vimentin purified in urea was kept in itssoluble (tetrameric) form by dialysis into solution D (seeMaterial and methods). The protein was than subjected tolimited chymotryptic digestion and fragments generatedwere applied to a pleABD/2a-Sepharose affinity column.

Elution and SDS/PAGE of bound proteins revealed that asingle major fragment of � 18 kDa was retained on thecolumn (Fig. 6). This fragment could be mapped to �Coil 1�,an N-terminal segment of the vimentin rod domain (aminoacid residue positions 124–276 of mouse vimentin, EMBLaccession no. M26251), using MALDI-TOF mass spectro-metric sequence analysis.

Discussion

Because of its expression in the form of several distinctisoforms, the ABD of plectin is unique among those of otheractin-binding protein family members [6]. The isoformcrystallized and analyzed here contains an extra five aminoacids (HWRAE; positions 28–32) encoded by exon 2a,a differentially spliced exon inserted between the commonexons 2 and 3. This insertion makes the connecting loop ofhelices a1 and a2 longer. Amino acid residues contained inthis loop are located closely to one (the first) of threesegments identified as direct docking sites for actin withindystrophin’s ABD [22] (Fig. 1). This insertion in pleABD/2a may result in higher flexibility of this region and thesurface exposure and amino acid composition of thissegment (facilitating charge–charge as well as hydrophobicinteractions) may improve binding properties of the ABD.In fact, we have previously shown that this isoform exhibitsa higher affinity to actin than several other isoforms withoutthe 2a sequence [6].

Our analysis revealed that both, mouse and humanpleABD/2a are structurally similar to the fimbrin ABD,although these domains share only little sequence identity.The fimbrin and plectin ABDs have a similar closedconformation, differing from the open conformation des-cribed for the ABDs of dystrophin and utrophin [21–24]. Itis interesting to note that until now, sequences correspond-ing to exons 2a and 3a of mouse plectin have been identifiedneither in other family members, nor in human plectin. Inthis view, insertion of exon 2a into the sequence ofhuman pleABD/2a as reported [24] seems without rationalexplanation.

Although the ABDs of fimbrin, utrophin and a-actininare related, they appear to have different effects on F-actinconformation upon binding and thus may use differentmechanisms of association [25,45,46]. Up to now a consen-

Fig. 6. Affinity-binding of proteolytically derived fragments of vimentin to pleABD/2a. Partial chymotryptic digestion of vimentin and affinity-

chromatography 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.

Fig. 5. 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 vimentin-

bound 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.


sus on the mode of binding (open or closed conformation ofABD) of utrophin and dystrophin to actin filaments has notbeen reached [47,48]. It has been reported that fimbrin [25]and human plectin ABDs [24] bind to actin filaments in theirclosed conformation. In view of these findings it is expectedthat pleABD/2a could adopt a closed as well as an openconformation in binding to actin, as the connecting segmentbetween the CH1 and CH2 domains is flexible and longenough to allow both conformations (as proposed also forhuman plectin [24]). The notion of pleABD/2a adopting anopen conformation is supported by similar characteristics ofcontact areas in the CH1 and CH2 structures of pleABD/2a, utrophin, and dystrophin. ABD of fimbrin is expectedto bind only in its closed conformation, in agreement withthe finding that 72% of its contact area (excluding theconnecting segment) is hydrophobic and there is nohydrogen bond shorter than 3.25 A. On the other hand,only 59% of the corresponding contact areas of pleABD/2aand utrophin are hydrophobic and there are five and sixhydrogen bonds, respectively. Moreover, the secondarystructure prediction of pleABD/2a suggested a helix to existbetween the CH1 and CH2 subdomains. The sites ofpleABD/2a responsible for binding to actin have not beenidentified so far. Assuming that they are similar to thosereported for dystrophin [22], the amino acid sequences13–22, 88–116 and 131–146 might be involved, slightlydiffering from the binding segments predicted for fimbrin[25] (Fig. 1).

The ABDs of dystrophin and utrophin crystallized asantiparallel dimers [21,22], whereas fimbrin and humanplectin ABDs crystallized in monomeric form [23,24]. WhenpleABD/2a was crystallized at pH 9, we also found twomolecules in the asymmetric unit [28]. However, conditionsat pH 6.5 led to the formation of crystals with only onepleABD/2a molecule in the asymmetric unit. The assess-ment of the contact area between molecules A and B inpleABD/2a crystals obtained at pH 9 (crystal form I)showed that these molecules do not interact strongly enoughto form dimers which could exist also in solution. Therefore,the crystallographic dimer observed in the asymmetric unitwas probably an artefact of crystallization rather than adimerization product.

Although CH subdomains forming a canonical ABDstructurally seem to be highly conserved, data are accumu-lating that show functional diversity of CH1 and CH2subdomains [8]. It was reported that proteins containingeither single CH subdomains (calponin), or more complexABDs (fimbrin) can interact with IF proteins. Calponinbinds to desmin, the major IF protein in smooth andskeletal muscle [17–19] and fimbrin interacts with andcolocalizes with vimentin in filopodia, retraction fibres,and podosomes at the ventral surface of cultured macro-phages [20]. In both cases there is evidence that bindingoccurred to IF subunit proteins in their nonfilamentousstate [17–20]. Fimbrin was unable to bind to polymerizedvimentin in cosedimentation assays and binding occurred ata stoichiometry of 1 : 4, suggesting that the IF protein wasin its tetrameric form [20].

As one may expect on the basis of their structuralsimilarity, the ABDs of fimbrin and plectin apparently alsohave a number of functions in common, including bindingto vimentin. Similar to fimbrin [20] the ABD of plectin failed

to cosediment with vimentin filaments (data not shown),suggesting that it, too, interacted with soluble IF proteinsubunits rather than with their filamentous polymers.Binding assays revealed that the major vimentin-bindinginterface was localized within the CH1 subdomain ofplectin. Since ple4–8 (pleABD/2a lacking half of the CH1domain) bound to vimentin, sequences preceding thoseencoded by exon 4 apparently did not contribute to thisinteraction. However, as fragments corresponding to thefirst half of plectin’s CH1 domain (exons 2–4) have not beenexamined in our studies, a role of this part in vimentin-binding can not be fully excluded.

Using an overlay assay we found strong binding ofplectin’s ABD to a fragment of vimentin comprising the rodand its N-terminal domains, but when examined individu-ally, both of these domains showed only weak binding.Using a similar method, it had been shown that a vimentinfragment lacking the C terminus (N410/VimNR) wascapable of binding to fimbrin, contrary to a fragmentlacking the initial 102 amino acid residues (102C/VimRC),suggesting that the fimbrin-binding site was located in theN-terminal head domain of vimentin [20]. The N-terminalvimentin fragment (VimN) and the rod fragment (VimR)used in our experiments ended and started, respectively, atPhe95. Consequently, the rod-containing fragment used inour experiments and the N-terminal fragment N410/Vim-NR used in [20] overlapped by a few amino acid residues.With this consideration, the results of our overlay assaywere consistent with the findings reported in [20]. Using asan alternative method affinity-binding of proteolytic frag-ments of vimentin in combination with mass spectrometry,we found that plectin bound to a � 18 kDa fragmentcorresponding to �Coil 1� [49], the N-terminal part of the a-helical rod domain of vimentin. This is in agreement withsimilar experiments in which the calponin-binding site wasrestricted to the N-terminal part of the desmin rod domain[19]. However, in the overlay assay, the rod domain ofvimentin (VimR) alone showed little binding to plectin. Asfragments obtained by partial proteolytic digestion ofproperly folded full-length vimentin are more likely topreserve the structure of the native protein than recombi-nantly prepared fragments, we assume the true plectinABD-binding site of vimentin to be localized in the N-terminal part of its rod domain. Binding to the evolutionaryhighly conserved CH domains could be a common featureof IF proteins in general. Likewise, considering that the a-helical coiled-coil structure of vimentin’s rod domain ishighly conserved in all IF protein family members, plectin’sABD may bind also to other IF proteins, such as desmin.

The functional significance of the plectin ABD–vimentininteraction remains elusive. The primary activity of theABD supposedly is actin-binding, a function demonstratedfor both fimbrin and plectin [14,50]. The CH domain ofcalponin, on the other hand, doesn’t seem to be requiredfor actin-binding as calponin interacts with actin via itsC-terminal domain [51]. Thus, with an IF protein-bindingsite residing in their N-terminal CH domains in addition totheir genuine actin-binding activities, fimbrin, plectin andcalponin, might influence the assembly and organization ofboth actin and IF cytoskeletal networks. The interaction offimbrin’sABDwith vimentin has been shown tobe adhesiondependent and it has been suggested that this complex is a


transient structure involved in early cell adhesion [20]. Insmooth muscle cells calponin may be an integral componentof desmin IFs in the vicinity of dense bodies [18]. Theinteraction between IF proteins, such as desmin or vimentin,and proteins containing one or more CH domains couldrepresent a highly conserved function related to the estab-lishment of cell adhesion structures. By sequestering solublevimentin at certain sites, such as focal adhesion contacts,plectin may favor and/or initiate IF network formation atthese sites by locally increasing IF protein concentrations.Once filament assembly has been initiated, plectin maystabilize and anchor the filaments at these sites via itsalternative C-terminal IF-binding site. Future experimentsshould show whether this model can be verified.

Acknowledgements

We thank our colleagues Kamaran Abdoulrahman, Branislav Nikolic,

Gernot Putz, Gunther Rezniczek, Daniel Spazierer and Ferdinand

Steinbock, for providing various reagents and for valuable discussions.

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, andCulture ResearchContract (G.W.).

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Actin-binding domain of mouse plectin

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