Báo cáo khoa học: Solution structure and internal dynamics of NSCP, a compact calcium-binding protein doc

Solution structure and internal dynamics of NSCP,
a compact calcium-binding protein
Ghada Rabah
1
, Razvan Popescu
1
, Jos A. Cox
2
, Yves Engelborghs
3
and Constantin T. Craescu
1
1 INSERM & Institut Curie, Centre Universitaire, Orsay, France
2De
´
partement de Biochimie, Universite
´
de Gene
`
ve, Suisse
3 Katholieke Universiteit, Leuven, Belgium
Calcium is a universal cellular secondary messenger
involved in the regulation of a large variety of vital
processes from the fertilization and development of a
cell to its death [1]. Its great versatility is mainly due
to multiple specific interactions within various mole-
cular networks, the precise response being finally deter-
mined by the local ion concentration as well as its time
and spatial evolution. Some proteins (such as enzymes
and chaperones) bind Ca
2+

and change their own bio-
logical activity, whereas others convey the Ca
2+
signal
through a functional change in another (target) mole-
cule. Calmodulin and troponin C, the best studied
members of the latter category, belong to the EF-hand
superfamily, which is characterized by a well-conserved
Ca
2+
-binding motif (helix–loop–helix). In addition to
the mediator, or sensor-type activity, proteins of this
family may also interact with the metal ion for buffer-
ing, uptake, or transport purposes [2]. The ion-binding
parameters (affinity, binding kinetics, selectivity, co-
operativity) and the structural response on ion binding
are strongly related to the biological role played by the
protein. Thus, Ca
2+
buffers (e.g. parvalbumin, calbin-
din D
9k
) generally have a high affinity (K
d
<10
)7
m),
may bind Mg
2+
equally well, and are conformationally

less sensitive to ion binding. In contrast, calcium sen-
sors have a lower affinity (K
d
¼ 10
)5
to 10
)7
m)
Keywords
calcium-binding proteins; molecular
dynamics; NMR; nuclear relaxation; solution
structure
Correspondence
C. T. Craescu, INSERM & Institut Curie-
Recherche, Centre Universitaire, Ba
ˆ
timents
110–112, 91405 Orsay, France
Fax: +33 1 69 07 53 27
Tel: +33 1 69 86 31 63
E-mail:
(Received 15 December 2004, revised 14
February 2005, accepted 24 February 2005)
doi:10.1111/j.1742-4658.2005.04629.x
The solution structure of Nereis diversicolor sarcoplasmic calcium-binding
protein (NSCP) in the calcium-bound form was determined by NMR spec-
troscopy, distance geometry and simulated annealing. Based on 1859 NOE
restraints and 262 angular restraints, 17 structures were generated with a
rmsd of 0.87 A
˚

from the mean structure. The solution structure, which is
highly similar to the structure obtained by X-ray crystallography, includes
two open EF-hand domains, which are in close contact through their
hydrophobic surfaces. The internal dynamics of the protein backbone were
determined by studying amide hydrogen ⁄ deuterium exchange rates and
15
N
nuclear relaxation. The two methods revealed a highly compact and rigid
structure, with greatly restricted mobility at the two termini. For most of
the amide protons, the free energy of exchange-compatible structural open-
ing is similar to the free energy of structural stability, suggesting that iso-
tope exchange of these protons takes place through global unfolding of the
protein. Enhanced conformational flexibility was noted in the unoccupied
Ca
2+
-binding site II, as well as the neighbouring helices. Analysis of the
experimental nuclear relaxation and the molecular dynamics simulations
give very similar profiles for the backbone generalized order parameter
(S
2
), a parameter related to the amplitude of fast (picosecond to nanosec-
ond) movements of N
H
-H vectors. We also noted a significant correlation
between this parameter, the exchange rate, and the crystallographic B fac-
tor along the sequence.
Abbreviations
MD, molecular dynamics; NSCP, Nereis diversicolor sarcoplasmic calcium-binding protein; SCP, sarcoplasmic calcium-binding protein.
2022 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
and undergo important ion-induced conformational

changes.
Generally, the precise mechanism underlying the
biological function of a protein is determined by its
structural and dynamic properties which, in turn,
depend on the gene-encoded amino-acid sequence. A
large number of EF-hand proteins are currently known
[3]. However, despite global sequence and structural
similarities, each member has specific functional prop-
erties, acquired during a long evolution period. A com-
parative analysis of a large diversity of sequences and
biological functions within the EF-hand family is
therefore required to better understand the structural
basis of the various biological functions.
Nereis diversicolor sarcoplasmic calcium-binding pro-
tein (NSCP) is an acidic calcium buffer protein, which
is very abundant in the sarcoplasmic reticulum from
the annelid N. diversicolor. It belongs to the sarcoplas-
mic calcium-binding protein (SCP) subfamily, also
including the invertebrate functional analogs of the
vertebrate parvalbumin [4]. Similar to other SCPs,
NSCP has four potential ion-binding sites, but only
three of them (sites I, III and IV) have a high affinity
for Ca
2+
or Mg
2+
[5].
The 3D crystal structure, solved at 2 A
˚
resolution

[6], revealed a globular structure in which the two
EF-hand pairs, constituting an EF-hand domain, are
close to each other, in contrast with the bi-lobal,
extended conformation of calmodulin or troponin C.
Spatial proximity of the binding sites makes functional
communication between them possible, measured in
terms of a strong positive co-operativity in Ca
2+
bind-
ing [7]. In addition, physicochemical experiments per-
formed in our laboratory revealed that metal binding
induces transition from a molten globule state into a
well-defined, and highly stable conformation [8,9]. A
rational understanding of these specific properties
requires structure and dynamic characterization in
solution of the various functionally relevant states.
To achieve this aim, we initiated a structural and
dynamics analysis of Ca
2+
-saturated NSCP in solu-
tion, based on NMR spectroscopy, nuclear relaxation
measurements, and molecular dynamics (MD) simula-
tions. Using purified wild-type samples, as well as
15
N-labeled samples overexpressed in Escherichia coli,
we have assigned the
1
H and
15
N resonances of the

protein (BioMagResBank, accession number 4129)
[10], and collected distance and angle restraints for
structural determination. The solution structure, based
on 1859 NOE distance restraints and additional experi-
mental and chemical information, is very similar to the
previously reported crystallographic structure [6]. In
order to better understand the great structural stability
of the holo form of NSCP, we investigated internal
MD using both experimental (hydrogen ⁄ deuterium
exchange kinetics, nuclear relaxation) and theoretical
(MD simulation) approaches. The results reveal the
NSCP protein as a compact molecule, with restricted
dynamics in the picosecond to nanosecond time scale
and no important exchange movements at the micro-
second or slower scales. The rapid internal dynamics
are satisfactorily described by the MD simulation,
which enabled us to calculate backbone order param-
eter profiles that were in close agreement with the
experimental parameters.
Results and Discussion
Solution structure
We have shown previously that apo-NSCP in solution
is highly disordered and gives poor NMR spectra,
characterized by a low chemical shift dispersion and
absence of NOE interactions [8]. In the presence of
saturating Ca
2+
ions, the spectra extend over 12 p.p.m.
in the proton dimension and exhibit many interproton
dipolar interactions, and the sample is stable enough

for long lasting 3D experiments. Spin systems for 171
of 174 residues (% 98%) were partially or completely
assigned [10]. Unassigned resonances correspond to a
peptide fragment situated at the C-terminus of the pro-
tein (D163–T165). Combined analysis of an ensemble
of NMR parameters in the Ca
2+
-bound state, inclu-
ding short-range and medium-range NOE interactions,
H
a
secondary chemical shifts,
3
J
HNHa
coupling con-
stants, and amide proton exchange rates, enabled us to
delineate eight a-helices consisting of fragments 3–15,
25–38, 45–59, 72–82, 90–103, 113–122, 130–137 and
147–159, representing 56% of the residues. The posi-
tion of the helices corresponds closely to those
observed in the crystal structure [6], but the helices are
often shorter by 1–3 residues. Four short b-strands
(22–24, 69–71, 110–112, 144–146) were also identified
by the low-field-shifted H
a
protons, strong d
aN
(i,i+1)
sequential connectivities and large

3
J
HNHa
couplings.
The strands could be grouped into two antiparallel
b-sheets based on the strong dipolar interactions
observed between H
a
and H
N
protons from opposite
chains.
A total of 1859 interproton distance restraints and
262 dihedral angle restraints were used to fold these
secondary-structure elements into a 3D conformation,
using distance geometry and simulated annealing com-
putations. F igure 1 A s hows t he back bone s uperimpositio n
of the final 17 structures, and Fig. 1B the ribbon repre-
sentation of the best representative of the ensemble for
G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2023
the Ca
2+
-bound NSCP form. In contrast with the
bilobal aspect of calmodulin, the prototype of the EF-
hand superfamily, NSCP exhibits a globular shape
characterized by a large contact area and multiple
side-chain contacts between the two EF-hand domains.
Consequently, residues from the two terminal frag-
ments are very close to each other. The structural

cohesion of the two EF-hand domains is mainly deter-
mined by a highly hydrophobic core including 15 Phe
and three Trp side chains contributed roughly equally
by the two molecular halves (Fig. 2). The structure of
individual EF-hand domains is close to the canonical
geometry [11], observed for the family of proteins ana-
lyzed so far.
The aqua and procheck-nmr programs [12] were
used to assess the quality of the restraints and to deter-
mine the geometry regularity of the final structures
(Table 1). More than 87% of (F, Y) angle pairs of the
17 final structures lie in the most-favored region of
the Ramachandran plot, and about 99% of them lie in
the allowed regions. The segment 162–165 is mostly
found in the disallowed regions of the Ramachandran
plot. These loop residues are not engaged in a detect-
able hydrogen bond and presumably undergo some
geometry constraint from the neighboring amino acids.
The global solution structure of NSCP is very close
to the previously determined crystal structure [6]. The
rmsd calculated for the heavy atoms in regular secon-
dary-structure elements is 1.14 A
˚
, with a large asym-
metry between the two halves: 1.05 A
˚
and 0.77 A
˚
for
the N-terminal and C-terminal halves, respectively.

This may partially reflect the lack of metal binding
in the second loop and the longer linker region
between EF-hand I and II. As in aequorin [13], these
features may result in increased flexibility of the first
half.
It was generally observed that the relative position
of the two helices in EF-hand motifs changes signifi-
cantly upon Ca
2+
binding [14], from an almost anti-
parallel configuration to a perpendicular arrangement.
In a domain containing a pair of EF-hand motifs, this
movement creates a large exposed hydrophobic surface
which, in the case of regulatory proteins, constitutes
the target binding site. The values of the interhelix
angles in NSCP, measured over the NMR ensemble
(Table 2), are similar to those observed in regulatory
EF-hand proteins, in the Ca
2+
-bound state, and are
centered around 90°. Compared with the calmodulin,
where the two lobes are spatially separated, the com-
pact SCPs exhibit a larger variability among the inter-
helix motifs, with significantly lower values for the first
two motifs. Indeed, these two EF-hand domains in
SCPs are more open, than in other Ca
2+
buffer pro-
teins, such as calbindin D
9k

[15] or parvalbumin [16].
These differences may be due to the compact structure
and the tight interactions between the two EF-hand
domains, inducing supplementary constraints on the
interhelical angles.
As can be seen in Table 2, some binding loops lost
the high affinity for the metal ion, but this still main-
A
B
C
Fig. 1. Global representation of the solution structure of
Ca
2+
-NSCP. (A) Backbone stereo view of the 17 final structures
superimposed based on the main-chain heavy atoms in regular
secondary-structure elements. The N-terminal EF-hand domain is
shown in red, the C-terminal EF-hand domain is shown in blue,
the linker is green, and the C-terminal fragment is in magenta.
(B) Ribbon representation of the best structure selected as the clo-
sest to the ensemble average. The color code is the same as in
(A). The binding loops are noted from I to IV, and the a-helices and
some residue positions are labeled to facilitate the chain-folding
pathway. The figure was prepared with
MOLSCRIPT [55] and RASTER3D
[56]. (C) Electrostatic potential calculated at the molecular surface
of the best structure of Ca
2+
-NSCP using the GRASP program [57].
On the left side the molecule is oriented as in (B), whereas on the
right side it is rotated through 180° around the vertical axis. The

positive and negative potential are conventionally coded in blue and
red, respectively.
Structure and dynamics of NSCP in solution G. Rabah et al.
2024 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
tains the corresponding motif in an open confor-
mation. The presence of Ca
2+
in the three active
EF-hand motifs is nonambiguously confirmed by the
chemical-shift signature of the H
b
protons in the Asp
residue occupying the first position in the binding loop
D
1
[17]. Indeed, the close proximity to the conserved
Phe residue in position )4 (relative to D
1
) accounts for
the large upfield shift of one of the b protons in D
1
:
1.58, 1.73, and 1.97 p.p.m. in EF-hand motif I, III,
and IV, respectively.
The chemical shift of the amide nitrogen in the resi-
due occupying position 8 of the Ca
2+
loop was shown
to be larger in the Ca
2+

-bound form (124–127 p.p.m.),
relative to the apo form (111–120 p.p.m.) of EF-hand
proteins [18], because of a decreased electronic density
around the amide nitrogen nucleus. This parameter
was therefore proposed as a sensitive probe for the
metal occupancy of a given motif. In NSCP(Ca
2+
)
3
the amide
15
N chemical shift at the corresponding
positions (I23, I70, I111, L145) are 124.7, 122.0, 123.1
and 120.8 p.p.m., respectively, with the metal-bound
motif IV showing a smaller value than the empty site
II. According to the above classification, only the first
site shows a chemical shift compatible with a bound
loop. These observations suggest that the nitrogen elec-
Fig. 2. Stereoview of the aromatic residue cluster in Ca
2+
-NSCP. Phe, Trp and Tyr side chains are shown in red, blue and green, respect-
ively.
Table 1. Experimental restraints and structural statistics for the 17
simulated annealing structures of (Ca
2+
)
3
-NSCP.
Restraint statistics
NOE restraints 1859

Intraresidue 491 26.4%
Sequential 559 30.1%
Medium range (2 £ |i-j| < 5) 342 18.4%
Long range (|i-j| ‡ 5) 467 25.1%
Hydrogen bond restraints 188
Dihedral angle restraints (F,Y) 262
Average no. of NOE restraint violations
> 0.5 A
˚
None
> 0.4 A
˚
0.12 ⁄ molecule
> 0.3 A
˚
0.35 ⁄ molecule
> 0.2 A
˚
2.35 ⁄ molecule
Average of NOE upper restraint violations 0.0045 A
˚
Averahe of NOE lower restraint violations 0.0009 A
˚
Average rmsd from the average structure (A
˚
)
Residues 3–15, 22–37, 46–57, 69–82,
89–102, 110–122, 129–137, 144–159
a
0.87 ± 0.13

Residues 1–174
a
1.57 ± 0.16
Ensemble Ramachandran plot
Residues in the most-favored region 87.1%
Residues in additional allowed regions 10.8%
Residues in generously allowed regions 1.4%
Residues in disallowed regions 0.8%
a
Backbone atoms (N, C¢,Ca).
Table 2. Statistics of the interhelix angles within EF-hand motifs in
NSCP, related EF-hand proteins and calmodulin. An asterisk marks
EF-hand motif that had lost Ca
2+
-binding capacity.
Helix
pair
Angle (°)
NSCP
a
(NMR)
NSCP
b
(X-ray)
BlSCP
c
(X-ray)
SeCaBP
d
(NMR)

Calmodulin
e
(X-ray)
A ⁄ B67±4 60 59 78 89
C ⁄ D 69 ± 6* 84* 82 101* 89
E ⁄ F 108 ± 5 110 111 107 101
G ⁄ H 101 ± 3 100 96* 113 95
a
This work, mean ± SD over the 17 molecule ensemble.
b
[6],
2SCP.pdb.
c
B. lanceolatum SCP [53], 2SAS.pdb.
d
Bacterial EF-hand
protein, calerythrin [54], 1NYA.pdb.
e
[20], 1CLL.pdb.
G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2025
tron density is not the unique determining factor for
its chemical shift, and question the utilization of this
NMR parameter as a probe for the metal binding to a
given site.
The electrostatic potential at the protein surface is
dominantly negative (Fig. 1C), in agreement with the
acidic character of the EF-hand proteins. It may be
noted that the surface area encompassing the Ca
2+

-
binding sites III and IV exhibits a more homogeneous
and intense negative potential, as compared with the
corresponding area of the first two binding loops. This
may contribute to the low cation-binding affinity of
site II.
The question may be raised as to how the sequence
and structure of EF-hand proteins, classified as cal-
cium buffers or transporters, account for the lack of
regulatory capacity, essentially expressed by a Ca
2+
-
modulated interaction with cellular targets. Structural
analysis of calmodulin complexes revealed that the
interaction interface is constituted by a large hydro-
phobic surface created by the opening of the two
EF-hand domains on Ca
2+
binding. In the case of
SCPs, the highly apolar surface of the two EF-hand
domains exhibit a greater affinity for each other [19],
yielding a compact globular fold, which precludes the
recognition and binding to the hydrophobic surface of
the target proteins. This is made possible by extensive
bending of the interdomain linker, which is usually
found to be almost linear in the crystal structure of
calmodulin [20]. However, a recent crystallographic
study showed that calmodulin can equally form a com-
pact structure [21], as suggested by previous biophysical
and biochemical studies in solution [22,23]. In fact, the

D ⁄ E interhelix angle, calculated as in [14], is very close
in the compact calmodulin (1PRW.pdb) and NSCP
(solution structure): )111° and )116°, respectively.
Sequence and structural comparison between cal-
modulin and three different compact EF-hand proteins
(NSCP, Botrychium lanceolatum SCP and Saccharo-
polyspora erythraea calcium-binding protein) may
reveal some factors contributing to the preference for
the globular shape. A Pro residue between the D and
E helices, which exists only in NSCP, could induce a
tight turn in this region and render the compact struc-
ture more stable. More significantly, the number of
long-chain hydrophobic residues is distinctly higher in
the domains that associate to form compact structures.
Thus, the total number of Phe, Trp, Leu and Ile resi-
dues is 25 in calmodulin and 38, 37, and 35 in NSCP,
B. lanceolatum SCP and S. erythraea calcium-binding
protein, respectively. Therefore, the collapse of the two
EF-hand halves, with the formation of a more stable
apolar core (Fig. 2), is preferred over the extended,
highly solvent-exposed structure. In addition, a 9–10
residue insertion in the C-helix of NSCP, B. lanceola-
tum SCP and S. erythraea calcium-binding protein,
including five hydrophobic side chains, ensures a larger
and tighter contact surface between the N-terminal
and C-terminal domains (Fig. 1B). This tendency to
form a very stable hydrophobic core is very well illus-
trated by the fact that the isolated N-terminal and
C-terminal halves of NSCP form homodimers, but
when mixed, they form a complex with a conformation

as of intact NSCP [19]. A more detailed structural and
thermodynamic investigation of the interdomain inter-
face in the compact EF-hand proteins should be very
useful for a quantitative explanation of the conforma-
tional preference.
Conformational flexibility studied by amide
exchange kinetics
Analysis of the amide exchange kinetics provides a
site-specific description of global or local conforma-
tional dynamics of a protein in solution. Description
of the exchange process in terms of protection factors
[24] enables us to eliminate the influence of the solu-
tion properties (pH, ionic strength, temperature, etc.),
and of the chemical environment of amide groups (the
sequence context). Therefore, the protection factors
may be directly related to the relative attenuation of
the hydrogen exchange rate in given main-chain posi-
tions of the native structure, relative to the random-
coil state. We were able to quantitate this parameter
for 72 out of the total of 169 amide protons and for
three indole amino groups from Trp side chains. Five
missing values corresponding to the amide protons
with high exchange rates (k
ex
>10
)2
min
)1
), K19,
F35, L49, M122, and V168, are indicated by the down-

ward arrows in Fig. 3. The intensity of the remaining
peaks could not be accurately measured due to overlap
in the HSQC spectra.
Most of the measured protection factors have relat-
ively high values (mean % 10
6.5
), and are associated
with the a-helices and b-strands, except for the empty
binding motif II (Fig. 3A). The strong protection of
the amide protons in Ca
2+
-saturated NSCP is in good
agreement with its high structural stability [9]. Thus,
the free energy of conformational opening estimated
here from hydrogen isotopic exchange measurements
are centered around 37.7 kJÆmol
)1
(9 kcalÆmol
)1
) for
the three bound EF-hands, which is close to the free
energy of the structural stability calculated from dena-
turation experiments [9]. This strongly suggests that
the conformational fluctuations, enabling the measured
proton exchange, correspond to global dynamics, and
Structure and dynamics of NSCP in solution G. Rabah et al.
2026 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
are highly similar to those accompanying the co-opera-
tive unfolding of the whole structure.
Clearly, the number of measurable amide protection

factors and their magnitude are smaller for the second,
unbound EF-hand motif, but also for the neighboring
helices (B and E). The unbound loop induces more
rapid conformational fluctuations that extend outside
the motif, in both directions of the main chain, as also
reflected in the crystallographic B factors of the N
atoms (Fig. 3).
The side-chain Trp protons exhibit remarkably high
protection factors, comparable to the backbone amide
protons in the more flexible helices and in the C-
terminal fragment (Fig. 3). Among the three Trp resi-
dues, the N
e1
proton of Trp4 belongs to a hydrogen
bond with the carbonyl oxygen of Phe158, observed
both in the NMR and the previous X-ray structure [6].
Owing to the deshielding effect of this interaction, the
proton chemical shift is significantly low-field-shifted
(10.52 p.p.m.) relative to the random coil value (10.22
p.p.m.). The corresponding proton in Trp170 may
form an aromatic hydrogen bond with the side chain
of Phe157, as suggested by the structure, and suppor-
ted by the large high-field shift of its proton resonance
(7.28 p.p.m.) induced by the phenyl ring current. The
low exchange rate of the indole protons in these two
residues may be explained by the protection provided
by the hydrogen-bond formation. In contrast, no
explanation is actually available for the exchange pro-
tection in Trp57, which is largely exposed to the sol-
vent, and shows no detectable intramolecular hydrogen

bond.
Internal MD studied by relaxation measurements
Computation of the principal components of the iner-
tia momentum, based on the NMR-derived solution
structure, gives (1.00 : 0.83 : 0.66). According to these
values, only a modest degree of anisotropy is expected,
which may not influence significantly the microdynamic
parameters (at least the order parameters) [25]. Owing
to cross-peak overlap, reliable analysis of peak inten-
sities and relaxation parameters was limited to 121
H
N
-N vectors, including 118 amide groups (out of
the total of 169 observable amide protons) and three
indole amino groups from the Trp side chains.
Fig. 3. Dynamics analysis by amide proton
exchange kinetics of Ca
2+
-NSCP. (Top)
1
H ⁄
2
H exchange kinetics expressed as the
logarithm of the protection factor [log (P)]
and the free energy of isotope exchange
(DG
ex
). The mean log (P) is indicated by the
horizontal line. Downward arrows designate
the amino protons with exchange kinetics

faster than 10
)2
min
)1
. The exchange
parameter of the Trp indole protons (W4,
W57 and W170) are shown at the end of
the sequence, by the grey bars. The secon-
dary-structure elements and occupation of
the binding loops are represented sche-
matically at the top of the figure. (Bottom)
crystallographic B factors of N
H
atoms
determined by the X-ray approach [6].
G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2027
Figure 4 shows the relaxation parameters (R1, R2,
NOE) and their uncertainty plotted as a function of
residue number. The dynamic analysis started with
the determination of the rotational correlation time
(s
c
) of the whole protein using a procedure designed
to minimize the effects of heterogeneous local move-
ments [26]. For a given arbitrary value of s
c
, the
dynamic parameters (S
2

and s
e
) are computed using
R1 and heteronuclear NOE for each amide vector
within the most rigid segments (84 sites) in the frame
of the Lipari-Szabo approach [27]. Then, R2 values
may be reconstructed (from the spectral density func-
tions) and compared with the experimental counter-
parts for the selected sites. The final value of the
correlation time, corresponding to the minimum of
the v
2
(R2) function, was 6.93 nsÆrad
)1
. Using a simp-
ler method based on the independence of the R2 ⁄ R1
ratio from S
2
and s
e
[28], we obtained a very close
value for s
r
(6.86 ±0.34 ns). The value of the rota-
tional correlation time is indicative of a mainly mono-
meric form of NSCP.
With the value for the global correlation time
obtained by the first method, we analyzed the relaxa-
tion parameters in terms of the simple or extended
Lipari-Szabo model-free methods [27,29]. A Monte-

Carlo simulation with 500 steps was used to estimate
the standard error of the microdynamic parameters.
The data for all the studied vectors (except amides in
A32 and S112) could be fitted to the simple Lipari-
Szabo procedure giving the generalized order param-
Fig. 4. Relaxation parameters (R1, R2, g) measured in Ca
2+
-saturated NSCP at 308 K. The elements of secondary structure and the occu-
pancy of Ca
2+
-binding sites are shown at the top of the first panel. The last three values correspond to the N
e1
-H vector in Trp side chains
(W4, W57, W170).
Structure and dynamics of NSCP in solution G. Rabah et al.
2028 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
eter (S
2
), the internal correlation time (s
e
) and the
exchange contribution to the transversal relaxation
(R2
ex
). The extended procedure of relaxation analysis
did not improve the fitting quality of any amide or
amine system.
The generalized order parameter S
2
at a given back-

bone site is a measure of the amplitude of the fast
(picosecond to nanosecond) movement of the H
N
-N
vector. Figure 5 shows the order parameters and their
standard deviations, estimated by Monte-Carlo simula-
tions, as a function of the residue number. The param-
eter varies between 0.66 and 0.93, with a mean value
of 0.83 ± 0.04, which is close to the average value
usually observed for native globular proteins [30].
Overall, the estimated order parameters indicate that
the backbone amide vectors undergo picosecond-to-
nanosecond movements of low amplitude, reflecting a
compact and rigid fold, with well-structured end frag-
ments. Larger S
2
values (from 0.84 to 0.93) are
grouped in the loop I, helix F and helix H, while the
intermotif linkers and the unoccupied Ca
2+
-binding
loop exhibit lower S
2
values (down to 0.66), attesting
to larger amplitude fast movements. The end frag-
ments are characterized by high S
2
values, meaning
that their movement is significantly restricted. Of the
four EF-hand motifs, the first and the fourth exhibit

the most restricted picosecond-to-nanosecond mobility
of the backbone vectors. The absence of Ca
2+
binding
to the second EF-hand induces an irregular pattern of
S
2
values in the loop residues that extends over the
neighboring helices. It is worth noting that the helices
characterized by a larger fast movement amplitude and
a high amide proton exchange (B, C, E) belong to the
interface between the two EF-hand domains.
The relatively high values (0.75–0.78) of S
2
observed
for the amino group of the Trp side chains indicate
that the fast movements of these indole moieties are
restricted to a similar extent to the backbone amide
vectors (Table 3). Inspection of the calculated structure
(Fig. 2) shows that the three indole groups do not have
comparable environments in the 3D structure: whereas
W4 and W57 are highly exposed to the solvent at
the protein surface, W170 is deeply embedded in the
hydrophobic core created by helices E, F and H. The
order parameters of these side chains appear to be
independent of this environmental context.
The large majority of the residues display fast rate
librational motions characterized by an internal corre-
lation time s
e

< 50 ps, with a dozen (mainly localized
in linker fragments) having a correlation time in the
range 50–100 ps (not shown). Five residues (V5, E40,
G67, S90, and D156) exhibit R2
ex
values between 1
and 2 s
)1
, reflecting microsecond–millisecond internal
motions in their environment. Again, they are associ-
ated with end fragments (V5, D156), linkers (E40, S90)
or the empty calcium-binding loop (G67).
Fig. 5. Generalized order parameters along
the sequence of Ca
2+
-saturated NSCP. The
experimental values obtained from the NMR
relaxation experiments, together with the
standard deviations, are shown in red. The
order parameters estimated from the MD
simulations are in black. The last three
values, at the end of the sequence, corres-
pond to the Trp4, Trp57, and Trp170 indole
N-H vectors (boxed).
Table 3. Experimental (S
2
NMR
) and calculated (S
2
MD

) order parame-
ters for the Trp side chains.
Trp S
2
NMR
S
2
MD
W4 0.78 0.83
W57 0.75 0.79
W170 0.78 0.83
G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2029
Comparison with the crystallographic B factor
The crystallographic B factor is considered to reflect
mainly the molecular flexibility at the atomic level, but
other factors related to static and dynamic disorders in
the crystal may give important contributions as well
[31]. Intuitively, it should be inversely correlated with
the order parameters estimated from the relaxation
measurements, but in practice the relationship is more
complex [32]. Overall, in NSCP the regions of high-
order parameters exhibit lower B factors for amide
nitrogens (Fig. 3). As the range of values for the order
parameter is about three times lower than that for the
B factor, the quantitative correlation between the two
parameters along the sequence is only moderate (the
correlation coefficient is )0.32). Discrepancies also
arise from the fact that S
2

reflects only fast motions,
whereas the B factor is sensitive to both fast and slow
movements [33]. In a similar approach for ribonuc-
lease, Mandel et al. [34] found similar low values
between )0.36 and )0.64, depending on the X-ray
structure considered. This variability illustrates the
dependence of the thermal factors on the crystalliza-
tion state and the intermolecular contacts within the
crystal.
MD simulation
Simulation of the protein internal dynamics under an
appropriate physical force field provide a detailed
atomic picture of the movements underlying the nuc-
lear relaxation parameters and the corresponding order
parameters. Only 1 ns (the second half) from the 2-ns
very stable trajectory of the Ca
2+
-saturated form of
NSCP was used in the theoretical analysis. The
mean ± SD temperature was 299.99 ± 4.47 K, and
the total energy was 1722.5 ± 21 kJÆmol
)1
(411.4 ±
5 kcalÆmol
)1
). Correlation functions for backbone
N
H
-H vectors were computed from the selected traject-
ory using an interval of 400 ps, which provides reliable

sampling of the fast (% 100 ps) motions [35]. Correla-
tion functions were computed from the trajectory for
168 residues from the total of 174 (the N-terminus and
the six prolines do not have an sp
2
N
H
-H bond).
The correlation functions of the internal motions,
C
I
(t) display different patterns along the sequence, and
only 132 could be considered completely convergent.
For 25 other residues, the correlation function exhibits
a slow decay towards a lower plateau and oscillatory
behavior, suggesting the presence of slower motions,
which may not be accounted for correctly by the
length of the present trajectory [35]. Finally, for 11 resi-
dues no plateau value could be reached within 400 ps.
The plateau values of the correlation functions (inclu-
ding both rapidly and slowly convergent functions),
which represent the theoretical order parameters S
2
MD
,
were computed for 157 amino-acid residues (Fig. 5).
A total of 151 of the 157 simulated S
2
MD
values are

larger than 0.7, confirming the high rigidity of the
NSCP backbone structure. As shown in Fig. 5, there
is a good correlation between the order parameter
profiles established by theoretical and experimental
approaches.
The average value of S
2
obtained by MD (S
2
ave
¼
0.80, average over 157 points) is slightly lower than
that calculated from nuclear relaxation data (S
2
ave
¼
0.83, average over 116 points), as also noted for apo-
neocarzinostatin [36]. It may be noted that there is a
remarkable parallelism between the two S
2
profiles at
the limits between secondary-structure elements and
linkers, where this parameter exhibits larger variations.
Amide vectors in the unoccupied binding loop II, in
the linker between helices F and G, as well as in the
last loop show markedly decreased order parameters
both in the relaxation experiments and the simulation,
permitting cross validation of the underlying MD
(Fig. 5). The MD simulations predict slightly larger
amplitude motions in the more flexible protein seg-

ments than estimated by relaxation measurements. It
must be stressed that the overall shift between the
NMR and MD values for the order parameter could
be decreased by choosing a slightly smaller value for
the initially estimated global correlation time [37].
Previous comparative studies on the simulation and
experimental dynamic parameters of globular proteins
[35–37] have always noted some differences in the
order parameters determined by the two methods. One
of the reasons is that both MD and NMR analysis
involve several approximations. In the case of the MD
simulations, these include the parameters of the empir-
ical force field, insufficient sampling because of the
finite length of the trajectories, and the simplified treat-
ment of the solvent. The main simplification in the
NMR approach concerns the relaxation data analysis,
usually performed under the assumption of a unique
overall correlation time and the independence of move-
ments on different time scales.
In addition to the amide vector dynamics, we also
analyzed the fast movements of the N
e1
-H vectors in
the three Trp side chains. The order parameters of the
picosecond motions of these bonds (Table 3) are only
slightly lower than the backbone values (as in the
relaxation results), indicating that the side chains also
move in a highly restricted regime [37]. Trp57 senses
the molecular environment of the empty binding site
and shows significantly larger amplitude than the other

Structure and dynamics of NSCP in solution G. Rabah et al.
2030 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
two tryptophans, both in simulation and experimental
results.
The good qualitative description of the fast dynam-
ics in the Ca
2+
-bound form of NSCP encouraged us
to use a simple dynamics simulation to investigate the
initial structural changes accompanying the holo to
apo transition. A 2-ns MD trajectory, starting with the
crystallographic co-ordinates of (Ca
2+
)
3
-NSCP [6]
from which the three metal ions were removed, was
generated at 300 K. As estimated from the temperature
and co-ordinate rmsd profiles, the apo and holo
dynamics are of comparable quality. Figure 6A com-
pares the two final simulated structures and the crystal
structure, using the pseudo-dihedral angles defined by
four successive C
a
atoms along the sequence. As may
be noted from the upper panel, in presence of the
bound Ca
2+
ions the final protein secondary structure
(blue symbols) shows no significant change relative to

the crystal conformation (red bars). In contrast, the
final structure of the apo simulation (Fig. 6A, lower
panel) shows significant secondary-structure differences
relative to the starting (holo-type) conformation,
mainly localized to the first binding loop, the N-side of
the C and D helices, and the C-end of the F helix.
Comparison of the tertiary structures including these
areas (Fig. 6B) shows that removing the metal ion
from the first site determines an opening of the binding
loop, and induces important structural changes in the
first half of the protein. The rearrangement of this
domain has remote consequences on the F helix, in the
second protein half, which may be accounted for by
the close contacts existing between the B and F helices
(Fig. 6B). Indeed, this interhelix space belongs to the
large interdomain surface which plays an important
role in the structural and functional coupling between
the two halves. The perturbation propagation path-
way, observed in the present simulation, includes the
higher flexibility secondary-structure elements, high-
lighted by the nuclear relaxation and proton exchange
experiments.
A longer, and more complete, MD simulation of
apo-NSCP, in an explicit water environment, should
provide valuable insight into the metal-induced con-
formational changes and the characteristics of the apo
molten globule state. This approach is currently being
taken in our laboratory.
Experimental procedures
Protein preparation and labeling

Wild-type NSCP was purified from the Nereis muscle by
the method of Cox & Stein [5], modified as described by
Engelborghs et al. [38]. Recombinant protein and
15
N labe-
ling was obtained by overexpression in Escherichia coli [39].
The NSCP ORF was amplified by PCR starting from the
plasmid pNDner04, cloned into the vector pET22b, and trans-
fected into the host E. coli strain BL21(DE3) ⁄ pDIA17 ⁄
pHSP234 (Novagen, Madison, WI, USA). (
15
NH
4
)
2
SO
4
(1 gÆL
)1
) was added to the minimal medium to obtain uni-
formly labeled samples.
NMR samples
NMR samples (1.0–1.2 mm) were prepared in deuterated
20 mm Tris ⁄ HCl buffer ⁄ 5mm CaCl
2
,pH6.5,in95%
1
H
2
O ⁄

5%
2
H
2
O or in 100%
2
H
2
O.
NMR spectroscopy
All NMR spectra were acquired at 308 K on a Varian
Unity-500 spectrometer, equipped with a triple-resonance
probe and a Z-field gradient. Standard methods were used
to obtain 2D NOESY and 3D
15
N-NOESY-HSQC spectra
[40,41], with mixing times of 100 ms. The 3D spectra were
acquired as 128 (t
1
) · 32 (t
2
) · 512 (t
3
) complex points with
a spectral width of 1500 Hz in the nitrogen dimension,
3200 Hz in the amide proton dimension, and 7000 Hz in
the all-proton dimension. Dihedral angle restraints were
obtained by analyzing the HMQC-J spectrum by the
method proposed by Wishart & Wang [42]. Data processing
and restraint collection were performed using felix97 soft-

ware (Accelrys, San Diego, CA, USA), running on a Silicon
Graphics Octane workstation.
15
N nuclear relaxation
The heteronuclear relaxation experiments were performed
at 308 K and 11.74 T (500 MHz proton resonance fre-
quency). The R1 relaxation rate was measured using the
inversion recovery method, modified to obtain decreasing
signal intensities as a function of the relaxation delay.
Measurement of the transverse relaxation rate (R2) was
based on the Carr–Purcell–Meiboom–Gill pulse sequence
with a delay between
15
N 180° pulses during the relaxation
period of 0.9 ms. Recycle delays of 2.5 s were used at the
beginning of R1 and R2 pulse sequences. Spectra for R1
measurements were acquired using 11.04 (· 2), 55.22,
165.66, 220.88 (· 2), 386.54, 552.2, 662.64, 828.3 and
1104.4 ms as relaxation delays. R2 data were recorded with
delays of 31.4, 47.1, 62.8, 78.5 (· 2), 125.6, 157, 188.4 and
219.8 ms. Steady-state
1
H-
15
N NOE was determined from
spectra pairs with and without proton saturation. The two
experiments start with a 5 s recycle delay, but during the
last 3 s of the saturation experiment the protons are irradi-
ated by 120° pulses every 5 ms. The pulse sequences used in
the present experiments were adapted from those kindly

G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2031
provided by Lewis Kay [43,44]. Peak picking and peak
height measurement were performed with the appropriate
routines of the felix97 package. Uncertainties in the peak
intensities were evaluated from the standard deviation of
the noise in a few rows from the spectra with various relax-
ation delays. Relaxation rates R1 and R2 and their uncer-
A
B
Fig. 6. Comparison of the 2-ns MD simulations of the holo and apo forms of NSCP. (A) The pseudo-dihedral angles between consecutive C
a
in the final structure obtained for (Ca
2+
)
3
-NSCP (upper panel) and apo-NSCP (lower panel). The red lines in both panels represent the calcula-
ted values for the crystallographic (Ca
2+
)
3
-NSCP structure, and the blue symbols represent the simulated structures. (B) Schematic represen-
tation of the two simulated final structures focused on the first half of the protein. The green sphere represents the calcium ion bound to
the first loop.
Structure and dynamics of NSCP in solution G. Rabah et al.
2032 FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS
tainties were obtained by fitting the peak intensities to a
single-exponential function with two parameters, using the
physica package. Errors in relaxation rates were estimated
from the uncertainties in the peak heights and in the expo-

nential fit or by Monte-Carlo simulations. The steady-
state NOEs were calculated as NOE ¼ I
sat
⁄ I
nonsat
(g ¼
NOE ) 1), where I
sat
and I
nonsat
are the steady-state peak
intensities measured with and without proton saturation,
respectively. NOE uncertainties were estimated from the
peak height uncertainty and the law of error propagation.
Hydrogen exchange
Amide proton exchange kinetics was performed by acquir-
ing a series of 2D HSQC spectra of a
15
N-labeled lyophi-
lized sample, freshly dissolved in
2
H
2
O. The progress of the
exchange process was followed by observing successive spec-
tra, recorded at 308 K, and measurement of the amide peak
intensity. The experimental points were fitted to a three-
parameter exponential function by a nonlinear least squares
routine. The protection factors for each backbone site were
calculated using the procedure proposed by Bai et al. [24],

designed to eliminate the dependence on the physicochemi-
cal properties of the solution and on the chemical environ-
ment of the site. For the indole N
e1
protons of the Trp
residues, only the temperature and the pH corrections were
measured. Under the EX2 exchange conditions (in which
the rate of conformational change is much larger than the
rate of proton exchange), the exchange rate (k
ex
) for an
amide proton is calculated from the relation [24]:
k
ex
¼ K
op
k
ch
where k
ch
is the chemical exchange rate in the given
physicochemical conditions, and K
op
the equilibrium con-
stant between the open (exchange-compatible) and closed
(exchange-incompatible) conformations. The free energy of
the opening process, enabling the isotopic exchange (DG
ex
),
is calculated from the formula:

DG
ex
¼ÀRT ln K
op
The decrease in the exchange rate of a proton in the native
structure, relative to the random coil (k
ch
⁄ k
ex
) defines the
so-called protection factor, P ¼ 1 ⁄ K
op
[24].
Structure determination
Spin systems for 171 of 174 residues (% 98%) were partially
or completely assigned [10]. Unassigned resonances corres-
pond to a polypeptide fragment situated in an irregular sec-
ondary-structure region (D163–T165) at the C-terminal site
of the sequence. A total number of 1859 NOE distance
restraints were identified in the 2D and 3D NOESY spec-
tra, corresponding to an average of 10.7 restraints per resi-
due. In addition, 188 hydrogen-bond restraints were
identified from the analysis of hydrogen-exchange
experiments, and 262 dihedral angle restraints were gener-
ated based on
3
J
HNHa
coupling constant measurements and
preliminary secondary-structure delineation. NOE distance

restraints were classified into three categories, 1.8–2.9, 2.9–
3.7 and 3.7–5.0 A
˚
except for d
aN
(i,i +2) and d
NN
(i,i +2)
distances in regular helical fragments which fall into 4.2–4.6
and 4.0–4.4 A
˚
, respectively. No restraints involving calcium
ions were available. However, several hydrogen bonds
within the Ca
2+
-binding loop, confirmed by the low hydro-
gen exchange rate and a large downfield chemical shift
[45,46], were considered for the binding loop I, III and IV.
The restraint statistics are detailed in Table 1.
The aqua and procheck-nmr programs [12] were used
to analyze the restraint violations and to estimate the preci-
sion and quality of the structures obtained.
Molecular dynamics
MD simulations were performed using the program
charmm and the extended atom force field PARAM19 [47]
in which the polar hydrogen atoms are treated explicitly.
The initial set of atomic co-ordinates was obtained from
the crystal structure of the NSCP [6] completed with the
polar hydrogen co-ordinates [48]. Co-ordinates for the apo
form of the NSCP were generated by removing the three

Ca
2+
ions from the holo-NSCP structure. Both apo-NSCP
and holo-NSCP structures were then energy-minimized
in vacuo by 6000 steepest descent steps, followed by 3000
Newton–Raphson steps.
The minimized structures were heated to 300 K for
12 ps, equilibrated at 300 K for 18 ps, and finally two pro-
duction runs of 2 ns were generated for both the holo and
apo forms. The nonbonded interactions were truncated to
zero by applying a switching function between 5 and 9 A
˚
[49]. The nonbond list was cut off at 10 A
˚
and updated
every 10 steps. An ‘r’ relationship was considered for the
dielectric constant (r is the interatomic distance). For the
MD simulations, the Newton equations of motion were
integrated using the VERLET algorithm with an integra-
tion step of 1 fs. The heavy atom–hydrogen bond lengths
were constrained with the SHAKE algorithm [50]. The
co-ordinates were saved every 100 fs to the trajectory file.
Correlation functions and order parameter
computation from MD trajectories
Assuming that internal motion and the overall tumbling are
independent, the reorientation of the N
H
-H vector, which
contributes to the
15

N relaxation, can be described by the
angular autocorrelation function:
CðtÞ¼ð1=5ÞC
o
ðtÞC
I
ðtÞ
where the correlation functions C
o
(t) and C
I
(t) are related
to the overall rotational tumbling of the molecule and to
the internal dynamics, respectively. The internal correlation
G. Rabah et al. Structure and dynamics of NSCP in solution
FEBS Journal 272 (2005) 2022–2036 ª 2005 FEBS 2033
functions describing the dynamics of an N
H
-H bond are
computed from the trajectory using an autocorrelation
function of the unit vector upon the direction of N
H
-H
atoms,
^
l
NH
ðsÞ [51,52]:
C
I

ðtÞ¼hP
2
ðcos h
NH
Þi ¼
3 cos
2
h
NH
ðtÞÀ1
2

t
¼
1
T
MD
À t
X
T
MD
Àt
s¼1
3½
^
l
NH
ðsÞ
^
l

NH
ðs þ tÞ
2
À 1
2
where P
2
(cosh
NH
) is the second rank Legendre polynomial,
^
l
NH
the unit vector along the N
H
-H direction, T
MD
the
total number of MD frames, and s the index of MD frames
for the computed correlation function (the maximum value
of the ‘t’ parameter should be lower than T
MD
⁄ 3).
The generalized order parameters (S
2
) were evaluated for
amide N
H
-H bonds and N
e1

-H bonds of Trp side chains,
by fitting the calculated C
I
(t):
S
2
¼ lim
t!1
C
I
ðtÞ¼ lim
t!1
< P
2
½
^
l
NH
ð0Þ
^
l
NH
ðtÞ >
for one-third of the whole trajectory [35] to a single expo-
nential (‘model-free’ approach).
Accession number
Co-ordinates corresponding to the 17 structures of the
Ca
2+
-saturated NSCP have been deposited in the Protein

Data Bank with the accession code 1Q80. The first structure
in this assembly, which is closest to the averaged
co-ordinates of the ensemble, was chosen as a representative
conformer. The file containing the proton assignment of the
NSCP domain has been deposited in the BioMagResBank
with the entry No. 4129.
Acknowledgements
This work was supported by the Centre National de la
Recherche Scientifique, the Institut National de la
Sante
´
et de la Recherche Me
´
dicale, the Institut Curie
and the Swiss National Science Foundation. We are
indebted to Lewis E. Kay for providing pulse sequences
for relaxation experiments. R.P. was a recipient of a
French Government fellowship, and fellowships from
the Universite
´
Paris 6, and Socie
´
te
´
de Secours des Amis
des Sciences. We thank Aurel Popescu for his contribu-
tion to the hydrogen exchange data analysis, Joe
¨
l Mis-
pelter for the help with relaxation analysis, and Liliane

Mouawad for useful discussions on MD.
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