The Solution Structure of the N-Terminal Domain of
Human Tubulin Binding Cofactor C Reveals a Platform
for Tubulin Interaction
MaFlor Garcia-Mayoral1, Raquel Castan ˜o2, Monica L. Fanarraga2, Juan Carlos Zabala2, Manuel Rico1,
1Departamento de Quı ´mica Fı ´sica Biolo ´gica, Instituto de Quı ´mica Fı ´sica Rocasolano, Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Madrid, Spain,
2Departamento de Biologı ´a Molecular, Instituto de Formacio ´n e Investigacio ´n Marque ´s de Valdecilla, Facultad de Medicina, Universidad de Cantabria, Santander, Spain
Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of a- and b-tubulin
monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process
it collaborates with other cofactors (TBC’s A, B, D, and E) and forms a supercomplex with TBCD, b-tubulin, TBCE and a-
tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is
found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-
terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-
stranded a-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and
disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a
testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues
from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.
Citation: Garcia-Mayoral MF, Castan ˜o R, Fanarraga ML, Zabala JC, Rico M, et al. (2011) The Solution Structure of the N-Terminal Domain of Human Tubulin
Binding Cofactor C Reveals a Platform for Tubulin Interaction. PLoS ONE 6(10): e25912. doi:10.1371/journal.pone.0025912
Editor: Annalisa Pastore, National Institute for Medical Research, Medical Research Council, United Kingdom
Received July 5, 2011; Accepted September 13, 2011; Published October 18, 2011
Copyright: ? 2011 Garcia-Mayoral et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the projects Consolider-Ingenio Centrosome-3D CSD2006-00023, Centrosome-CM S-GEN-0166-2006, BQU2008-0080,
BFU2007-64882, BFU2010-18948 and the IFIMAV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
In recent years, a great effort has been made to elucidate the
complex series of events occurring during the a- and b- tubulin
folding pathways that lead to the final release of ab native
heterodimers incorporated in microtubules [1,2]. In mammals, this
process is initiated by the cytosolic chaperonin CCT (also known as
c-cpn or TriC) binding to the newly synthesised a- and b-tubulin
polypeptides  assisted by the molecular chaperone protein
prefoldin that, after various ATP-hydrolysis-dependent cycles,
produces quasi-native tubulin intermediates. In contrast to actin
and c-tubulin that can be completely folded by the exclusive action
of chaperonins, the intermediates of a- and b-tubulin need to be
further processed to reach their final active conformation, a process
that requires a set of five different tubulin binding cofactors (TBCA,
TBCB, TBCC, TBCD, and TBCE). TBCB associates with a-
tubulin folding intermediates and is then displaced by TBCE.
TBCA and TBCD interact in a similar way with quasi-native b-
tubulin. An additional tubulin binding cofactor, TBCC , is
necessary to complete the process by forming a supercomplex with
TBCD, b-tubulin, TBCE, and a-tubulin that, following GTP-
hydrolysis-dependent cycles, releases the native ab-tubulin hetero-
dimers. The stimulated hydrolysis of GTP by b-tubulin acts as a
switch for the release of native tubulin heterodimers from the
supercomplex . The discovery of this pathway has driven much
of the effort to the study of the implication of these proteins in the
folding/dimerization of tubulin.
Recent results have shown that tubulin binding cofactors also
participate in the proteostasis of the tubulin dimer through their
intrinsic ability to dissociate the tubulin heterodimer [1,2]. This
ability to dissociate the tubulin heterodimer in a controlled way is a
mechanism that certain types of cells exploit to regulate key
cytoskeletal processes, such as controlling their microtubule densities,
or the trimming of the distal microtubule tips at the axonal growth
cone terminal in macrophages and neurons respectively. TBCC is
regarding its function in vivo have been published.
TBCC is organized into three different domains (N-term,
CARP and C-term) (Fig. 1A). The C-terminal domain constitutes
the hallmark of the TBCC protein family and its structure was
recently solved by Saito, K. et al. (2007, PDB: 2YUH). This
domain shares ,29% sequence identity over half of the length of
Retinitis Pigmentosa 2 protein (RP2) and both proteins stimulate
the GTPase activity of native tubulin with the cooperation of
TBCD. In contrast to TBCC, RP2 has no tubulin heterodimer-
ization capacity . This domain is also present in TBCCD1
(TBCC-domain containing 1), a protein that localizes at the
centrosome and basal bodies of primary and motile cilia, required
for centrosome and Golgi Apparatus (GA) positioning in human
cells [7,8]. The TBCC C-terminal domain has a conserved
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arginine (R262) also present in RP2 (R118) postulated to act as an
arginine-finger in the GTP hydrolysis of tubulin in similar manner
as the arginine-finger in RasGAP . Like the corresponding
mutation in RP patients, substitution of R262 of TBCC abolishes
its GTPase activating protein (GAP) activity suggesting a role in
regulation of microtubule polymerization in vivo .
Although the N-terminal domain is expected to interact with
other spectrin-like domains , no functional roles have yet been
In this work we have demonstrated that TBCC is found at the
centrosome and we have used NMR spectroscopy to determine
the solution structure and the interactions with the ab-tubulin
dimer of its N-terminal domain (TBCC N-terminal domain:
TBCC is found at the centrosome
To study TBCC function, we investigated the subcellular
distribution of the endogenous protein in HeLa cells with a novel
affinity antiserum purified against the human recombinant protein.
The primary antibody recognizing human TBCC used was affinity
purified as previously described  against both, the full length
protein (Fig. 1, 2 and 3) or the TBCC N-terminal domain (Fig. 4) to
select TBCC N-terminal recognizing immunoglobulins from the
antiserum. A commercial anti-TBCC monoclonal antibody (Ab-
nova Corporation) was used to validate the TBCC centrosomal
immunostaining pattern. These antibodies recognised a unique
protein band corresponding to TBCC in western blots (Fig. 1B).
Doubly immunostained cells revealed a dot-like cytoplasmic
labelling accompanied by a prominent and irregular centrosomal
spot ofTBCC(Fig.2A).A centrosomalimmunostaining patternwas
also observable in metaphase cells where both spindle poles
displayed TBCC accumulation (Fig. 2A right, arrows).
We next overexpressed TBCC in order to investigate TBCC
subcellular localization. We observed accumulates of this cofactor
at spindle pole bodies and occasional multipolar spindles (Fig. 2B).
These results match those observed by Hage-Sleiman et al.  in
MCF7 cells (human mammary adenocarcinoma), where a G2-M
phase blockage in TBCC overexpressing cells has been reported.
Figure 1. Specificity of polyclonal and monoclonal anti-TBCC antibodies. A) Human TBCC protein family. Schematic representation of the
functional domains ascribed to human TBCC, RP2 and TBCCD1. The human proteins also possess a CARP domain present in CAPs (cyclase-associated
proteins) . TBCCD1 is related to TBCC and RP2 which functionally overlaps with TBCC . The C-terminus known as the TBCC domain is shown in
light blue, the CARP domain in magenta and the N-terminus domain (alpha module) in green. B) Both, the purified rabbit polyclonal anti-TBCC
produced in our laboratories (left) and the commercial mouse monoclonal antibody also used in this study (right, see Methods) recognised a single
band in whole cell extracts.
Structure of the N-Terminal Domain of TBCC
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Based on these findings, we hypothesize that TBCC is localised
at the centrosome. We compared TBCC colocalization with
classical centrosomal markers, such as c-tubulin (not shown) or
Nedd1, and as Fig. 2C (left) shows, TBCC produced an
overlapping immunostaining pattern thus supporting our hypoth-
esis. But since centrosomal proteins are typically recognized by
colocalization with centrosomal/centriolar markers after microtu-
bular destruction by cold and nocodazole, we destroyed the
microtubule cytoskeleton to corroborate the above hypothesis.
Fig. 2C (right) shows that TBCC was still detectable at the
centrosome of cold and nocodazole treated HeLa cells, partially
colocalizing with the centrioles labelled with an anti-acetylated
tubulin antibody. Moreover, HeLa cells displaying a primary
cilium (Fig. 2D) exhibited TBCC at the base of the basal body
(mother centriole) rather than the daughter centriole (arrow).
Subsequently, we silenced TBCC gene expression with a pool of
four synthetic RNAs recognizing different segments of the TBCC
mRNA and specifically designed to knockdown the human TBCC
gene with no off-target effect (see Methods). As Fig. 3A shows, a
noticeable reduction in cell numbers was clearly observed after 72 h
treatment with TBCC RNAi. TBCC gene downregulation
produced a broad range of mitotic spindle defects and mitotic
failure (Fig. 3B, ) typically reported for most centrosomal proteins
. On the other hand, the severe depletion observed for this
protein in whole HeLa cell extracts was however not accompanied
by a marked reduction in a- and b-tubulin levels (Fig. 3C). A
quantitative and morphological study of these cultures revealed a
high proportion of cells blocked at mitosis as soon as 24 h after
RNAi treatment(Fig. 3D), a resultwhich wasfurther supportedby a
reduced number of cells undergoing anaphase and telophase, and a
higher apoptotic rate compared to controls. Moreover, less than
20% of the mitotic cells in TBCC RNAi treated cultures displayed
standard bipolar metaphases, while almost 30% displayed evident
aberrant mitotic figures, mostly multipolar spindles. Longer RNAi
incubation times (48 and 72 h) as shown above, produced a massive
rise in cell death. These data support the hypothesis that TBCC is a
key protein in centrosomal function at mitosis.
The TBCC N-terminal domain is masked at the
polyclonal antiserum against the N-terminal domain of TBCC.
Figure 2. TBCC is located at the centrosome. A) Confocal microscopy image of TBCC localization on interphase (left) and mitotic (right) HeLa
cells. TBCC is mostly a cytoplasmic protein but concentrates at the centrosomes of HeLa cells (arrows). B) TBCC overexpression produces an increase
of TBCC immunostaining at the spindle poles (arrows) and a higher rate of mitotic aberration defects such as multipolar spindles. C) (left) HeLa cells in
prophase exhibiting a clear TBCC colocalization with the protein Nedd1, a classical centrosomal marker. High resolution confocal microscopy images
of both centrosomes (#1,#2) are shown. (right) Confocal-microscopic image of HeLa cells where the microtubule cytoskeleton has been destroyed
by cold and nocodazole treatment. Double-immunostaining against acetylated tubulin and TBCC revealed how, under these conditions, TBCC
colocalizes with the centrioles that typically exhibit acetylated tubulin. D) (left) Triple immunostained HeLa cell displaying a primary cilium (arrow).
(right) High resolution confocal image of the primary cilium and daughter centriole (arrow) immunostained with anti-acetylated tubulin (blue
channel) and TBCC (green channel). These images show that TBCC is mostly localised at the base of the primary cilium, around the basal body.
Structure of the N-Terminal Domain of TBCC
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Figure 3. TBCC depletion leads to mitotic failure and apoptosis. A) (left) HeLa cell culture treated with control RNAi for 72 h. A cell
confluence of almost 90% is achieved. (right) Identical culture treated with TBCC RNAi for 72 h. TBCC gene interference produces a rise in cell death
leading to a conspicuous cell depletion in the culture. B) Confocal-microscopy projection image of 72 h RNAi treated HeLa cell where a multipolar
mitotic spindle is shown (spindle poles indicated by arrows). C) Western blot confirmation of TBCC silencing on whole cell lysates (50 mg/lane total
protein). TBCC expression was compared to total a- and b-tubulins. TBCC depletion did not noticeably affect tubulin levels at this post-transfection
time point. D) Distribution of the different mitotic cell stages observed in TBCC RNAi treated cultures and controls at different time points after TBCC
RNAi treatment. These data show that TBCC RNAi treatment blocks cells mostly at metaphase, leading to a high rate of apoptotic cells. Data represent
mean values and bars standard errors.
Figure 4. The TBCC N-terminal domain is embedded at the centrosome. A) Mitotic HeLa cell doubly-immunostained with the anti-TBCC
antibody purified against the TBCC N-terminal domain, and tubulin. TBCC (arrows) is not detected at the centrosome by the antibody purified against
the N-terminus of TBCC (immunoglobulins recognizing the C-terminus are removed). This result suggests that the TBCC N-terminal domain
centrosomal epitopes are masked in the centrosome. B) (left) High resolution confocal images of HeLa cells transfected with the TBCC N-terminal
domain. Overexpression of the TBCC N-terminal domain produces accumulates of this protein at the perinuclear-centrosomal region (inset, arrow).
(right) Confocal microscopy projection image of a mitotic HeLa cell transfected with the TBCC N-terminal domain and doubly immunostained against
tubulin and TBCC. TBCC N-terminal domain overexpression produces mitotic aberration defects such as multipolar spindles, similar to those observed
for the full-length construct.
Structure of the N-Terminal Domain of TBCC
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Unexpectedly, the same antisera, when purified against the TBCC N-
terminal domain, produced a similar cytoplasmic immunostaining
pattern but did not label the centrosome (Fig. 4A, arrows). These
differences suggest that the TBCC N-terminal domain is masked at
In the view of the above results, we decided to study a TBCC
truncation mutant containing the N-terminal domain overex-
pressed in HeLa cells. In contrast to the cytoplasmic pattern
observed for the full-length polypeptide, the TBCC N-terminal
domain produced a dot-like pattern, distributed at the perinuclear-
centrosomal region (Fig. 4B left). As observed for the full-length
construct, TBCC N-terminal domain overexpression was also
associated with a number of metaphase aberrations (Fig. 4B right).
These results confirm a role for TBCC at the centrosome and
support the hypothesis that the TBCC N-terminal domain is
masked within this organelle. These data led us to study in more
detail the TBCC N-terminal domain.
Structure of the TBCC N-terminal domain
Fig. 5A shows the superposition of the 20 conformers of the
TBCC N-terminal domain determined by NMR. The structure is
a left-handed 3-stranded a-helix bundle composed of 3 antiparallel
and almost coaxial a-helices: a2, N56-R77; a3, V81-S101; a4,
A107-L131 connected by short linkers: loop 2, A78-S80; loop 3,
V102-A106. The N-terminal portion (residues 25–55) of this
domain has not a defined orientation relative to the protein core
and shows regions with partial helix formation (Fig. 5B). In
particular, residues E33-K44 and N49-E55 adopt helical confor-
mations with populations of ,60 and ,38%, respectively as
estimated on the basis of their conformational shifts . No
NOEs connect these nascent helices to the rest of the protein. The
entire N-terminal region is structurally disordered relative to the
domain and samples all the available conformational space. The
structured part of the protein (residues 56–131), is well-defined
with low pairwise RMSD values (Table 1). Average interhelical
angles of 170u between helix a2 and a3, 6u between helix a2 and
a4, and 173u between helix a3 and a4 are obtained for the
ensemble. The compact helix bundle confers the molecule a rod-
like shape with a volume of 11000 (,17617638) A˚3and a global
accessible surface area of 6400 A˚2. Helical wheel projections
(Fig. 5C) show that the sequences of the three helices conforming
the TBCC’s bundle fulfil the characteristic heptad pattern of left-
handed coiled coils .
The side chains of a significant number of hydrophobic residues
are deeply buried in the protein core, pointing to the interior of the
helix bundle (Fig. 5C, 5D). Among them, F60, F64, L75, A78,
L84, A87, L91, L94, I98, L105, G113, A116, L117, L120, L124
possess ASA values below 5%. These residues participate in
hydrophobic interactions that contribute to stabilizing the helical
bundle by forming an extended hydrophobic platform along the
helix axis. These interactions include the aromatic-aromatic
contacts between F60 and F64 in an edge-to-face fashion, and
many aromatic-aliphatic and aliphatic-aliphatic contributions. For
example, close contacts (,5 A˚) in the upper part of the bundle
involve the aromatic rings of F60 and F64 with the aliphatic chains
of V61, L94, L97, I98, V102, L105, A116, and L117 (Fig. 5C, 5D,
yellow). In the lower part of the bundle, interactions involve V71,
L74, L75, A87, A88, L91, L94, L120, A123, L124 also from the
three helices (Fig. 5C, 5D, magenta). At the bottom A78, V81,
L84, L131 belong to loop 2 and the N- and C-termini of helices a3
and a4, respectively (Fig. 5C, 5D, green). Close to the disordered
part, the aromatic rings of F103 and F104 interact with the methyl
groups of V102, L105 and A106, all located in loop 3 (Fig. 5C, 5D,
orange). Y108 is also close to A106 (,4.6 A˚). In contrast to
hydrophobic interactions, electrostatic interactions are much less
abundant. A salt bridge connecting the side-chains of E67 and
R90, that links helices a2 and a3 (Fig. 5C, 5D, red), is present. The
side chains of residue pairs E76-R127, E79-R83, E82-R83, E126-
R127, and E126-R129 are relatively close to each other and may
form favourable charge-charge interactions.
The surface of the TBCC N-terminal domain is highly charged
(Fig. 6A). Interestingly, two contiguous regions differing in 90u
rotation concentrate longitudinally charges of opposite signs while
on the two remaining faces there is a more random distribution.
Such a distribution would favour protein-protein interactions with
partners having the appropriate charge complementarities. Also,
remarkably, the 30-residue N-terminal region is very rich in
positive charges, except for a central patch of negatively charged
residues (E33, E35, E39, and E41). The 30-residue N-terminal
region concentrates 80% of charged and polar residues with
ASAs$30% and these features are likely to be important for the
interaction with tubulin as discussed below.
Dynamics of the TBCC N-terminal domain
We have measured the heteronuclear NOEs to get information
on the local backbone flexibility of the TBCC N-terminal domain
in the ns-ps time-scale (Fig. 7). The dynamics of helices a2, a3, and
a4 is quite restricted although several residues located at both
termini of helix a2 (N56, S57, F60, R77) as well as those in loop 2
(A78-S80), L105 in loop 3, residues G130 and L131 at the end of
helix a4, and the C-terminus are more flexible. Interestingly, the
30-residue N-terminal region is highly dynamic; all residues
display lower than average heteronuclear NOEs. These data
corroborate the disordered nature of the N-terminal region, and
shows that its high flexibility is responsible for the absence of long
Interaction of the TBCC N-terminal domain with tubulin
We tested whether the TBCC N-terminal domain is able to
interact directly with ab-tubulin heterodimer and with two
peptides of 16 (residues 435–450, 9 residues charged, 8 negatively
and 1 positively charged) and 20 (residues 412–431, includes helix
H12, 5 residues negatively charged) residues derived from the C-
terminus of the b6-tubulin subunit (class III, ). Region 412–
431 is highly conserved in tubulins and the last 10–15 residues of
their C-terminus represent the most variable region although it is
always negatively charged and contains several Glu residues.
Secondary structure predictions and circular dichroism experi-
ments suggested that while the region 412–431 forms an a-helix,
the last 10–15 C-terminal residues lacks ordered structure 
independently of the isotype. This last region of b-tubulin is known
to interact with many microtubule associated proteins (MAPs) and
the electrostatic contacts with the stretches of negatively charged
residues have been reported to play a crucial role in the interaction
[18,19,20]. At this regard, the 16-aminoacid peptide was chosen to
specifically include the acidic region. We monitored the changes
induced in the
terminal domain in the presence and absence of unlabelled ab-
tubulin and b-tubulin peptides (Fig. 8). Severe spectral broadening
most likely accompanied by the consequent loss of signals would
be expected upon the formation of the large TBCC-tubulin
complex. For this reason we have explored the interaction with
low TBCC/tubulin ratios, conditions that can be followed by
NMR. Important changes in the position and the intensity of
many peaks are observed upon ab-tubulin titration that map to a
large portion of the surface of the TBCC N-terminal domain
(Fig. 8A and Fig. 6B). Interestingly, at the lowest amounts of
tubulin, there was a set of residues whose signals considerably
15N-HSQC spectra of the labelled TBCC N-
Structure of the N-Terminal Domain of TBCC
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broadened, including N56, F104, A107, and S57, which almost
disappear. All observed changes map to an interaction surface that
includes the disordered and flexible 30-residue N-terminal
segment, as well as residues from the three helices, and the loops
(Fig. 6B). Such a large contact surface is probably indicative of
significant conformational changes occurring upon binding. Many
of the residues affected are charged and polar, particularly those in
the 30-residue N-terminal segment. Examples of these residues in
helices a2–a4 and loops are: N56, S57, E73, E76, R77, E79, E82,
E85, E86, R90, N99, D100, Q114, and E126. Also, important
hydrophobic groups from V61, F64, A69, V71, L84, A87, L91,
F103, F104, A106, A107, A122, A123, and A125, participate in
binding. Most of these residues are also perturbed by the
interaction with the 16-residue peptide corresponding to the
tubulin sequence 435–450 (Fig. 8B and Fig. 9) but no spectral
changes occur with the 20-residue peptide corresponding to the
Figure 5. Solution structure of the TBCC N-terminal domain. A) Superposition of the 20 lowest-energy conformers. B) Ribbon display of a
representative conformer of the family showing the limits of the helical segments and one of the possible orientations of the N-terminal tail (in green)
with respect to the protein core. Two regions with helical propensity (33–44) and (49–55) are labelled. C) Hydrophobic contacts in the interior of the
bundle. Different colours are used along the helix axis for the upper N-terminal side (yellow), the lower C-terminal side (magenta), the bottom part
including loop 2 (green), the top part comprising loop 3 next to the disordered N-terminal region (orange). The salt bridge between E67 and R90 is
highlighted in red. D) Distribution of aliphatic/aromatic and charged/polar residues along the helices. The hydrophobic side-chains are concentrated
at the helical interfaces favouring the molecular packing, and most polar and charged residues are in contact with the solvent. Colour code as in C).
Structure of the N-Terminal Domain of TBCC
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tubulin sequence 412–431 (Fig. 8C), suggesting that the sequence
region covered by the latter peptide does not participate directly in
To date not much literature is available on the biological
processes involving TBCC. It is known to be involved in the last
step of the pathway leading to correctly folded ab-tubulin,
however the molecular details of the mechanistic aspects remain
elusive. This protein is necessary for life in higher eukaryotes
[21,22,23,24,25] and has been implicated in cancer cell prolifer-
ation control , but so far no reports regarding its function in
vivo have been published.
Comparison of TBCC with other tubulin cofactor
From the set of five cofactors assisting tubulin folding, structural
data are available for only two of them, cofactors A and B of
different species. Both form stable complexes with b-tubulin and
a-tubulin folding intermediates, respectively, in the presence of
GTP  following their release from the ab-tubulin-CCT binary
complex . Human cofactor A, the first cofactor discovered to
participate in the process [28,29], is the one whose structure most
closely resembles the N-terminal domain of TBCC although it has
not sequence similarity and lacks disordered regions (Fig. 9). Its X-
ray structure revealed a helical bundle of three antiparallel helices
 similar to that of the TBCC N-terminal domain, although
their lengths are quite different. The crystal structure of the TBCA
yeast homolog in S. cerevisiae, Rbl2p, shares a similar overall fold
but crystallizes as an inverted dimer with the two long helices
associated into a four-helix bundle . On the other hand, the
crystal structure of A. thaliana TBCA has recently been reported
. It forms a monomeric three-helix bundle where the a-helical
residues are important for b-tubulin affinity as was previously
shown for the human ortholog . A search for structural
homologs of TBCA yielded the cytoskeletal proteins a-spectrin
and a-actinin. The length of the rod units is identical in TBCA,
BAG1 domain (Fig. 9) and spectrin/actinin repeats . It is
noteworthy that recently, PRC1, a nonmotor microtubule binding
protein belonging to the MAP65 protein family, has been found to
establish crosslinks in dynamic cytoskeletal networks. These
proteins have an N-terminal coiled-coil domain, a C-terminal
regulatory domain and a central region that mediates microtubule
binding. This last domain also resembles spectrin repeats
structurally . While MAP65 proteins bind to microtubules,
TBCA binds to the b-tubulin subunit.
Biological role of the flexible disordered N-terminal
segment of the TBCC N-terminal domain
Interestingly, some of the cofactor structures have disordered
regions for which roles in intermolecular interactions have been
proposed. The crystal structure of the CAP-Gly domain of TBCB
in C. elegans, F53F4.3, shows a completely different fold with three
antiparallel b-sheets . However, despite this different topology
with respect to the TBCC N-terminal domain, it also has a flexible
a-helix at the N-terminus preceded by 17 disordered residues
which were proposed to participate in intermolecular interactions
. The TBCC homolog RP2 has a chiefly disordered 33 residue
segment at its N-terminus that was also proposed to participate in
Using NMR chemical shift perturbation mapping, we have
shown that the region of the TBCC N-terminal domain involved
in tubulin binding includes the flexible 30-residue N-terminal
segment, which remarkably concentrates the largest number of
charged and polar residues. The calculated pI value for this region,
9.7, reflects a markedly cationic character suggestive of a possible
involvement in electrostatic interactions with the C-terminal
anionic domains of tubulin. A pair of adjacent Arg and Lys
residues have been shown to be essential for the ability of the
microtubule destabilizing domain of centrosomal protein CPAP to
bind to microtubules . Also, site directed mutagenesis
corroborated the importance of positively charged residues in
the binding of CLIP170 . At this respect, it is of note that the
stretch 42–47 of the TBCC N-terminal domain flexible segment
(RRKQKR) has a high local positive charge density and would be
a good candidate for target interactions. Moreover, we have
provided evidence that the 30-residue N-terminal segment of the
TBCC N-terminal domain is highly mobile and disordered in the
free protein according to NMR relaxation measurements. High
degrees of disorder and unfolded segments are a common feature
of many centrosomal proteins  suggesting that this might be an
intrinsic requirement for biological function. Nowadays there is
considerable evidence that microtubule associated proteins are
unstructured in solution [39,40,41] and become ordered upon
binding to protofilament surface . Although the 30-residue N-
terminal region of the TBCC N-terminal domain is globally
disordered with respect to the protein core, non-negligible helical
tendencies exist particularly in the E33-K44 and N49-E55
segments. This flexible segment might fold into a defined tertiary
structure with increased helix content upon tubulin binding in a
similar way as proposed for the predominantly helical 23-residue
region of the mainly unstructured PN2-3 fragment of centrosomal
protein CPAP .
Table 1. Structural Statistics of the 20 Best NMR Structures of
the TBCC N-terminal domain.
NOE Distance and Dihedral Constraints
No. of short-range distances (|i-j|#1) 990
No. of medium-range distances (1,|i-j|,5)412
No. of long-range distances (|i-j|$5)290
No. of angular restraints (Q, y) 95
No. of total restraints 1692
Average CYANA target function value0.49
Maximum distance violation (A˚)0.25
Maximum dihedral angle violation (u) 2.87
Average AMBER energy (kcal/mol)
Bond lengths from ideal geometry0.010860.0006
Bond angles from ideal geometry 1.9960.02
Pairwise backbone (26–135)7.6362.75
Pairwise heavy atom (26–135)8.7962.87
Pairwise backbone (56–131) 0.6560.13
Pairwise heavy atom (56–131)1.5360.14
Ramachandran Plot Analysis (%)
Most favored regions94.7
Additional allowed regions5.3
Generously allowed regions0
Structure of the N-Terminal Domain of TBCC
PLoS ONE | www.plosone.org7 October 2011 | Volume 6 | Issue 10 | e25912
Interaction of the TBCC N-terminal domain with tubulin
It is well known that NMR spectroscopy provides a fast method
for analysing weak protein–protein interactions  and is
therefore highly convenient for the study of transient protein
complexes that are difficult to detect by other methods.
Specifically, monitoring chemical shift perturbation is the most
widely used NMR method to map protein interfaces . The
tubulin binding region mapped in this study for the TBCC N-
terminal domain (Fig. 6B and Fig. 9) partially agrees with that
suggested for TBCA or BAG1 proteins with b-tubulin and the
ATPase domain of Hsc70, respectively. In the present study, the
interaction has been corroborated with synthetic peptides covering
relevant regions of the b-tubulin sequence (412–431 and 435–450)
and with the tubulin heterodimer. In our study, the sequence of
the peptide 435–450 was derived from the human b6 tubulin
isotype (Class III) found in humans by Sullivan and Cleveland
. Although there might be some small differences (one residue)
with respect to the sequences in the databases, the classical stretch
of negatively charged residues is present in all isotypes. Also,
TBCC should be promiscuous regarding b-tubulin isotype binding
because there is just one TBCC gene in the genome and it
contains a single exon. We do not know the actual TBCC partner
at the centrosome but it is noticeable that TBCC binds to different
tubulin isotypes in the supercomplex formed with TBCD and
TBCE during dimer formation in the postchaperonin tubulin
pathway . The fact that the interacting region with tubulin
mapped along the helical bundle in this study (Fig. 6B) is the same
as that detected with the isolated peptide (Fig. 9), strongly suggests
that the interactions are physiologically relevant.
The interaction site for BAG1, also based on NMR titration
data with synthetic peptides that mimic some helices of the
ATPase Hsc70 subdomains, was assigned predominantly to the
central regions of helices a2 and a3 (Fig. 9), on the same face of its
conserved domain . For TBCA, although interacting regions
Figure 6. Surface properties of the TBCC N-terminal domain. A) The electrostatic surface is represented for four views corresponding to 90u
rotations. The distribution of the negatively charged (red), positively charged (blue), and nonpolar residues (white) defines a highly charged surface,
with two 90u-rotated faces concentrating mainly negative and positive charges (left), and the other two with more random charge distribution (right).
B) Two 180u rotated views of the mapped chemical shift perturbation data. Residues affected by the interaction with ab-tubulin dimer are coloured in
yellow in the helices and in green for the N-terminal disordered segment.
Structure of the N-Terminal Domain of TBCC
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e25912
were mapped along the three helices using binding assays of b-
tubulin with a cellulose-bound TBCA peptide library, the role of
residues in helix a2 was found the more relevant . Although
TBCA, BAG1 and the TBCC N-terminal domain share a similar
fold topology, their helix lengths and sequences greatly differ,
making it difficult the identification of structurally equivalent
residues for comparisons. However, our NMR data reveal a large
number of residues at the contact surface along the main axis of
the helical bundle suggesting that the recognition of tubulin by the
TBCC N-terminal domain probably has an orientation similar to
that of TBCA and BAG1. This similarity is shown in Fig. 9, where
the proposed interacting surfaces for the three proteins are
displayed in a similar orientation. Despite the different sequences
involved, a large variety of amino acid types seems to be perturbed
upon binding suggesting a complex network of intermolecular
contacts responsible for the interaction with tubulin.
A significant difference is provided by the flexible N-terminal
region of TBCC also participating in binding, which is neither
present in TBCA nor in BAG1. A reasonable explanation for the
concomitant changes observed in the flexible N-terminal segment,
and nearby residues, would be a structural reorganization in which
this region adopts an ordered and defined orientation within the
helix bundle mediating molecular recognition. This process is most
likely driven by electrostatic interactions. Interestingly, a theoret-
ical coiled-coil structure, which includes residues in the natively
disordered N-terminal end (from P26 to E55), is predicted by the
program COILS . In fact, the region comprising residues
Q30-N56 has a high probability to adopt a helix coiled-coil
structure just preceding the experimentally determined helix a2.
In principle, two kinds of non-mutually exclusive elements of
interaction have been postulated in disordered protein segments:
molecular recognition features and preformed elements. Molecu-
lar recognition features are short regions that undergo a disorder
to order transition that is induced by binding to their partners
. On the other hand, preformed elements are regions with
some percentage of secondary structure population that are
present in the free unstructured form and usually represent the first
interacting elements, that grow and become more stable upon
interacting with their partners . In our case, the Q30-N56
region of the N-terminal domain of TBCC has the characteristics
of a preformed structure, an helix, that is present although not
100% populated when isolated and that would adopt a more
ordered coiled-coil structure upon binding. In this regard more
work will be necessary to test this hypothesis.
In summary, we show that TBCC is a protein implicated in
centrosomal stability particularly at mitosis. TBCC expression
changes in human cells produce several mitotic spindle defects
leading to mitotic failure and apoptosis. These results demonstrate
that TBCC is a crucial protein in the control of the eukaryotic cell
cycle, and support the hypothesis that this tubulin binding cofactor
could be implicated in genomic instability and cancer. Our data
show how TBCC interacts with components of the centrosome by
its N-terminal domain, which is masked within this organelle. We
have also shown that the structure of the TBCC N-terminal
domain solved by NMR adopts a spectrin-like fold and with a
flexible and disordered N-terminal segment. This segment is
highly charged and participates in tubulin interaction. The tubulin
binding region of the structured coiled coil region resembles those
proposed for TBCA and BAG1 proteins.
Materials and Methods
Expression and purification of TBCC N-terminal domain
Human TBCC cDNA (accession number U61234) was
obtained from Dr N. Cowan (New York University, Medical
Center, New York, USA). The TBCC N-terminal domain was
generated by PCR (codons 25–135, corresponding to the sequence
showing high homology between species) and cloned into pET3a
Figure 7. TBCC N-terminal domain heteronuclear NOEs for local backbone flexibility. Residues in the 30-residue N-terminal segment have
lower than average NOE values, indicative of high backbone mobility in the ns-ps time-scale. Some flexibility is also found at the C-terminus of the
domain and residues at the interhelical connecting loops. The dynamics of the helices a2, a3, a4 is more restricted.
Structure of the N-Terminal Domain of TBCC
PLoS ONE | www.plosone.org9 October 2011 | Volume 6 | Issue 10 | e25912
vector (Invitrogen). The TBCC N-terminal domain was expressed
in the E. coli BL21(DE3)/pLysS strain (Life Technologies, SA,
Spain) using the T7 expression system  and purified from 15 L
culture (in 46500 mL batches). Upon reaching optical cell
densities of <0.7 at 600 nm, the cells were centrifuged at
3,0006 g in a JA-25.50 rotor for 15 min and pelleted. The cells
were then washed with phosphate buffer and pelleted again at
room temperature. The cell pellet was resuspended in J volume
of minimal medium containing
glucose and was incubated to allow the recovery of growth. Protein
15NH4Cl alone or with
expression was induced after 1 h by addition of isopropylthio-b-D-
galactoside to a concentration of 1 mM. After a 3 h incubation
period the cells were harvested. Cells were pelleted by centrifu-
gation, washed, and stored frozen at 270uC. Pellets were
resuspended in 10–20 mL of 50 mM Tris pH 8.0 with protease
inhibitors. Cells were then ruptured by sonication (3 bursts of
10 seconds). After centrifugation at 25,0006g in a JA-25.50 rotor
for 30 min at 4uC, the supernatant was loaded onto a HiTrap Q
(5 mL, GE Healthcare) equilibrated with Tris-HCl pH 8 contain-
ing 10 mM KCl. The flow-through was passed through the same
Figure 8. TBCC N-terminal interaction assays with ab-tubulin dimer and C-terminal b-tubulin peptides. Superposition of15N-HSQC
spectra of the TBCC N-terminal domain free (blue) and in the presence of ab-tubulin heterodimer (yellow). Selected perturbed residues are labelled,
with green labels corresponding to amino acids in the N-terminal disordered region. B) Superposition of15N-HSQC spectra of the TBCC N-terminal
domain free (blue) and in the presence of an excess of the 16-residue C-terminal b-tubulin peptide EMYEDDEEESESQGPK (magenta). Selected
perturbed residues are labelled. C) Superposition of15N-HSQC spectra of the TBCC N-terminal domain free (blue) and in the presence of excess of the
20-residue C-terminal b-tubulin peptide ESNMNDLVSEYQQYQDATAD (grey). No significant perturbations are observed.
Figure 9. Comparison of the interacting face of TBCA, BAG1 and TBCC N-terminal domain. Ribbon displays of the similarly oriented
spectrin-like domains of TBCA (left), BAG1 (middle), and TBCC (right) with the residues involved in the interaction with b-tubulin, the ATPase domain
of Hsc70, and the 16-residue C-terminal b-tubulin peptide EMYEDDEEESESQGPK (435–450), respectively, shown in strong colours.
Structure of the N-Terminal Domain of TBCC
PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25912
column for a second time, and then applied to a high-resolution
Mono-S column (5/50 GL). The TBCC N-terminal domain was
eluted with a linear gradient of 10–500 mM KCl in Tris-HCl
pH 8. Fractions containing the TBCC N-terminal domain were
pooled, diluted 10 fold with 20 mM phosphate buffer pH 6
containing 1 mM TCEP and concentrated by ultrafiltration with
Amicon Ultra 10 filters. Protein purity was determined by SDS-
Cell biology procedures
Passage 10 human cervical carcinoma HeLa cells cultures
(obtained from EMBO laboratories stocks, Heidelberg, Germany)
were fixed in chilled (220uC) methanol or 4% paraformaldehyde,
and further permeabilized in phosphate-buffered saline (PBS)–
0.1% Triton X-100. For centrosomal immunostaining, microtu-
bules were depolymerized with 2 mM nocodazole and 4uC
treatments for 30 min. Anti-a-tubulin (B512) and anti-acetylated
tubulin antibodies were both from Sigma (Aldrich). Human
TBCC N-terminal domain was generated by PCR and inserted
into the pcDNA3 vector (Invitrogene, Life Technologies). RNA
interference was performed with a pool of four siRNA fragments
targetinghuman TBCC (ONTARGETplus
DHARMACON, CO, USA). These siRNAs have been designed
and tested to have no off-target effects. TBCC silencing was
confirmed 24, 48, and 72 h after RNAi treatment by western
blotting on quantified total cell extracts compared to and non
target RNAi control. Morphological cell quantification (Fig. 3) was
performed on live cultures to prevent cell loss during washes.
Counts were performed for three different culture plates of three
different experiments and controls. Statistical analysis of data and
graphing were performed using the SigmaPlot 8.0 software (Systat
Software, Richmond, CA). Confocal-microscopic images were
obtained using a Zeiss LSM-510 confocal microscope with a
6361, 40 lens.
NMR, sample preparation and experiments
For NMR experiments, the
terminal domain sample was prepared in 90:10 H2O:D2O and
D2O solutions of KH2PO4/K2HPO4 buffer 20 mM, 1 mM
TCEP, 20 mM KCl, 1 mM EDTA, with protease inhibitors,
pH 6.0 at final concentrations in the range 0.5–1 mM. DSS was
used for spectra referencing.
Microtubule proteins were prepared from calf brains by
repeated cycles of assembly– disassembly using a temperature-
dependent procedure. Native tubulin heterodimers were purified
following our published protocols and their viability was checked
by non-denaturing electrophoresis, as described [49,50]. The
solution of ab-tubulin dimer was dissolved in 50 mM MES buffer,
1 mM EGTA, 0.25 mM MgCl2, pH 6.7. The concentration of the
stock solution was ,30 mM. Two concentrated solutions of
peptides derived from the C-terminal end of the b6-tubulin (Class
435EMYEDDEEESESQGPK450)  were prepared in water to
final concentrations of 7 mM and 10 mM, respectively.
15N-labelled TBCC N-
Spectral assignment was done using sets of standard 2D and 3D
experiments as reported . For the backbone
measurement, experiments with and without proton saturation
were acquired simultaneously in an interleaved manner and split
during processing into separate spectra for analysis. A relaxation
delay of 7.5 s was used. The NOE values were obtained from the
ratio intensities of the resonances in both spectra.
The TBCC N-terminal domain interaction with ab-tubulin was
followed by comparing the
TBCC N-terminal domain, with that obtained after the addition
of ,100 mL of the TBCC N-terminal domain to the tubulin stock
solution. Both the chemical shift and line width changes were
analysed. To test the interaction with the b-tubulin peptides, the
appropriate volumes of the concentrated solutions were added to
the TBCC N-terminal domain sample to get approximately 1:1
protein:peptide stoichiometries. Changes of peak intensity and
position were monitored. In all cases the pH was checked at the
final points of the titrations.
All the experiments were recorded at 25uC on a Bruker AV 800
NMR spectrometer equipped with a cryoprobe. The spectra were
processed with Bruker Topspin (Bruker, Germany) and spectral
analysis was performed with Sparky3 . Molmol  was used
for molecular display.
15N-HSQC spectrum of the free
The structure calculation of the TBCC N-terminal domain was
performed with CYANA  using the automatic NOE
assignment facility combined with lists of manually assigned
NOEs. In total there were 1692 upper distance constraints, 870 of
which were manually assigned. Backbone dihedral angle con-
straints were determined, for each residue except for the segments
M25-R32, K46-Q48, P133-K135, and A78, E79, and L131, from
chemical shift values using TALOS+ . Initially 100 conformers
were generated that were forced to satisfy experimental data using
a standard automatic CYANA protocol . The 20 conformers
with the lowest final CYANA target function values were selected
and subjected to 2,000 steps of energy minimization using the
generalized Born continuum solvation model  implemented in
AMBER9  with a non-bonded cutoff of 10 A˚. The AMBER
energy was 27,000 kcal/mol with an electrostatic contribution
term of 26,300 kcal/mol. Final structure quality was checked with
PROCHECK-NMR  and the coordinates have been
deposited in the PDB under the accession number 2l3l. Statistics
of the calculation are summarized in Table 1.
We thank Dr. D. V. Laurents for English style suggestions.
Conceived and designed the experiments: JCZ MR MB. Performed the
experiments: MFG-M RC MLF. Analyzed the data: JCZ MR MB MFG-
M RC MLF. Contributed reagents/materials/analysis tools: MFG-M RC
MLF. Wrote the paper: JCZ MR MB MFG-M RC MLF.
1. Lewis SA, Tian G, Cowan NJ (1997) The alpha- and beta-tubulin folding
pathways. Trends Cell Biol 7: 479–484.
2. Lo ´pez-Fanarraga M, A´vila J, Guasch A, Coll M, Zabala JC (2001) Review:
postchaperonin tubulin folding cofactors and their role in microtubule dynamics.
J Struct Biol 135: 219–229.
3. Gao Y, Thomas JO, Chow RL, Lee GH, Cowan NJ (1992) A cytoplasmic
chaperonin that catalyzes beta-actin folding. Cell 69: 1043–1050.
4. Tian G, Huang Y, Rommelaere H, Vandekerckhove J, Ampe C, et al. (1996)
Pathway leading to correctly folded beta-tubulin. Cell 86: 287–296.
5. Fontalba A, Paciucci R, Avila J, Zabala JC (1993) Incorporation of tubulin
subunits into dimers requires GTP hydrolysis. J Cell Sci 106(Pt 2): 627–
6. Bartolini F, Bhamidipati A, Thomas S, Schwahn U, Lewis SA, et al. (2002)
Functional overlap between retinitis pigmentosa 2 protein and the tubulin-
specific chaperone cofactor C. J Biol Chem 277: 14629–14634.
7. Goncalves J, Nolasco S, Nascimento R, Lopez Fanarraga M, Zabala JC, et al.
(2010) TBCCD1, a new centrosomal protein, is required for centrosome and
Golgi apparatus positioning. EMBO Rep 11: 194–200.
Structure of the N-Terminal Domain of TBCC
PLoS ONE | www.plosone.org 11October 2011 | Volume 6 | Issue 10 | e25912
8. Feldman JL, Marshall WF (2009) ASQ2 encodes a TBCC-like protein required Download full-text
for mother-daughter centriole linkage and mitotic spindle orientation. Curr Biol
9. Scheffzek K, Ahmadian MR, Kabsch W, Wiesmuller L, Lautwein A, et al.
(1997) The Ras-RasGAP complex: structural basis for GTPase activation and its
loss in oncogenic Ras mutants. Science 277: 333–338.
10. Grynberg M, Jaroszewski L, Godzik A (2003) Domain analysis of the tubulin
cofactor system: a model for tubulin folding and dimerization. BMC
Bioinformatics 4: 46.
11. Lo ´pez-Fanarraga M, Carranza G, Bellido J, Kortazar D, Villegas JC, et al.
(2007) Tubulin cofactor B plays a role in the neuronal growth cone. J Neurochem
12. Hage-Sleiman R, Herveau S, Matera EL, Laurier JF, Dumontet C (2010)
Tubulin binding cofactor C (TBCC) suppresses tumor growth and enhances
chemosensitivity in human breast cancer cells. BMC Cancer 10: 135–148.
13. Mikule K, Delaval B, Kaldis P, Jurcyzk A, Hergert P, et al. (2007) Loss of
centrosome integrity induces p38-p53-p21-dependent G1-S arrest. Nat Cell Biol
14. Garcı ´a-Mayoral MF, Castan ˜o R, Zabala JC, Santoro J, Rico M, et al. (2010)1H,
Tubulin Cofactor C. Biomol NMR Assign 4: 219–221.
15. Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF, et al. (2003) VADAR: a
web server for quantitative evaluation of protein structure quality. Nucleic Acids
Res 31: 3316–3319.
16. Lupas A (1996) Coiled coils: new structures and new functions. Trends Biochem
Sci 21: 375–382.
17. Sullivan KF, Cleveland DW (1986) Identification of conserved isotype-defining
variable region sequences for four vertebrate beta tubulin polypeptide classes.
Proc Natl Acad Sci U S A 83: 4327–4331.
18. Al-Bassam J, Ozer RS, Safer D, Halpain S, Milligan RA (2002) MAP2 and tau
bind longitudinally along the outer ridges of microtubule protofilaments. J Cell
Biol 157: 1187–1196.
19. Zhu ZC, Gupta KK, Slabbekoorn AR, Paulson BA, Folker ES, et al. (2009)
Interactions between EB1 and microtubules: dramatic effect of affinity tags and
evidence for cooperative behavior. J Biol Chem 284: 32651–32661.
20. Devred F, Barbier P, Douillard S, Monasterio O, Andreu JM, et al. (2004) Tau
induces ring and microtubule formation from alphabeta-tubulin dimers under
nonassembly conditions. Biochemistry 43: 10520–10531.
21. Reuter G, Szidonya J (1983) Cytogenetic analysis of variegation suppresors and
a dominant temperature-sensitive lethal in region 23–26 of chromosome 2L in
Drosophila melanogaster. Chromosoma 88: 277–285.
22. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, et al. (2003) Systematic
functional analysis of the Caenorhabditis elegans genome using RNAi. Nature
23. Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, et al. (2004) Toward improving
Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi
library. Genome Res 14: 2162–2168.
24. So ¨nnichsen B, Koski LB, Walsh A, Marschall P, Neumann B, et al. (2005) Full-
genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.
Nature 434: 462–469.
25. Kirik V, Mathur J, Grini PE, Klinkhammer I, Adler K, et al. (2002) Functional
analysis of the tubulin-folding cofactor C in Arabidopsis thaliana. Curr Biol 12:
26. Llosa M, Aloria K, Campo R, Padilla R, Avila J, et al. (1996) The beta-tubulin
monomer release factor (p14) has homology with a region of the DnaJ protein.
FEBS Lett 397: 283–289.
27. Melki R, Rommelaere H, Leguy R, Vandekerckhove J, Ampe C (1996) Cofactor
A is a molecular chaperone required for beta-tubulin folding: functional and
structural characterization. Biochemistry 35: 10422–10435.
28. Gao Y, Melki R, Walden PD, Lewis SA, Ampe C, et al. (1994) A novel
cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.
J Cell Biol 125: 989–996.
29. Campo R, Fontalba A, Sa ´nchez LM, Zabala JC (1994) A 14 kDa release factor
is involved in GTP-dependent beta-tubulin folding. FEBS Lett 353: 162–166.
30. Guasch A, Aloria K, Perez R, Avila J, Zabala JC, et al. (2002) Three-
dimensional structure of human tubulin chaperone cofactor A. J Mol Biol 318:
31. Steinbacher S (1999) Crystal structure of the post-chaperonin beta-tubulin
binding cofactor Rbl2p. Nat Struct Biol 6: 1029–1032.
15N resonance assignments of the N-terminal domain of human
32. Lu L, Nan J, Mi W, Li LF, Wei CH, et al. (2010) Crystal structure of tubulin
folding cofactor A from Arabidopsis thaliana and its beta-tubulin binding
characterization. FEBS Lett 584: 3533–3539.
33. Subramanian R, Wilson-Kubalek EM, Arthur CP, Bick MJ, Campbell EA, et al.
(2010) Insights into antiparallel microtubule crosslinking by PRC1, a conserved
nonmotor microtubule binding protein. Cell 142: 433–443.
34. Li S, Finley J, Liu ZJ, Qiu SH, Chen H, et al. (2002) Crystal structure of the
cytoskeleton-associated protein glycine-rich (CAP-Gly) domain. J Biol Chem
35. Ku ¨hnel K, Veltel S, Schlichting I, Wittinghofer A (2006) Crystal structure of the
human retinitis pigmentosa 2 protein and its interaction with Arl3. Structure 14:
36. Hsu WB, Hung LY, Tang CJ, Su CL, Chang Y, et al. (2008) Functional
characterization of the microtubule-binding and -destabilizing domains of CPAP
and d-SAS-4. Exp Cell Res 314: 2591–2602.
37. Mishima M, Maesaki R, Kasa M, Watanabe T, Fukata M, et al. (2007)
Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its
autoinhibition. Proc Natl Acad Sci U S A 104: 10346–10351.
38. Cormier A, Clement MJ, Knossow M, Lachkar S, Savarin P, et al. (2009) The
PN2-3 domain of centrosomal P4.1-associated protein implements a novel
mechanism for tubulin sequestration. J Biol Chem 284: 6909–6917.
39. Schweers O, Schonbrunn-Hanebeck E, Marx A, Mandelkow E (1994) Structural
studies of tau protein and Alzheimer paired helical filaments show no evidence
for beta-structure. J Biol Chem 269: 24290–24297.
40. Mukrasch MD, von Bergen M, Biernat J, Fischer D, Griesinger C, et al. (2007)
The ‘‘jaws’’ of the tau-microtubule interaction. J Biol Chem 282: 12230–12239.
41. Voter WA, Erickson HP (1982) Electron microscopy of MAP 2 (microtubule-
associated protein 2). J Ultrastruct Res 80: 374–382.
42. Takeuchi K, Wagner G (2006) NMR studies of protein interactions. Curr Opin
Struct Biol 16: 109–117.
43. Zuiderweg ER (2002) Mapping protein-protein interactions in solution by NMR
spectroscopy. Biochemistry 41: 1–7.
44. Briknarova ´ K, Takayama S, Brive L, Havert ML, Knee DA, et al. (2001)
Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat
shock protein. Nat Struct Biol 8: 349–352.
45. Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein
sequences. Science 252: 1162–1164.
46. Vacic V, Oldfield CJ, Mohan A, Radivojac P, Cortese MS, et al. (2007)
Characterization of molecular recognition features, MoRFs, and their binding
partners. J Proteome Res 6: 2351–2366.
47. Fuxreiter M, Simon I, Friedrich P, Tompa P (2004) Preformed structural
elements feature in partner recognition by intrinsically unstructured proteins.
J Mol Biol 338: 1015–1026.
48. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA
polymerase to direct expression of cloned genes. Methods Enzymol 185: 60–89.
49. Avila J, Soares H, Fanarraga ML, Zabala JC (2008) Isolation of microtubules
and microtubule proteins. Curr Protoc Cell Biol Chapter 3: Unit 3 29.
50. Fanarraga ML, Carranza G, Castano R, Nolasco S, Avila J, et al.
Nondenaturing electrophoresis as a tool to investigate tubulin complexes.
Methods Cell Biol 95: 59–75.
51. Goddard TD, Kneller DG Sparky 3, University of California, San Francisco.
52. Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: a program for display and
analysis of macromolecular structures. J Mol Graph 14: 51–55, 29–32.
53. Gu ¨ntert P (2004) Automated NMR structure calculation with CYANA. Methods
Mol Biol 278: 353–378.
54. Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid method for
predicting protein backbone torsion angles from NMR chemical shifts. J Biomol
NMR 44: 213–223.
55. Hawkins GD, Cramer CJ, Truhlar DG (1995) Pairwise solute descreening of
solute charges from a dielectric medium. Chem Phys Lett 246: 122–129.
56. Case DA, Darden TA, Cheatham ITE, Simmerling CL, Wang J, et al. (2006)
AMBER 9, University of California, San Francisco.
57. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM
(1996) AQUA and PROCHECK-NMR: programs for checking the quality of
protein structures solved by NMR. J Biomol NMR 8: 477–486.
58. Freeman NL, Field J (2000) Mammalian homolog of the yeast cyclase associated
protein, CAP/Srv2p, regulates actin filament assembly. Cell Motil Cytoskeleton
Structure of the N-Terminal Domain of TBCC
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