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

Actin-binding domain of mouse plectin
Crystal structure and binding to vimentin
Jozef S
ˇ
evc
ˇ
ı
´
k
1
, L’ubica Urba
´
nikova
´
1
,Ju
´
lius Kos
ˇ
t’an
1,2
, Lubomı
´
r Janda
2
and Gerhard Wiche
2
1
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic;
2
Institute 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, dif-
fering 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 experi-
ments revealed an additional vimentin-binding site of plec-
tin, 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 plectin-
deficient 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 2 and 3, and 3 and 4, respectively. Thus,
not only do 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. Interest-
ingly, 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:
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 ( />(Received 21 December 2003, revised 26 February 2004,
accepted 18 March 2004)
Eur. J. Biochem. 271, 1873–1884 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04095.x
dystrophin and utrophin with F-actin through direct
binding to CH domains, although the physiological rele-
vance of this is not clear [11,12]. Furthermore, CH domains
contain specific binding sites for phosphoinositides and PIP
2
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 conforma-
tion, 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 crystal-
line 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 affinity-
binding 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 5 and extending
to half of exon 9 (ATC CGG); and ple4–8 (pDS19) cDNA
starting with the first in-frame ATG in exon 4 and extending
close to the end of exon 8 (GCA CAG). The differences
were that ple4–8 cDNA was subcloned into pBN120
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 5 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 sub-
cloning 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)aswellas
ple1cABD/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 tag
was purified from bacterial lysates in soluble form using
several matrices (Phenyl-Sepharose 6 fast flow, DEAE
Sepharose CL-6B, Superdex 75
TM
). Recombinant full-
length vimentin encoded by pFS129 was isolated from
lysed bacterial pellets following the inclusion body prepar-
ation procedure [29]. Final pellets were dissolved in 5 m
M
Tris/HCl, pH 7.5, 8
M
urea, 1 m
M
EDTA, 10 m
M
2-merca-

ptoethanol, 0.4 m
M
phenylmethanesulfonyl fluoride (solu-
tion A) and after centrifugation (40 000 g,15min,4°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 frac-
tions were pooled, diluted 1 : 5 (v/v) with 50 m
M
sodium
formate, pH 4.0, 8
M
urea, 10 m
M
2-mercaptoethanol,
0.4 m
M
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].

1874 J. S
ˇ
evc
ˇ
ı
´
k et al.(Eur. J. Biochem. 271) Ó FEBS 2004
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 m
M
KH
2
PO
4
,
8m
M
NaH
2
PO
4
, 137 m
M
NaCl, 2.6 m
M
KCl, pH 7.4
(solution C). Blots were overlaid with full-length mouse

vimentin or ple1cABD/2a (both at concentrations of
5 lgÆmL
)1
)in4.3m
M
Na
2
HPO
4
,1.4m
M
KH
2
PO
4
,
137 m
M
NaCl, 2.7 m
M
KCl, 1 m
M
EGTA, 2 m
M
MgCl
2
,
0.1% (v/v) Tween 20, 1 m
M
dithiothreitol, pH 7.5. After 1 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 1 : 5000 (v/v), or
isoform-specific affinity-purified rabbit antibodies to plectin
1c [31], diluted 1 : 1000 (v/v), in combination with secon-
dary 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 2
M
urea in solution
D(10m
M
Tris/acetate, pH 8.3, 0.1 m
M
EDTA, 5 m
M
2-mercaptoethanol). Each dialysis step was performed for
30 min at room temperature, followed by dialysis against
solution 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 was
stopped by the addition of
L
-chloro-3-[4-tosylamido]-
4-phenyl-2-butanone (TPCK) (final concentration of

100 lgÆmL
)1
). The digest was then immediately loaded
onto a pleABD/2a Sepharose column equilibrated with
10 m
M
Tris/HCl, pH 7.5, 0.5 m
M
MgCl
2
,0.2m
M
dithio-
threitol, 25 m
M
NaCl, 50 lgÆmL
)1
TPCK (solution E).
Bound protein was eluted with a linear gradient of
25–400 m
M
NaCl in solution E.
Crystallization
Two crystalline forms (I and II) were prepared by the
hanging-drop vapor diffusion method. Monoclinic crystals
(form I), belonging to the P2
1
space group with two
molecules in the asymmetric unit, were grown from 4 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 mgÆmL
)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 P2
1
2
1
2
1
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 equili-
bration and the precipitate was removed by centrifugation
(10 000 g, 5 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 mgÆmL
)1
)andprecip-
itant [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 precipi-
tant 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 1 (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 V
M
of 2.5 A
˚
3

ÆDa
)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 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 P2
1
P2

1
2
1
2
1
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)
merge
a
(%) 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.

Ó 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 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 refine-
ment with the program
REFMAC
5 [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 showed
that 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 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
REFMAC
5.
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 R
free
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 coeffi-
cient of 39 and 63%, respectively. Refinement was per-
formedinthesamewayasthatofstructure I. Refinement
statistics of both structures, average temperature factors,
and target deviations against stereochemical restraints are
given in Table 2. 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
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 1 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 orthorhom-
bic 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 1 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)

Table 2. Refinement statistics.
Parameter structure I structure II
Resolution limits (A
˚
) 30.0–2.0 50.0–2.0
No. of reflections all/R
free
set 42 811/2286 15 889/848
R
work
(%) 15.1 19.7
R
free
(%) 19.4 29.9
R
all
(%) 15.3 20.2
ESU based on R
free
(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 (A
˚
2
)
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 (A
˚
3
) 0.22 (0.20) 0.21 (0.20)
Planar groups (A
˚
) 0.01 (0.02) 0.02 (0.02)
Main-chain bond B-values (A
˚
2
) 2.16 (1.50) 2.38 (1.50)
Main-chain angle B-values (A
˚
2
) 3.44 (2.00) 3.57 (2.00)
Side-chain bond B-values (A
˚
2
) 4.70 (3.00) 5.27 (3.00)
Side-chain angle B-values (A

˚
2
) 7.20 (4.50) 7.26 (4.50)
1876 J. S
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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 confor-
mation in which the CH1 and CH2 subdomains were facing
each other through helices a1anda5, 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 A
˚
2

, corres-
ponding to  3% (380 A
˚
2
)ofthesurfaceofeachisolated
molecule (11 800 A
˚
2
). 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 R
free
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
PRO-

CHECK
[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 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).
Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1877
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 tempera-
ture 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 R
free

factors were 20.2
and 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)ofpleABD/2a (red) with fimbrin ABD
(green). Figures were generated using the
program
MOLSCRIPT
[52].
1878 J. S
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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 tempera-
ture. Refinement parameters of structure II were slightly
worse compared to those of structure I,whichwas
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 5 A
˚
). Excluding the residues

for which distances between corresponding Ca atoms
exceeded 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 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 3. 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
Fig. 3. 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.
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 atoms
a
rmsd (A
˚
)
IA/IB 218 0.23
IA/II 211 0.44
IB/II 212 0.48
IA/HP 203 0.55
IA/utrophin
b
192 0.73
IA/dystrophin
b
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).
Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1879
superpositions (reflecting the degree of structural similarit-
ies) 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
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 (Vim-
RC). 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.
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.
1880 J. S
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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.
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 spectro-
metric sequence analysis.
Discussion
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 2 and 3. This insertion makes the connecting loop of
helices a
1
and a
2
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 des-
cribed for the ABDs of dystrophin and utrophin [21–24]. It
is interesting to note that until now, sequences correspond-
ing 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. 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.
Ó FEBS 2004 Structure and vimentin-binding of plectin ABD (Eur. J. Biochem. 271) 1881

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 assess-
ment of the contact area between molecules A and B in
pleABD/2a crystals obtained at pH 9 (crystal form I)
showed that these molecules do 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 accumu-
lating 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 macro-
phages [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 1 : 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
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 4 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 vimentin-
binding 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 individu-
ally, 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/Vim-

NR 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 frag-
ments 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 a-
helical 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 recombi-
nantly prepared fragments, we assume the true plectin
ABD-binding site of vimentin to be localized in the N-
terminal 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 a-
helical 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
1882 J. S
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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 estab-
lishment 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, Gu

¨
nther Rezniczek, Daniel Spazierer and Ferdinand
Steinbo
¨
ck, 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, and Culture Research Contract (G.W.).
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