Báo cáo khoa học: Crystal and solution structure, stability and post-translational modifications of collapsin response mediator protein 2 pdf

Crystal and solution structure, stability and
post-translational modifications of collapsin
response mediator protein 2
Viivi Majava
1
, Noora Lo
¨
ytynoja
1
, Wei-Qiang Chen
2
, Gert Lubec
2
and Petri Kursula
1
1 Department of Biochemistry, University of Oulu, Finland
2 Department of Pediatrics, Medical University of Vienna, Austria
Axonal growth cone guidance is a tightly regulated
process central to both nervous system development
and its repair after injury. One of the proteins shown
to play a specific role in growth cone guidance is the
collapsin response mediator protein 2 (CRMP-2) [1–3],
also known as dihydropyriminidase-related protein 2
(DRP-2, DPYSL-2), unc-33 like protein 2 (Ulip-2) and
turned on after division 64 kDa (TOAD-64).
CRMP-2 is a member of the family of collapsin
response mediator proteins, which is comprised of five
related proteins in humans [4]. The crystal structure of
human CRMP-2 has previously been determined to a
resolution of 2.4 A
˚

[5] from crystals grown in the pres-
ence of calcium ions. Structurally, CRMP-2 is homolo-
gous to the dihydropyriminidases (DHP), with a major
part of its 3D structure being formed as a (ba)
8
barrel,
but it has no characterized catalytic activity of its own,
nor have any specific small-molecule ligands for
CRMP-2 been identified. However, interactions
between CRMP-2 and other proteins, such as tubulin
[6], Sra-1 [7], Numb [8], a2-chimaerin [9] and phospho-
lipase D [10], have been described.
CRMP-2 is a homotetramer, but it can also form
heterotetramers with other members of the
CRMP ⁄ DHP structural family [11]. It is possible that
its functional mechanism in neuronal development
relates, at least partly, to its ability to bind to and
regulate other homologous proteins in the family.
Recently, CRMP-2 has been highlighted as a target for
drug development against nervous system disorders,
Keywords
divalent cations; nervous system; oligomeric
status; protein structure; small-angle X-ray
scattering
Correspondence
P. Kursula, Department of Biochemistry,
University of Oulu, P.O. Box 3000,
FIN-90014 Oulu, Finland
Fax: +358 8 5531141
Tel: +358 44 5658288

E-mail: petri.kursula@oulu.fi
Database
The coordinates and structure factors have
been deposited in the Protein Data Bank
under the accession code 2VM8
(Received 28 April 2008, revised 14 July
2008, accepted 17 July 2008)
doi:10.1111/j.1742-4658.2008.06601.x
The collapsin response mediator protein 2 (CRMP-2) is a central molecule
regulating axonal growth cone guidance. It interacts with the cytoskeleton
and mediates signals related to myelin-induced axonal growth inhibition.
CRMP-2 has also been characterized as a constituent of neurofibrillary
tangles in Alzheimer’s disease. CD spectroscopy and thermal stability
assays using the Thermofluor method indicated that Ca
2+
and Mg
2+
affect
the stability of CRMP-2 and prevent the formation of b-aggregates upon
heating. Gel filtration showed that the presence of Ca
2+
or Mg
2+
promoted the formation of CRMP-2 homotetramers, and this was further
proven by small-angle X-ray scattering experiments, where a 3D solution
structure for CRMP-2 was obtained. Previously, we described a crystal
structure of human CRMP-2 complexed with calcium. In the present study,
we determined the structure of CRMP-2 in the absence of calcium at 1.9 A
˚
resolution. When Ca

2+
was omitted, crystals could only be grown in the
presence of Mg
2+
ions. By a proteomic approach, we further identified
a number of post-translational modifications in CRMP-2 from rat brain
hippocampus and mapped them onto the crystal structure.
Abbreviations
CRMP-2, collapsin response mediator protein 2; DHP, dihydropyriminidase; SAXS, small-angle X-ray scattering.
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4583
such as epilepsy [12], Alzheimer’s disease and nerve
injury [3]. Its association with depression and schizo-
phrenia has also been studied [13,14]. CRMP-2 also
forms a part of the signal transduction cascade related
to axonal growth inhibition brought about by myelin
components, such as the myelin-associated glycopro-
tein [15], a mechanism that prevents the regeneration
of myelinated axons in the central nervous system.
Non-neuronal expression for CRMP-2 has also been
reported [16] and, recently, it has been identified as a
putative marker for colorectal carcinoma [17].
The best characterized function for CRMP-2 relates
to its interactions with cytoskeletal components, espe-
cially tubulin [6,18]. CRMP-2 is able to regulate the
formation of microtubules and, accordingly, it is
highly concentrated in growing axons. CRMP-2 binds
to tubulin heterodimers, and its overexpression in
neurons promotes axonal growth and branching [6].
Extensive post-translational modifications have been
detected for CRMP-2 [19–25]; mainly, this has con-

cerned phosphorylation of the C-terminal tail, which is
predicted to be unfolded. In addition, CRMP-2 has
been characterized as a major target for oxidation in
the aging brain [23,26–29]. Changes in CRMP-2 post-
translational modifications have also been suggested to
play a role in Alzheimer’s disease [19–21,26–29].
In the present study, we describe the crystal struc-
ture of human CRMP-2 in the absence of calcium.
Instead of calcium, magnesium ions were required to
grow the crystals, resulting in a 1.9 A
˚
structure of
CRMP-2 being obtained. The effects of Ca
2+
and
Mg
2+
on the CRMP-2 structure were also analysed by
CD spectroscopy, the Thermofluor method, small-
angle X-ray scattering (SAXS) and gel filtration. At a
20 mm concentration, both CaCl
2
and MgCl
2
stabilize
the protein and promote the formation of tetramers.
The structure was further analysed by mapping post-
translational modifications, as detected using advanced
proteomics methods [30], onto the 3D structure of the
folded core domain of CRMP-2.

Results
CD spectroscopic analysis of CRMP-2
conformation and stability in solution
To characterize the effects of divalent cations on
CRMP-2 structure and stability, CD spectroscopy was
carried out in the presence of NaCl, CaCl
2
and MgCl
2
.
The CD spectra of CRMP-2 in all tested conditions
were similar, indicating the expected presence of both
a and b secondary structures. However, in the presence
of Ca
2+
and Mg
2+
, the CD signal was significantly
stronger, suggesting a higher average content of sec-
ondary structure in solution. NaCl had no effect on
the CD spectrum (Fig. 1A).
Temperature scans of the samples revealed an
intriguing phenomenon upon denaturation. In buffer
alone, CRMP-2 underwent a structural transition at
approximately 50 °C, which resulted in an increase in
ellipticity at 220 nm (Fig. 1B); this is opposite to the
effect generally expected upon protein denaturation.
The ellipticity did not significantly decrease, even at
temperatures approaching 100 °C (data not shown). A
CD spectrum recorded at 90 °C shows that the transi-

tion involved a complete loss of helical structure and a
significant increase in the amount of b structure
(Fig. 1C). After cooling down, the spectrum at room
temperature indicated that the observed structural
transition into a b-aggregate was irreversible (data not
shown). A sample analysed in the presence of 50 mm
NaCl behaved essentially the same (data not shown).
In the presence of Ca
2+
or Mg
2+
, however, heat
denaturation proceeded as expected, with a sharp
decrease in ellipticity at 220 nm at the melting temper-
ature and complete loss of secondary structure
(Fig. 1B,C). Increasing the concentration of these ions
from 20 to 200 mm resulted in a decrease by several
degrees in the T
m
(Fig. 1B and Table 1), indicating
destabilization of CRMP-2 by a high concentration of
divalent cations.
Test for heat stability of CRMP-2 using the
Thermofluor method
A series of conditions were screened in 96-well format,
by measuring the fluorescence of SyproOrange, in
order to further characterize the stability of CRMP-2
in the presence and absence of divalent cations. The
results clearly indicate that, although 20 mm CaCl
2

and MgCl
2
stabilize the protein slightly, a 200 mm
concentration destabilizes the protein significantly
(Fig. 1D,E and Table 1). The same effect was observed
in two different buffers, phosphate and Hepes, both
adjusted to pH 7.5. The results are summarized in
Table 1.
Divalent cations promote the tetramerization
of CRMP-2
The oligomerization status of CRMP-2 was analysed
by gel filtration in the presence and absence of 20 mm
CaCl
2
and MgCl
2
. It is evident that, in the absence of
divalent cations, only approximately 50% of CRMP-2
is in the tetrameric state. The rest of CRMP-2 is in its
monomeric form. In the presence of either Ca
2+
or
Structural properties of CRMP-2 V. Majava et al.
4584 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
Mg
2+
, however, the protein is almost completely
tetrameric (Fig. 2A), with only a minor fraction of
monomer being detectable.
The structure of CRMP-2 in the absence of

calcium ions
The replacement of calcium with magnesium in
CRMP-2 crystallization gave rise to a novel ortho-
rhombic crystal form, which diffracted X-rays signifi-
cantly better than the monoclinic form previously
obtained in the presence of Ca
2+
[5]. Thus, the human
CRMP-2 structure could be refined to a resolution of
1.9 A
˚
(Fig. 2B and Table 2). As a slight drawback, a
significant pseudotranslational symmetry component
was present in the new crystal form (see below), which
lead to unusually high R factors in refinement. The
anomaly of the crystal form is given rise to by the
noncrystallographic symmetry axes present in
the CRMP-2 tetramer.
A

B

C
D E
Fig. 1. Folding and stability of CRMP-2. (A)
CD spectra for CRMP-2 in different buffers.
The spectra were measured at 23.4 °Cas
described in the Experimental procedures.
The samples are: 0 ⁄ 50 m
M NaCl (thin ⁄ thick

black line); 200 ⁄ 20 m
M CaCl
2
(thin ⁄ thick red
line); and 200 ⁄ 20 m
M MgCl
2
(thin ⁄ thick blue
line) in 10 m
M Hepes (pH 7.3). (B) Melting
curves based on the change in molar elliptic-
ity as a function of temperature. Sample
colours are as described in (A). (C) Spectra
for CRMP-2 after heating denaturation,
measured at 90 °C. The samples contain
200 m
M CaCl
2
(red), 200 mM MgCl
2
(blue),
or no additives (black) in 10 m
M Hepes
(pH 7.3). In the absence of divalent cations,
CRMP-2 forms a b-aggregate. (D) Thermo-
fluor stability assay. Eight replicate samples
are shown at a single condition with 50 m
M
phosphate buffer + 20 mM CaCl
2

. The
curves were normalized such that the maxi-
mum is 1 and the minimum is 0. (E) Super-
position of averaged Thermofluor curves
from samples under the conditions: phos-
phate buffer (thin black line); phos-
phate + 150 m
M NaCl (thick black line);
phosphate + 20 m
M CaCl
2
(thick red line);
phosphate + 200 m
M CaCl
2
(thin red
line); phosphate + 20 m
M MgCl
2
(thick
blue line); and phosphate + 200 m
M MgCl
2
(thin blue line).
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4585
As previously described [5], CRMP-2 forms a homo-
tetramer by an arrangement with 222 symmetry (i.e. a
‘dimer of dimers’) (Fig. 3B). We have also previously
suggested that this type of oligomeric assembly is

responsible for dimer formation between homodimers
of CRMP-1 and CRMP-2, resulting in a hetero-
tetramer [5]. The protein used in both the current and
previous [5] structural studies contains a His-tag,
which is disordered in the crystal structure; thus, it is
highly unlikely that the oligomeric status or stability of
CRMP-2 would be affected by the affinity tag.
Analysis of the packing
Pseudotranslation with the vector 0,0.175,0.5 (fraction
26.3%) was clearly observed in the data obtained from
the orthorhombic crystal form, indicating that a frac-
tion of the tetramers in the unit cell are related to each
other by this vector. This was confirmed by the solved
structure, in which, for each of the four tetramers
in the unit cell, there is another one related by
pseudotranslation. Pseudotranslation affects refinement
statistics by incorporating a large amount of weak
reflections and, thus, leads to the crystallographic R
factors during refinement being higher than generally
expected for the used resolution range. Taking this
into account, the observed R
cryst
and R
free
values from
this crystal form are acceptable. An analysis of the
data using rstats software [31] also confirmed that
the weak reflections in the data had systematically high
crystallographic R factors (data not shown). A better
quality indicator in such a case is the correlation

coefficient, and these values indicate that the CRMP-2
model is accurate (Table 2).
Comparison with the structure in the presence
of calcium
The high-resolution structure obtained from the ortho-
rhombic crystal form grown in the presence of Mg
2+
,
but in the absence of Ca
2+
, was compared with the
Table 1. T
m
values from CD spectroscopy and Thermofluor assays.
In both cases, the T
m
was taken as the point of steepest ascent of
the measured curve. The number of replicates for each condition in
the Thermofluor assays is given in parentheses.
Assay T
m
CD (all in 10 mM Hepes, pH 7.3)
20 m
M CaCl
2
49.5
200 m
M CaCl
2
44

20 m
M MgCl
2
48.5
200 m
M MgCl
2
45.5
Thermofluor
50 m
M Hepes (pH 7.5) 46.1 ± 0.9 (8)
150 m
M NaCl 47.6 ± 0.4 (7)
20 m
M CaCl
2
47.3 ± 0.4 (6)
200 m
M CaCl
2
40.6 ± 2.0 (6)
20 m
M MgCl
2
47.5 ± 0 (6)
200 m
M MgCl
2
44.9 ± 0.5 (7)
50 m

M NaPO
4
(pH 7.5) 46.8 ± 0.8 (6)
150 m
M NaCl 47.5 ± 0 (8)
20 m
M CaCl
2
47.5 ± 0 (8)
200 m
M CaCl
2
39.8 ± 0.8 (6)
20 m
M MgCl
2
47.5 ± 0 (6)
200 m
M MgCl
2
40.3 ± 1.1 (7)
A
B
Fig. 2. Divalent cations and the oligomeric structure of CRMP-2.
(A) Analysis of oligomeric state by size exclusion chromatography.
The samples contained either no additives (black), 20 m
M CaCl
2
(red), or 20 mM MgCl
2

(blue). The elution volumes of molecular
mass markers (in kDa) are indicated above the graph. (B) The crys-
tal structure of CRMP-2 and the locations of detected divalent
cations. Ca
2+
ions (from the previous structure) [5] are shown in
red and Mg
2+
(from the current structure) are shown in blue, and
the different subunits of CRMP-2 are colour-coded. Only the ions
bound to the two monomers in front are visible in this view.
Structural properties of CRMP-2 V. Majava et al.
4586 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
previous structure determined in the presence of Ca
2+
.
The Ca rmsd between the new structure and the pre-
vious one is 0.2–0.35 A
˚
, depending on which chains of
the tetramer are compared. The rmsd of Ca positions
for the whole tetramer is 0.32 A
˚
, as determined by ssm
[32].
The electron density clearly indicates that no large
ion is bound to the previously observed calcium site [5]
in the new crystal form. This observation further
confirms the presence of a Ca
2+

ion in the previous
structure because the main difference between the crys-
tallization conditions is the change of Ca
2+
to Mg
2+
.
The solvent environment of the new crystal structure
was analysed to identify putative Mg
2+
ions. The only
location that shows an electron density reminiscent of
hydrated Mg
2+
, as well as a suitable coordination
environment, is approximately the same site where
Ca
2+
was bound in the earlier structure, at the mouth
of the central pocket in the (ba)
8
barrel (Fig. 2B). It
should be noted that Mg
2+
is not easy to distinguish
from a water molecule based on electron density
alone because it has the same number of electrons as
oxygen.
Table 2. Crystallographic data collection and structure refinement.
Space group P2

1
2
1
2
1
Unit cell parameters 86.5, 126.2, 209.8 A
˚
Data collection
Resolution (A
˚
) 20–1.90 (2.00–1.90)
Completeness (%) 94.2 (83.7)
R
merge
(%) 12.1 (46.6)
<I ⁄ rI> 11.4 (3.8)
Redundancy 6.0 (5.5)
Structure refinement
R
cryst
(%) 25.6
R
free
(%) 32.5
rmsd bond lengths (A
˚
) 0.016
rmsd bond angles (°) 1.6
Correlation coefficient 0.915
Correlation coefficient (free) 0.847

A
B
C
Fig. 3. Small-angle X-ray scattering. (A)
Superposition of the measured scattering
curves in the presence (red) and absence
(green) of 20 m
M CaCl
2
, as well as the theo-
retical scattering curve calculated from the
crystal structure (black) using
CRYSOL. (B)
Distance distribution functions for CRMP-2
in the presence (red) and absence (green) of
CaCl
2
. (C) Ab initio models from DAMMIN
(spheres) superimposed on the crystal struc-
ture (ribbons). Red spheres indicate the
SAXS structure in the presence of Ca
2+
, and
green spheres indicate the structure in its
absence.
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4587
Considering the effects of Ca
2+
on CRMP-2 oligo-

merization and stability, the solvent shell of the earlier
CRMP-2 crystal structure (Protein Data Bank entry
2GSE) [5] was carefully re-analysed to locate any
calcium ions, as indicated by an electron density too
strong for a water molecule and a good coordination
environment for calcium, that might explain such
effects. Indeed, two novel calcium sites were identified
in each CRMP-2 monomer. These are located between
the backbone carbonyls of residues 128 and 464 and
near the side chain of Gln245, which is located at an
oligomerization interface (Fig. 2B). In the structure
determined in the presence of Mg
2+
, these sites do not
contain Mg
2+
ions.
Small-angle X-ray scattering
The oligomeric status and solution structure of
CRMP-2 were studied using synchrotron SAXS in
the presence and absence of Ca
2+
. In line with the
above results, SAXS indicated a tetrameric structure
for the sample measured in the presence of CaCl
2
(Fig. 3 and Table 3). The estimated molecular mass
of 191 kDa, based on a standard sample of BSA,
also clearly shows the predominant presence of a
tetrameric form (expected molecular mass of

220 kDa). The crystal structure could easily be fitted
into ab initio models built based on the distance
distribution function calculated from the SAXS data
(Fig. 3B,C). Similar results were also obtained in the
absence of calcium, indicating that the major form
of CRMP-2 is a tetramer also in the absence of
Ca
2+
, in the high protein concentration conditions
employed (10 mgÆmL
)1
). However, when calculating a
theoretical scattering curve based on the crystal struc-
ture, the fit between the measured data and the crystal
structure is better in the presence of calcium (Fig. 3A),
indicating a possible heterogeneity in the sample with-
out calcium. Indeed, the software oligomer could fit
the data without calcium better when given a combina-
tion of calculated scattering curves for a tetramer and a
dimer or a monomer. This was not true for the sample
containing CaCl
2
(data not shown). In line with this,
ab initio model building with dammin resulted in a
better fit to the crystal structure for the sample in
the presence of CaCl
2
, indicating the possibility of
subtle calcium-dependent movements of the subunits,
or the presence of dimeric or monomeric forms of

CRMP-2 in the sample without calcium. These
observations could also explain the success of
crystallization experiments only in the presence of Ca
2+
or Mg
2+
.
Analysis of ion binding by surface plasmon
resonance
To obtain an idea of the extent and affinity of diva-
lent cation binding by CRMP-2, we carried out a
surface plasmon resonance experiment where CRMP-
2 was immobilized and incubated in the presence of
different concentrations of MgCl
2
, CaCl
2
, BaCl
2
and
KCl (Fig. 4). The results indicate that the divalent
cations produce a similar strong response, whereas
potassium chloride only gives a weak signal. The
observed strong signal suggests the presence of many
binding sites, the binding involving hydrated ions
and ⁄ or ordering of solvent on the protein surface.
The estimated overall K
d
values for the binding of
the cations onto the CRMP-2 protein surface are

15–20 mm for the divalent cations and > 100 mm
for potassium.
The detection of several isoforms of CRMP-2
in rat hippocampus using 2D electrophoresis
and MS
When identifying all the protein spots from rat hippo-
campus on 2D gels [30], 26 spots were identified as
CRMP-2 (Fig. 5). To shed light on the post-transla-
tional modifications present in CRMP-2, all these
spots were picked, digested with trypsin and analysed
by nano-LC-ESI-MS ⁄ MS. The results are summarized
in the Supporting information (Table S1). It is
noteworthy that the deamidation of Asn356 was
detected in eight spots, and that oxidation of methio-
nines 64, 152, 168, 362, 375 and 437 was detected in at
least five spots each. We also detected phosphorylation
of Thr509 in three spots (one of these spots also
showed phosphorylation of Ser522).
Table 3. SAXS results. The calculated values were obtained from
samples of 10 mgÆmL
)1
, and the crystal structure values for R
g
,
excluded volume and D
max
are as given by CRYSOL. The theoretical
I(0) was calculated for the crystal structure based on the I(0) of
BSA. The SAXS excluded volumes are the average volumes of the
ab initio models obtained from

DAMMIN.
Sample R
g
(nm) I(0)
D
max
(nm)
Excluded
volume
(nm
3
)
Molecular
mass
(kDa)
CRMP-2 +
CaCl
2
3.65 ± 0.001 716 ± 0.4 10.5 310 191
CRMP-2 3.75 ± 0.004 708 ± 0.4 11.5 284 189
Crystal
structure
(homotetramer)
3.69 825 12.5 261 220
Structural properties of CRMP-2 V. Majava et al.
4588 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
Mapping of the post-translational modifications
onto the crystal structure of CRMP-2
A number of different post-translational modifications
were identified for the various isoforms detected for

adult rat brain hippocampal CRMP-2 (see above).
These modifications were mapped onto the 3D struc-
ture of human CRMP-2 (Fig. 6). The sequence identity
between human and rat CRMP-2 is very high, with
only seven residues out of 572 being nonconserved,
and all the detected modification sites being conserved.
All the post-translational modifications are located
outside the core of the (ba)
8
barrel fold, mainly in
looplike structures on the protein surface. The sites are
concentrated on specific regions in 3D space, especially
when considering the sites that were detected in several
spots of the 2D gel (see Discussion).
Discussion
The effects of divalent cations on the stability
and oligomeric status of CRMP-2
CRMP-2 forms homotetramers [5,11] and, in the present
study, these assemblies are demonstrated to be stabilized
A
B
Fig. 4. Surface plasmon resonance analysis of ion binding by
CRMP-2. (A) Kinetic analysis of Ca
2+
binding by immobilized
CRMP-2. For clarity, only injections with 1, 5, 10 and 50 m
M CaCl
2
are shown. (B) A comparison of the responses obtained with differ-
ent ions at 20 m

M concentration. Ca
2+
, red; Mg
2+
, blue; Ba
2+
, light
blue; K
+
, orange.
Fig. 5. 2D gel electrophoretic analysis of rat brain hippocampal
proteins; spots identified as CRMP-2 are highlighted. The details on
the selected spots are given in Table S1. In the inset, the region
containing the CRMP-2 spots is shown in more detail.
Fig. 6. Mapping of post-translational modifications onto the 3D
structure of CRMP-2. In this stereo view, the major oxidation sites
are shown in magenta, minor oxidation sites are shown in yellow,
major deamidation sites are shown in green, and minor deamida-
tion sites are shown in blue.
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4589
by divalent cations. The heat stability of CRMP-2 was
shown, by two independent methods, to decrease signifi-
cantly when the divalent cation concentration was raised
from 20 to 200 mm. This indicates that the effect on
CRMP-2 stability is dual: at low concentration, both
Ca
2+
and Mg
2+

stabilize the protein, whereas, at higher
concentrations, the effect is the opposite.
Divalent cations have previously been shown to
similarly affect protein stability in a concentration-
dependent manner [33–40]. For example, reported
examples include glucose oxidase [35], RNase T1 [36]
and papain [34], and the results can often be explained
by two different modes of binding of divalent cations
to proteins. At lower concentrations, the hydrated ions
act mainly to order and stabilize the solvent shell
around the protein molecule. At a higher concen-
tration, the ions will bind directly to the protein
surface, resulting in destabilization.
The structure of CRMP-2
The crystal structure of human CRMP-2 was refined at
1.9 A
˚
resolution, and the overall structure is very simi-
lar to the earlier lower resolution structure obtained in
the presence of calcium [5], also demonstrating the same
oligomeric assembly into a homotetramer. We have also
shown by SAXS studies that the homotetrameric struc-
ture of CRMP-2 is maintained in solution, especially in
the presence of calcium. The good fit between our
experimental solution and crystal structures indicates
that the crystal structure is an accurate representation
of the CRMP-2 tetramer in solution. Moreover, the
conformation obtained by ab initio modelling resembles
the crystal structure more closely in the presence of
calcium than in its absence. This hints at the possibility

of subtle conformational changes within the tetramer
that can be induced by divalent cations. In principle, in
the presence of calcium, the CRMP-2 tetramer appears
to be slightly more compact than in its absence, as
demonstrated by the smaller R
g
and D
max
values for the
Ca
2+
-containing sample in conjunction with the I(0)
and excluded volumes, which are higher (Table 3).
These differences could also be caused, at least partly,
by the presence of small amounts of dimeric CRMP-2
in the absence of calcium.
Using CD spectroscopy, we have shown that
CRMP-2 undergoes a transition to a b-aggregate upon
heating in the absence of divalent cations. Interest-
ingly, CRMP-2 has been characterized as a constituent
of the paired helical filaments of neurofibrillary tangles
in Alzheimer’s disease [41,42]. Future studies should
aim to investigate any possible relationships between
the folding and denaturation behaviour of CRMP-2
and neuronal fibril formation in Alzheimer’s disease
brains.
Post-translational modifications and previously
characterized mutations of CRMP-2
A number of post-translational modifications were
detected in CRMP-2 from rat brain, including several

sites of deamidation, oxidation and phosphorylation
(Table S1). Although both of the phosphorylation sites
that were detected (i.e. Thr509 and Ser522) were in the
C-terminal tail region, which is predicted to be struc-
turally disordered, most of the other modifications
could be mapped onto the 3D structure. Thr509 and
Ser522 have been both characterized as major phos-
phorylation sites in CRMP-2 [9,42]. The reason why
some of the other previously characterized phosphory-
lation sites on the C-terminal tail were not detected
could be due to the fact that hippocampal tissue from
an adult animal was used. This aspect, however, was
not investigated further in the present study because
we were specifically interested in the post-translational
modifications that could be mapped onto the folded
domain of CRMP-2. Previously, modifications of
residues within the folded region have not been charac-
terized in detail, except for the detection of one phos-
phorylation site at Ser465 [43].
Three ‘hotspot’ regions for post-translational modifi-
cations can be visualized in the 3D structure of CRMP-
2 (Fig. 6). The first concerns the main deamidation site
and four of the main oxidation sites. The second one
contains the remaining two main oxidation sites and
two minor deamidation sites, and the third one a minor
deamidation site and three minor oxidation sites. The
reasons for the concentration of the modifications to
these areas in 3D space are currently not known, but
they could include the accessibility of these areas
towards modifications, their mobility and the relation

of such modifications towards CRMP-2 function.
In light of the finding that CRMP-2 is one of the
neuronal proteins that accumulate high levels of iso-
aspartate [23], it is interesting to observe a number
of deamidated asparagine residues in CRMP-2. The
formation of isoaspartate is an important source of
protein damage under physiological conditions, and is
linked to the deamidation of Asn residues. The deami-
dation of Asn356, detected in eight of the 26 character-
ized CRMP-2 spots, could be related to isoaspartate
accumulation on brain CRMP-2.
The e204 mutation in the Caenorhabditis elegans
unc-33 gene [44] results in a protein where a conserved
aspartate residue is mutated into an asparagine; the
corresponding residue in CRMP-2 is Asp71 [44]. In a
Structural properties of CRMP-2 V. Majava et al.
4590 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
yeast two-hybrid assay, the mutation prevented unc-33
oligomerization [45]. The mutant was originally
isolated by identifying its involvement in paralysis
resulting from defective axon growth [44], and similar
effects on axonal growth have subsequently been
found with the corresponding D71N mutant of
CRMP-2 [7]. In the 3D structure, Asp71 is buried
within the folded protein, close to the interface
between the small and large lobes of CRMP-2, but far
from the tetramerization interfaces. Its side chain is
buried, being close to that of Arg361, but no hydrogen
bond ⁄ salt bridge is formed between the two side
chains. These two buried charges apparently neutralize

each other and, in the D71N mutant, the charged
Arg361 is expected to have an isolated buried charge,
which may destabilize the protein.
Conclusions
CRMP-2 is a central regulator of axonal growth cone
guidance, and its function is likely to involve both
homo- and heterotetramerization, as well as post-trans-
lational modifications. Using a number of biochemical
and biophysical methods, we have gained a more
detailed view than was previously available for the
structure and properties of the CRMP-2 protein, both
in solution and in the crystal state. Divalent cations
have a drastic effect on both the stability and oligo-
meric state of CRMP-2, and a number of different
post-translationally modified isoforms of CRMP-2
could be identified in the brain. Our data, including
the mapping of post-translational modifications onto
the structure of CRMP-2, open up new possibilities for
studying the function and interactions of CRMP-2,
which still remain enigmatic.
Experimental procedures
Protein purification
The purification of human CRMP-2 has been described
previously [5]. The protein batches for the present study
were obtained from the SGC Stockholm laboratory. A con-
struct containing residues 1–490 was used for the surface
plasmon resonance experiments, and other experiments
were carried out with a construct containing residues
13–490 of CRMP-2. The His tags were not removed prior
to any experiments.

CD spectroscopy
CRMP-2 was extensively dialysed against 10 mm Hepes
(pH 7.3); subsequent dilutions were made in the dialysis
buffer, which was also used for the measurement of a
buffer control by CD. CD spectra were measured in the
wavelength range 195–250 nm, on a Jasco J-810 spectro-
polarimeter (Jasco, Tokyo, Japan). The protein concen-
tration was 3 lm, and a 1 mm cuvette was used. After
measuring each spectrum at 23.4 °C, a temperature scan at
a fixed wavelength of 220 nm was run between 30 and
70 °C to obtain a melting curve. After the temperature
scan, CD spectra were further recorded for the samples,
both at 90 °C and after cooling back to room temperature.
The effects of the following additives on the behaviour of
CRMP-2 were studied: 20 or 200 mm CaCl
2
, 20 or 200 mm
MgCl
2
, and 50 mm NaCl. CD spectra and a temperature
scan were recorded using exactly the same parameters and
procedures for all samples. Of note, the sample gained a
gel-like appearance upon heating in the absence of divalent
cations, but not in their presence.
Stability analysis by the Thermofluor method
The thermal stability of CRMP-2 was analysed in 96-well
format by following the fluorescence from SYPRO Orange
(Invitrogen, Carlsbad, CA, USA) as a function of tempera-
ture (i.e. the so-called Thermofluor or thermal shift assay
method) [46,47]. The experiment was carried out using a

7500 Real Time PCR System apparatus (Applied Biosys-
tems, Foster City, CA, USA) and the temperature was
scanned from 20 to 90 °C with 1 °C increments, with moni-
toring of fluorescence at 542 nm. Eight replicates each from
12 different conditions were randomly placed on the 96-well
PCR plate. Occasionally, curves with abnormal shapes were
observed; such curves were excluded from the analysis,
most likely resulting from incomplete sealing of an individ-
ual well on the 96-well PCR plate.
Size exclusion chromatography
The oligomeric status of CRMP-2 was analysed by gel fil-
tration on a Superdex 200 HR 10 ⁄ 30 column coupled to an
A
¨
KTApurifier (GE Healthcare, Uppsala, Sweden). An iden-
tical sample (0.67 mgÆmL
)1
of 200 lL) was run with the
same protocol in 10 mm Hepes (pH 7.5), 100 mm NaCl,
and in the same buffer containing either 20 mm CaCl
2
or
20 mm MgCl
2
. Sample elution was followed at 280 nm.
Molecular masses were estimated by comparing the sample
elution volumes with those observed for standard proteins
run on the same column.
Crystallization, data collection and structure
solution

Human CRMP-2 was crystallized essentially as described
previously [5], while simultaneously screening for substitutes
for CaCl
2
that could promote crystallization. In the absence
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4591
of CaCl
2
, the only tested additive producing crystals was
MgCl
2
. Crystals (crystal form 1) without CaCl
2
were grown
at 20 °C in hanging drops over a well solution consisting of
15% poly(ethylene glycol) 10 000, 0.1 m Tris (pH 9), 0.2 m
MgCl
2
and 10 mm MgI
2
. Another crystal form (crystal
form 2) was similarly grown in the absence of MgI
2
(pH 8.5).
Prior to data collection, the crystals were briefly soaked
in a cryoprotectant solution (well solution supplemented
with 20% glycerol) and cooled to 100 K in a stream of
gaseous nitrogen. Diffraction data were collected at MAX-
Lab (Lund, Sweden) beamline I911-2 [48]. Data processing

was performed using xds [49] and xdsi [50]. An analysis
of the data revealed the presence of pseudotranslational
symmetry in crystal form 1 (26.3%, fractional translation
0,0.175,0.5). However, space group determination for
crystal form 2 was not straightforward, and this mono-
clinic crystal was found to be pseudomerohedrally
twinned; for this reason, crystal form 2 was discarded
from the analysis.
The previously determined structure of human CRMP-2
[5] was used as a template in molecular replacement. Four
monomers of CRMP-2 were found within the asymmetric
unit using molrep [51], and refinement and model building
were carried out iteratively using refmac [52], phenix.
refine [53,54] and coot [55]. The coordinates and structure
factors were deposited in the Protein Data Bank under the
accession code 2VM8.
Surface plasmon resonance
The binding of Ca
2+
,Mg
2+
,Ba
2+
and K
+
by CRMP-2
was analysed by surface plasmon resonance on a Biacore
3000 apparatus (Biacore AB, Uppsala, Sweden) by immo-
bilizing CRMP-2 on a CM5 chip and passing 0, 1, 5, 10,
20, 50 and 100 mm solutions of CaCl

2
, MgCl
2
, BaCl
2
and KCl over the chip. In the immobilization, 10 mm
socium acetate buffer (pH 4.5) was used. During the
binding experiment, the running buffer contained 10 mm
Hepes (pH 7.5), 100 mm NaCl and 0.004% surfactant
P20 (Biacore AB), in addition to the salts being tested.
The experiments were carried out at 25 °C, with a flow
rate of 30 lLÆmin
)1
. For regeneration of the surface
between injections, the ions were allowed to dissociate
freely into the binding buffer. A control channel on
the chip was similarly treated, with the exception that no
protein was immobilized onto it. The data were fitted
against a 1 : 1 binding model using biaevaluation
software (Biacore AB).
Small-angle X-ray scattering
For SAXS, CRMP-2 was dialysed into a buffer contain-
ing 10 mm Hepes (pH 7.5) and 100 mm NaCl. SAXS
data for CRMP-2 in the presence and absence of 20 mm
CaCl
2
, at concentrations in the range 1–10 mgÆmL
)1
, were
measured on the EMBL Hamburg ⁄ DESY beamline X33,

and the corresponding buffer was always used for a
blank experiment. Programs from the atsas software
package [56] were used for data analysis, essentially as
described previously [57]. The measured data were further
processed using primus [58]. The molecular mass was
estimated by comparing the forward scattering I(0) with
that of a standard solution of BSA. The distance distri-
butions were obtained using gnom [59] and further used
for ab initio modelling in dammin [60]. An averaged
model was generated from several runs using damaver
[61], and the SAXS model and the crystal structure were
superimposed with supcomb [62]. The possible oligomeric
assemblies were also studied using oligomer [58], after
evaluating the solution scattering of each possible
component using crysol [63].
2D electrophoresis and MS
Materials
Immobilized pH gradient strips and buffers were purchased
from Amersham Biosciences, a part of GE Healthcare (Mil-
waukee, WI, USA). Reagents for polyacrylamide gel prepa-
ration were purchased from Bio-Rad Laboratories
(Hercules, CA, USA). Chaps was obtained from Roche
Diagnostics (Mannheim, Germany), urea was obtained
from AppliChem (Darmstadt, Germany), thiourea was ob-
tained from Fluka (Buchs, Switzerland), 1,4-dithioerythritol
and EDTA were obtained from Merck (Darmstadt,
Germany) and tributylphosphine was obtained from Pierce
Biotechnology (Rockford, IL, USA).
Sample preparation
Twenty-three-month-old rat hippocampus tissue was pow-

derized in liquid nitrogen and suspended in 2 mL of sam-
ple buffer [20 m m Tris, 7 m urea, 2 m thiourea, 4%
Chaps, 10 mm 1,4-dithioerythritol, 1 mm EDTA and
1mm phenylmethanesulfonyl fluoride, containing 1 tablet
CompleteÔ (Roche Diagnostics) and 0.2% (v ⁄ v) phos-
phatase inhibitor cocktail (Calbiochem, San Diego, CA,
USA)]. The suspension was sonicated for approximately
30 s and centrifuged at 15 000 g for 60 min at 12 °C.
Desalting was performed with an Ultrafree-4 centrifugal
filter unit (Millipore, Bedford, MA, USA), with a molec-
ular mass cut-off of 10 kDa. The protein concentration
of the supernatant was determined by the Bradford
assay.
2D gel electrophoresis
Samples of 750 lg of protein were applied on immobilized
nonlinear pH gradient (pH 3–10) strips. Focusing started at
Structural properties of CRMP-2 V. Majava et al.
4592 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
200 V, and the voltage was gradually increased to 8000 V
at 4 VÆmin
)1
and kept constant for a further 3 h (approxi-
mately 150 000 Vh in total). Separation in the second
dimension was performed on a 10–16% gradient SDS ⁄
PAGE. After protein fixation for 12 h in 50% methanol
and 10% acetic acid, the gels were stained with colloidal
Coomassie blue (Novex, San Diego, CA, USA) for 8 h,
and the excess of dye was removed with distilled water.
Molecular masses were determined by running standard
protein markers (Bio-Rad Laboratories), covering the range

10–250 kDa. pI values were used as provided by the
supplier of the immobilized pH gradient strips.
In-gel digestion
Spots of interest were excised and washed sequentially
with 10 mm ammonium bicarbonate and 50% acetonitrile
in 10 mm ammonium bicarbonate. After washing, the gel
plugs were shrunk by the addition of acetonitrile and
dried in a SpeedVac (Eppendorf, Hamburg, Germany).
The dried gel pieces were reswollen with 40 ngÆl L
)1
tryp-
sin (sequencing grade; Promega, Madison, WI, USA) in
digestion buffer (5 mm octyl b-d-glucopyranoside and
10 mm ammonium bicarbonate) and incubated for 4 h at
30 °C. Extraction was performed first with 10 lLof1%
trifluoroacetic acid in 5 mm octyl b-d-glucopyranoside,
and then using 10 lL of 0.1% trifluoroacetic acid, 4%
acetonitrile. Both peptide extracts were pooled and
concentrated in a SpeedVac until the volume reached
8 lL.
Protein identification and characterization by
nano-LC-ESI-MS ⁄ MS
Six microlitres of the extracted sample were used for
nanoLC-ESI-MS ⁄ MS investigation. The HPLC used
comprised an Ultimate 3000 system (Dionex Corporation;
Sunnyvale, CA, USA) equipped with a PepMap C-18
analytic column (75 lm · 150 mm). The gradient was
(A = 0.1% formic acid in water, B = 80% aceto-
nitrile ⁄ 0.08% formic acid in water) 4% B to 60% B from 0
to 30 min, 90% B from 30 to 35 min, and 4% B from 35

to 60 min. Peptide spectra were recorded over the mass
range of m ⁄ z 350–1600, and MS ⁄ MS spectra were recorded
in an information dependent data acquisition over the mass
range of m ⁄ z 50–1600. Repeatedly, one MS spectrum was
recorded followed by two MS ⁄ MS spectra on the QSTAR
XL instrument (Applied Biosystems); the accumulation time
was 1 s for MS spectra and 2 s for MS ⁄ MS spectra. The
collision energy was automatically set, according to the
mass and charge state of the peptides chosen for fragmenta-
tion. Doubly or triply charged peptides were chosen for
MS ⁄ MS experiments due to their good fragmentation char-
acteristics. MS ⁄ MS spectra were interpreted using mascot
software (Matrix Science Ltd, London, UK) and searched
against the SwissProt 51.0 database to identify the protein
spot. The search parameters were set: a mass tolerance of
500 p.p.m. for MS tolerance, 0.2 Da for MS ⁄ MS tolerance,
one missing cleavage site, fixed modification of carbamido-
methyl, and variable modification of methionine oxidation.
Positive protein identifications were based on a significant
Mowse score [64]. After the protein was identified, an
error-tolerant search was performed to detect unspecific
cleavage and unassigned modifications. Protein identifica-
tion and modification returned from mascot were manually
examined and filtered to create a confirmed protein identifi-
cation and modification list.
Acknowledgements
P. K. is an Academy Research Fellow (Academy of
Finland). The work was supported by grants from the
Finnish MS Foundation and the Department of Bio-
chemistry, Oulu University (V. M.). The support of

Ylva Lindqvist and Gunter Schneider in the early
stages of this work and enlightening discussions with
Inari Kursula and Maxim Petoukhov are gratefully
acknowledged. We thank the Stockholm node of the
SGC for providing materials for this study and the
beamline staff at EMBL Hamburg and MAX-Lab for
enabling smooth data collection. The measurement of
synchrotron SAXS data at EMBL Hamburg and the
crystallographic data collection at MAX-Lab were
both supported by the European Community –
Research Infrastructure Action under the FP6
‘Structuring the European Research Area’ Programme
(through the Integrated Infrastructure Initiative
‘Integrating Activity on Synchrotron and Free Elec-
tron Laser Science’), contract RII3-CT-2004-506008
(IA-SFS).
References
1 Kamata T, Subleski M, Hara Y, Yuhki N, Kung H,
Copeland NG, Jenkins NA, Yoshimura T, Modi W &
Copeland TD (1998) Isolation and characterization of a
bovine neural specific protein (CRMP-2) cDNA homol-
ogous to unc-33, a C. elegans gene implicated in axonal
outgrowth and guidance. Brain Res Mol Brain Res 54,
219–236.
2 Schmidt EF & Strittmatter SM (2007) The CRMP
family of proteins and their role in Sema3A signaling.
Adv Exp Med Biol 600, 1–11.
3 Yoshimura T, Arimura N & Kaibuchi K (2006) Molec-
ular mechanisms of axon specification and neuronal
disorders. Ann NY Acad Sci 1086, 116–125.

4 Charrier E, Reibel S, Rogemond V, Aguera M, Thom-
asset N & Honnorat J (2003) Collapsin response media-
tor proteins (CRMPs): involvement in nervous system
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4593
development and adult neurodegenerative disorders.
Mol Neurobiol 28, 51–64.
5 Stenmark P, Ogg D, Flodin S, Flores A, Kotenyova
T, Nyman T, Nordlund P & Kursula P (2007) The
structure of human collapsin response mediator pro-
tein 2, a regulator of axonal growth. J Neurochem
101, 906–917.
6 Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura
T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A,
Hotani H et al. (2002) CRMP-2 binds to tubulin hete-
rodimers to promote microtubule assembly. Nat Cell
Biol 4, 583–591.
7 Kawano Y, Yoshimura T, Tsuboi D, Kawabata S,
Kaneko-Kawano T, Shirataki H, Takenawa T &
Kaibuchi K (2005) CRMP-2 is involved in kinesin-1-
dependent transport of the Sra-1 ⁄ WAVE1 complex and
axon formation. Mol Cell Biol 25, 9920–9935.
8 Nishimura T, Fukata Y, Kato K, Yamaguchi T,
Matsuura Y, Kamiguchi H & Kaibuchi K (2003)
CRMP-2 regulates polarized Numb-mediated endo-
cytosis for axon growth. Nat Cell Biol 5, 819–826.
9 Brown M, Jacobs T, Eickholt B, Ferrari G, Teo M,
Monfries C, Qi RZ, Leung T, Lim L & Hall C (2004)
Alpha2-chimaerin, cyclin-dependent kinase 5 ⁄ p35, and
its target collapsin response mediator protein-2 are

essential components in semaphorin 3A-induced
growth-cone collapse. J Neurosci 24, 8994–9004.
10 Lee S, Kim JH, Lee CS, Kim JH, Kim Y, Heo K, Ihara
Y, Goshima Y, Suh PG & Ryu SH (2002) Collapsin
response mediator protein-2 inhibits neuronal phospho-
lipase D(2) activity by direct interaction. J Biol Chem
277, 6542–6549.
11 Wang LH & Strittmatter SM (1997) Brain CRMP
forms heterotetramers similar to liver dihydropyrimidin-
ase. J Neurochem 69, 2261–2269.
12 Beyreuther BK, Freitag J, Heers C, Krebsfanger N,
Scharfenecker U & Stohr T (2007) Lacosamide: a
review of preclinical properties. CNS Drug Rev 13,
21–42.
13 Bisgaard CF, Jayatissa MN, Enghild JJ, Sanchez C,
Artemychyn R & Wiborg O (2007) Proteomic investiga-
tion of the ventral rat hippocampus links DRP-2 to
escitalopram treatment resistance and SNAP to stress
resilience in the chronic mild stress model of depression.
J Mol Neurosci 32, 132–144.
14 Nakata K, Ujike H, Sakai A, Takaki M, Imamura T,
Tanaka Y & Kuroda S (2003) The human dihydro-
pyrimidinase-related protein 2 gene on chromosome
8p21 is associated with paranoid-type schizophrenia.
Biol Psychiatry 53, 571–576.
15 Mimura F, Yamagishi S, Arimura N, Fujitani M,
Kubo T, Kaibuchi K & Yamashita T (2006) Myelin-
associated glycoprotein inhibits microtubule assembly
by a Rho-kinase-dependent mechanism. J Biol Chem
281, 15970–15979.

16 Tahimic CG, Tomimatsu N, Nishigaki R, Fukuhara A,
Toda T, Kaibuchi K, Shiota G, Oshimura M & Kuri-
masa A (2006) Evidence for a role of collapsin response
mediator protein-2 in signaling pathways that regulate
the proliferation of non-neuronal cells. Biochem Biophys
Res Commun 340, 1244–1250.
17 Wu CC, Chen HC, Chen SJ, Liu HP, Hsieh YY,
Yu CJ, Tang R, Hsieh LL, Yu JS & Chang YS (2008)
Identification of collapsin response mediator protein-2
as a potential marker of colorectal carcinoma by com-
parative analysis of cancer cell secretomes. Proteomics
8, 316–332.
18 Kimura T, Watanabe H, Iwamatsu A & Kaibuchi K
(2005) Tubulin and CRMP-2 complex is transported via
kinesin-1. J Neurochem 93, 1371–1382.
19 Kanninen K, Goldsteins G, Auriola S, Alafuzoff I &
Koistinaho J (2004) Glycosylation changes in Alzhei-
mer’s disease as revealed by a proteomic approach.
Neurosci Lett 367, 235–240.
20 Petratos S, Li QX, George AJ, Hou X, Kerr ML,
Unabia SE, Hatzinisiriou I, Maksel D, Aguilar MI &
Small DH (2008) The b-amyloid protein of Alzheimer’s
disease increases neuronal CRMP-2 phosphorylation by
a Rho-GTP mechanism. Brain 131, 90–108.
21 Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S,
Yamashita N, Nakamura F, Takei K, Ihara Y,
Mikoshiba K et al. (2005) Semaphorin3A signalling
is mediated via sequential Cdk5 and GSK3beta
phosphorylation of CRMP2: implication of common
phosphorylating mechanism underlying axon guidance

and Alzheimer’s disease. Genes Cells 10, 165–179.
22 Yoshimura T, Kawano Y, Arimura N, Kawabata S,
Kikuchi A & Kaibuchi K (2005) GSK-3beta regulates
phosphorylation of CRMP-2 and neuronal polarity.
Cell 120, 137–149.
23 Zhu JX, Doyle HA, Mamula MJ & Aswad DW
(2006) Protein repair in the brain, proteomic analysis
of endogenous substrates for protein L-isoaspartyl
methyltransferase in mouse brain. J Biol Chem 281,
33802–33813.
24 Arimura N, Inagaki N, Chihara K, Menager C,
Nakamura N, Amano M, Iwamatsu A, Goshima Y &
Kaibuchi K (2000) Phosphorylation of collapsin
response mediator protein-2 by Rho-kinase. Evidence
for two separate signaling pathways for growth cone
collapse. J Biol Chem 275, 23973–23980.
25 Arimura N, Menager C, Kawano Y, Yoshimura T,
Kawabata S, Hattori A, Fukata Y, Amano M,
Goshima Y, Inagaki M et al. (2005) Phosphorylation
by Rho kinase regulates CRMP-2 activity in growth
cones. Mol Cell Biol 25, 9973–9984.
26 Castegna A, Aksenov M, Thongboonkerd V, Klein JB,
Pierce WM, Booze R, Markesbery WR & Butterfield
DA (2002) Proteomic identification of oxidatively
modified proteins in Alzheimer’s disease brain. Part II:
Structural properties of CRMP-2 V. Majava et al.
4594 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS
dihydropyrimidinase-related protein 2, alpha-enolase
and heat shock cognate 71. J Neurochem 82, 1524–1532.
27 Choi J, Forster MJ, McDonald SR, Weintraub ST,

Carroll CA & Gracy RW (2004) Proteomic identifica-
tion of specific oxidized proteins in ApoE-knockout
mice: relevance to Alzheimer’s disease. Free Radic Biol
Med 36, 1155–1162.
28 Pamplona R, Dalfo E, Ayala V, Bellmunt MJ, Prat J,
Ferrer I & Portero-Otin M (2005) Proteins in human
brain cortex are modified by oxidation, glycoxidation,
and lipoxidation. Effects of Alzheimer disease and
identification of lipoxidation targets. J Biol Chem 280,
21522–21530.
29 Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce
WM, Klein JB, Merchant M, Markesbery WR &
Butterfield DA (2006) Redox proteomics identification
of oxidized proteins in Alzheimer’s disease hippocampus
and cerebellum: an approach to understand pathologi-
cal and biochemical alterations in AD. Neurobiol Aging
27, 1564–1576.
30 Chen WQ, Kang SU & Lubec G (2006) Protein pro-
filing by the combination of two independent mass
spectrometry techniques. Nat Protoc 1, 1446–1452.
31 Collaborative Computational Project, Number 4 (1994)
The CCP4 suite: programs for protein crystallography.
Acta Crystallogr D Biol Crystallogr 50, 760–763.
32 Krissinel E & Henrick K (2004) Secondary-structure
matching (SSM), a new tool for fast protein structure
alignment in three dimensions. Acta Crystallogr D Biol
Crystallogr 60, 2256–2268.
33 Xu X, Chen J, Zhang L, Wang S, Shen D & Liu Q
(2007) Calcium ion-induced stabilization and refolding
of agkisacutacin from Agkistrodon acutus venom stud-

ied by fluorescent spectroscopy. J Fluoresc 17, 215–
221.
34 Kaul P, Sathish HA & Prakash V (2002) Effect of metal
ions on structure and activity of papain from Carica
papaya. Nahrung 46, 2–6.
35 Akhtar MS, Ahmad A & Bhakuni V (2002) Divalent
cation induced changes in structural properties of the
dimeric enzyme glucose oxidase: dual effect of dimer
stabilization and dissociation with loss of cooperative
interactions in enzyme monomer. Biochemistry 41,
7142–7149.
36 Deswarte J, De Vos S, Langhorst U, Steyaert J & Loris
R (2001) The contribution of metal ions to the confor-
mational stability of ribonuclease T1: crystal versus
solution. Eur J Biochem 268, 3993–4000.
37 Gotwals PJ, Chi-Rosso G, Ryan ST, Sizing I, Zafari
M, Benjamin C, Singh J, Venyaminov SY, Pepinsky RB
& Koteliansky V (1999) Divalent cations stabilize the
alpha 1 beta 1 integrin I domain. Biochemistry 38,
8280–8288.
38 Ebel C, Faou P, Kernel B & Zaccai G (1999) Relative
role of anions and cations in the stabilization of
halophilic malate dehydrogenase. Biochemistry 38,
9039–9047.
39 Rozhkov SP (1997) Stabilization of albumin by
CaCl
2
- and MgCl
2
-regulated interaction of macromol-

ecules: a study by the spin label method. Biofizika 42,
1020–1028.
40 Coolbear T, Whittaker JM & Daniel RM (1992) The
effect of metal ions on the activity and thermostability
of the extracellular proteinase from a thermophilic
Bacillus, strain EA.1. Biochem J 287, 367–374.
41 Yoshida H, Watanabe A & Ihara Y (1998) Collapsin
response mediator protein-2 is associated with neuro-
fibrillary tangles in Alzheimer’s disease. J Biol Chem
273, 9761–9768.
42 Gu Y, Hamajima N & Ihara Y (2000) Neurofibrillary
tangle-associated collapsin response mediator protein-2
(CRMP-2) is highly phosphorylated on Thr-509, Ser-
518, and Ser-522. Biochemistry 39, 4267–4275.
43 Vosseller K, Hansen KC, Chalkley RJ, Trinidad JC,
Wells L, Hart GW & Burlingame AL (2005) Quantita-
tive analysis of both protein expression and serine ⁄ thre-
onine post-translational modifications through stable
isotope labeling with dithiothreitol. Proteomics 5,
388–398.
44 Brenner S (1974) The genetics of Caenorhabditis elegans.
Genetics 77, 71–94.
45 Tsuboi D, Hikita T, Qadota H, Amano M &
Kaibuchi K (2005) Regulatory machinery of UNC-33
Ce-CRMP localization in neurites during neuronal
development in Caenorhabditis elegans. J Neurochem
95, 1629–1641.
46 Cummings MD, Farnum MA & Nelen MI (2006)
Universal screening methods and applications of
ThermoFluor. J Biomol Screen 11, 854–863.

47 Ericsson UB, Hallberg BM, Detitta GT, Dekker N &
Nordlund P (2006) Thermofluor-based high-throughput
stability optimization of proteins for structural studies.
Anal Biochem 357, 289–298.
48 Mammen CB, Ursby T, Thunnissen M & Als-Nielsen J
(2004) Bent diamond crystals and multilayer based
optics at the new 5-station protein crystallography
beamline ‘Cassiopeia’ at MAX-lab. AIP Conf Proc 705,
808–811.
49 Kabsch W & Sander C (1983) Dictionary of protein
secondary structure: pattern recognition of hydrogen-
bonded and geometrical features. Biopolymers 22, 2577–
2637.
50 Kursula P (2004) XDSi – a graphical interface for the
data processing program XDS. J Appl Crystallogr 37,
347–348.
51 Vagin A & Teplyakov A (1997) MOLREP: an auto-
mated program for molecular replacement. J Appl
Crystallogr 30, 1022–1025.
52 Murshudov GN, Vagin AA & Dodson EJ (1997)
Refinement of macromolecular structures by the
V. Majava et al. Structural properties of CRMP-2
FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4595
maximum-likelihood method. Acta Crystallogr D Biol
Crystallogr 53, 240–255.
53 Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger
TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini
JC, Sauter NK & Terwilliger TC (2002) PHENIX:
building new software for automated crystallographic
structure determination. Acta Crystallogr D Biol

Crystallogr 58, 1948–1954.
54 Afonine PV, Grosse-Kunstleve RW & Adams PD
(2005) A robust bulk-solvent correction and anisotropic
scaling procedure. Acta Crystallogr D Biol Crystallogr
61, 850–855.
55 Emsley P & Cowtan K (2004) Coot: model-building
tools for molecular graphics. Acta Crystallogr D Biol
Crystallogr 60, 2126–2132.
56 Konarev PV, Petoukhov MV, Volkov VV & Svergun
DI (2006) ATSAS 2.1, a program package for small-
angle scattering data analysis. J Appl Crystallogr 39,
277–286.
57 Majava V, Petoukhov M, Hayashi N, Pirila
¨
P, Svergun
D & Kursula P (2008) Interaction between the C-termi-
nal region of human myelin basic protein and calmodu-
lin: analysis of complex formation and solution
structure. BMC Struct Biol 8, 10.
58 Konarev P, Volkov V, Sokolova A, Koch M & Svergun
D (2003) PRIMUS – a Windows-PC based system for
small-angle scattering data analysis. J Appl Crystallogr
36, 1277–1282.
59 Svergun D (1992) Determination of the regularization
parameter in indirect-transform methods using percep-
tual criteria. J Appl Crystallogr 25, 495–503.
60 Svergun DI (1999) Restoring low resolution structure
of biological macromolecules from solution
scattering using simulated annealing. Biophys J 76,
2879–2886.

61 Volkov V & Svergun D (2003) Uniqueness of ab initio
shape determination in small angle scattering. J Appl
Crystallogr 36, 860–864.
62 Kozin M & Svergun D (2001) Automated matching of
high- and low-resolution structural models. J Appl
Crystallogr 34, 33–41.
63 Svergun D, Barberato C & Koch M (1995) CRYSOL –
a program to evaluate X-ray solution scattering of
biological macromolecules from atomic coordinates.
J Appl Crystallogr 28, 768–773.
64 Pappin DJ, Hojrup P & Bleasby AJ (1993) Rapid iden-
tification of proteins by peptide-mass fingerprinting.
Curr Biol 3, 327–332.
Supporting information
The following supplementary material is available:
Table S1. Identification and characterization of rat
CRMP2 (DPYL2_rat) by MS ⁄ MS spectrum.
This supplementary material can be found in the
online version of this article.
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
material supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Structural properties of CRMP-2 V. Majava et al.
4596 FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS