Tài liệu Báo cáo Y học: Structural and biochemical characterization of neuronal calretinin domain I– II (residues 1– 100) Comparison to homologous calbindin D28k domain I–II (residues 1 –93) pdf

Structural and biochemical characterization of neuronal calretinin
domain I–II (residues 1–100)
Comparison to homologous calbindin D
28k
domain I–II (residues 1–93)
Małgorzata Palczewska
1
, Patrick Groves
1
, Attila Ambrus
2,
*, Agata Kaleta
1
, Katalin E. Ko¨ve
´
r
3
, Gyula Batta
4
and Jacek Kuz
´
nicki
1,5
1
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland;
2
Department of Biochemistry and Molecular Biology, University of Debrecen, Hungary;
3
Department of Inorganic Chemistry,
and
4

Research Group for Antibiotics, Department of Chemistry, University of Debrecen, Hungary;
5
International Institute of Molecular and Cell Biology, Warsaw, Poland
This study characterizes the calcium-bound CR I–II
domain (residues 1–100) of rat calretinin (CR). CR, with
six EF-hand motifs, is believed to function as a neuronal
intracellular calcium-buffer and/or calcium-sensor. The
secondary structure of CR I–II, defined by standard NMR
methods on
13
C,
15
N-labeled protein, contains four helices
and two short interacting segments of extended structure
between the calcium-binding loops. The linker between the
two helix–loop–helix, EF-hand motifs is 12 residues long.
Limited trypsinolysis at K60 (there are 10 other K/R
residues in CR I–II) confirms that the linker of CR I–II is
solvent-exposed and that other potential sites are protected
by regular secondary structure.
45
Ca-overlay of glutathione
S-transferase (GST)–CR(1–60) and GST–CR(61–100)
fusion proteins confirm that both EF-hands of CR I –II have
intrinsic calcium-binding properties. The primary sequence
and NMR chemical shifts, including calcium-sensitive glycine
residues, also suggest that both EF-hand loops of CR I – II bind
calcium. NMR relaxation, analytical ultracentrifugation,
chemical cross-linking and NMR translation diffusion
measurements indicate that CR I–II exists as a monomer.

Calb I–II (the homologous domain of calbindin D
28k
) has the
same EF-hand secondary structures as CR I–II, except that
helix B is three residues longer and the linker has only four
residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald,
P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur.
J. Biochem. 262, 933 –938]. In contrast, Calb I–II binds one
calcium cation per mono-meric unit and exists as a dimer.
Despite close homology and similar secondary structures,
CR I– II and Calb I–II probably have distinct tertiary
structure features that suggest different cellular functions for
the full-length proteins.
Keywords: calretinin; calcium; calbindin D
28k
; EF-hand;
NMR secondary structure.
Calretinin (CR) and calbindin D
28k
(Calb) are homologous
calcium-binding EF-hand proteins with 59% sequence
identity (rat forms) [1–3]. The proteins contain six
helix–loop–helix motifs (EF-hands) in which the loops
carry the calcium-binding ligands. The rat sequences are
271 (CR) and 261 (Calb) amino acids long [4,5]. Both
proteins have distinct, predominantly neuronal cellular
distributions [3,6 –9] and Calb is additionally found in the
digestive system [10]. Both proteins are used as markers for
a subset of neurons and several neurodegenerative diseases
[11–13]. Immunohistochemistry of CR is used to distin-

guish adenocarcinomas from mesothelioma [14,15], with
CR playing a role in the early stages of mesothelioma
[16]. In contrast, Calb is found in subpopulations of neuro-
endocrine phenotypes of some carcinoids and small-cell
carcinomas [17].
EF-hand proteins act as intracellular calcium-sensors,
linked to protein signaling cascades, and/or calcium-buffers
[3]. For example, calmodulin binds to more than 100 different
proteins in a calcium-specific manner, while parvalbumin is
considered important in maintaining nontoxic levels of free
intracellular calcium through its buffering ability [3]. The
function of CR is unclear 2 there is evidence for both
buffer and sensor roles (reviewed in [18]) but no definite
target protein has yet been identified for CR to support a
sensor role. Calb appears to play a buffer role in neurons
[19] and possibly facilitates calcium uptake through the
digestive system [10]. However, there is evidence that
intestinal brush border membrane alkaline phosphatase
Correspondence to J. Kuznicki, Department of Molecular and Cellular
Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur
Street, 02-093 Warsaw, Poland. Fax: 1 48 22 822 53 42,
Tel.: 1 48 22 659 31 43, E-mail: or to G. Batta,
Research Group for Antibiotics, Department of Chemistry, Egyetem te
´
r.
1, University of Debrecen, PO Box 70, H-4010 Debrecen, Hungary.
Fax: 1 36 52 512 914, Tel.: 1 36 52 512 900 extn 2370,
E-mail:
*Present address: The University of Arizona, Department of Chemistry,
1306 E University, Tucson, AZ 85721– 0041, USA.

Note: a web page is available at
/>(Received 28 June 2001, revised 24 September 2001, accepted
4 October 2001)
Abbreviations: CR, calretinin (rat isoform, unless otherwise stated); CR
I–II, calretinin residues 1–100 consisting of the first pair of EF-hand
motifs; Calb, calbindin D
28k
(rat isoform, unless otherwise stated); Calb
I–II, calbindin D
28k
1–93 consisting of the first pair of EF-hand motifs;
Calb III–IV, calbindin D
28k
79–193, consisting of the second pair of
EF-hand motifs; CSI, chemical shift index; GST, glutathione
S-transferase; DOSY, diffusion ordered spectroscopy.
Eur. J. Biochem. 268, 6229–6237 (2001) q FEBS 2001
and caspase-3 might serve as functional targets for Calb
[20,21].
The biochemical characterizations of CR [22–25] and
Calb [26–29] suggest both proteins undergo significant
calcium-dependent structural changes. In particular,
calcium-modulated exposure of hydrophobic surfaces,
perhaps the most important sensor characteristic, has been
reported for both CR and Calb [22,24,30]. The biochemical
properties of CR and Calb are similar to those of
calmodulin, the best-known EF-hand calcium sensor. It is
difficult to localize the biochemical properties of CR and
Calb to particular domains but this information can be
obtained by studying protein fragments. For example, the

structure of the calcium-bound first domain of Calb,
comprising the first two EF-hands (Calb I –II, residues
1–93), was recently reported [31]. Calb I –II binds a single
calcium and forms a homodimer [31]. We have expressed
and purified the homologous domain of CR (CR I–II,
residues 1–100) [23,32]. This has allowed us to analyze the
biochemical and structural properties of CR I–II and to
compare them with Calb I–II. Our data show that CR I–II
and Calb I –II share virtually the same EF-hand structures.
However, a shorter helix B in CR I–II accentuates a longer
linker loop between the EF-hands of CR I–II compared to
Calb I–II. In contrast to Calb I –II, CR I–II shows no
tendency to dimerize and both EF-hands of CR I –II bind
calcium. We conclude that the significant structural and
biochemical differences between CR I–II and Calb I–II
are related to a region of poor sequence identity (residues
54–79 of CR). We expect the differences between CR I–II
and Calb I – II will be present in the full-length proteins and
that these differences may play a role in the distinct cellular
functions of CR and Calb.
EXPERIMENTAL PROCEDURES
Expression and purification of CR and CR fragments
Recombinant CR and CR fragments were expressed from
Escerichia coli as glutathione S-transferase (GST) fusion
proteins and were purified as described previously [23,33].
GST–CR(1–60) and GST–CR(61–100) were prepared
using the same techniques as described for the preparation
of other GST–CR fragments [23,33]. Extracellularly
expressed CR I–II using Pichia pastoris was purified by
DEAE chromatography [32] and the proteins eluted were

desalted by dialysis. The resulting protein solutions were
lyophilized to dryness. Reconstituted protein concentrations
were established using the Bradford method, with BSA as
standard [34]. CR I–II from P. pastoris was used in all
experiments unless stated otherwise.
15
N-labeled CR I–II was produced from P. pastoris
using 98%
15
N-ammonium sulfate (Martek Biosciences)
and the same protocols as for unlabeled material [32].
13
C,
15
N-labeled CR I–II was prepared as for
15
N-labeled
material but with 5 g
:
L
21
glucose (day 1) and 2 g
:
L
21
98%
13
C-glucose (day 2) (Martek Biosciences) as the sole carbon
source during the growth phase and the addition of 0.5%
13

C-methanol each day (Euris-top, France) during the
expression phase (additional 5 days). Protein purification
followed the same steps as for unlabeled protein.
Sequence/structure analysis
Protein sequences for rat CR and rat Calb were retrieved
from GenBank (accession numbers X66974 and M31178).
The relevant parts of the N-terminal modified CR I–II and
Calb I–II sequences were aligned with SeqWeb (Oxford
Molecular Group) using the
BESTFIT algorithm.
NMR determination of secondary structure
A standard method of assigning protein backbone HN, NH,
CA, HA, CB, HB and CO chemical shifts was used, based
on protocols described in Cavanagh et al. [35] and Sattler
[36]. The sample contained 1 m
M
13
C,
15
N-labeled CR I–II,
50 m
M Tris pH 7.7, 25 mM NaCl, 10 mM CaCl
2
. The
experiments included HNCO, HN(CA)CO, HNCA,
HN(CO)CA, HNCACB, CBCANH, CBCA(CO)NH
(H)CC-TOCSY(CO)NH, H(CC)-HEHAHA(CO)NH,
HNHA, and HSQC. The experiments were processed
using
FELIX (MSI, San Diego). The AUTOASSIGN program

[37] was used to obtain automatic assignments. All
assignments were manually checked for residue type and
compared to Calb I –II assignments [31] in homologous
regions using
FELIX (MSI software) or SPARKY [38]. Full
experimental details are included in BMRB entry 5156.
Spectra at pH 5.6 were more complete but of poorer
quality (more overlapping peaks in the area of the HSQC
corresponding to unstructured peptide). The assignment at
pH 7.7 was extended by a series of experiments at pH 6.7 as
few of the signals undergo large pH dependent changes in
chemical shift. A restricted set of triple resonance data was
collected on a sample in which the pH was lowered to 6.7,
leading to a more complete assignment.
Limited trypsinolysis of CR I–II
Tryptic digests of CR I–II were investigated using
conditions and protocols previously described for CR [39].
Briefly, 40 mg protein in 80 mL150m
M KCl, 50 mM
Tris pH 7.5 was subjected to 1 : 100 trypsin (w/w) for
0–50 min. Uncleaved (as control) and cleaved proteins
were then separated by Tris/tricine/PAGE on 15%
acrylamide gels [40]. The identity of the CR polypeptides
were confirmed by MALDI-TOF MS (CR (1–60), 7382 Da
with 7377 Da expected, and CR(61–99) 4570 Da with
4572 Da, unknown loss of last residue) and N-terminal
amino acid sequencing (GSMAG for CR(1–60), residues
22to1 3, and SDNFG for CR(61–100), residues 61–65).
Calcium-binding to the CR II EF-hand motif
Samples of GST, CR, GST–CR I, GST–CR II and standards

were separated on 10% acrylamide Tris/tricine/gels in the
presence of SDS and then electroblotted to nitrocellulose
(0.45 mm, Bio-Rad) in 20% methanol, 48 m
M Tris, 39 mM
glycine, 0.37% SDS buffer (pH 8.3) for 60 min at 0.1 A.
The method of Maruyama et al. [41] was followed with the
blot incubated in 2 m
M
45
Ca (10.5 Ci
:
g
21
Ca; ICN) in 5 mM
MgCl
2
,60mM KCl, 10 mM imidazole pH 6.8 for 10 min
followed by washing in 50% ethanol (1 min). Subseqeuntly,
blots were subjected to autoradiography on Hyperfilm
(Amersham Life Science) and developed after 24 h.
6230 M. Palczewska et al. (Eur. J. Biochem. 268) q FEBS 2001
Measurement of correlation time and translation
diffusion of CR I–II
The
15
NT
1
,T
2
and NOE relaxation data in a 1.5-mM

15
N-labeled CR I–II sample with 10% D
2
O (for field lock),
33 m
M NaCl, 10 mM CaCl
2
,50mM sodium acetate pH 5.4
at 310 K were measured in a series of two-dimentional
heteronuclear correlated spectra using the sensitivity-
enhanced gradient pulse schemes of Farrow et al. [42].
Spectral widths were 13 and 28 p.p.m. in F
2
and F
1
,
respectively, on a Bruker DRX 500 spectrometer. The
1
H
carrier frequency was placed on the water resonance and
at 118 p.p.m. for nitrogen. An in-house written
MATLAB
routine was applied to fit NMR relaxation data to
experimental parameters.
A sample of unlabeled 0.25 m
M CR I–II was prepared
in 50 m
M deuterated Tris, 25 mM NaCl pH 8.1. The
stimulated-echo with longitudinal eddy current delay
(STE-LED) method was applied to obtain diffusion ordered

spectroscopy (DOSY) spectra at 278 K [43,44]. We imple-
mented Bruker’s LEDGS2S pulse sequence [45] with a
combined watergate and presaturation sequence which
allowed measurements in H
2
O/D
2
O solution. The mean of
the diffusion profile arising from CR I–II protons between 0
and 4 p.p.m. was determined. The relative diffusion rate
measurements were carried out using known proteins as
external references or internal TSP [3-(trimethylsilyl)pro-
pionic acid, sodium salt]. Calibration proteins included
ubiquitin (8.7 kDa), Spo0F (14.4 kDa) and HP-RNAse
dimer (24.7 kDa).
Analytical ultracentrifugation of CR I–II
Experiments were carried out on a Beckman XL-A
instrument in a sedimentation equilibrium experiment at
39 000 g as described for Calb I–II [31]. Analytical
ultracentrifugation was performed at 293 K with a sample
concentration of 0.233 mg
:
mL
21
, < 20 mM as calculated
by a molar extinction coefficient of 1 ¼ 11 460 cm
21
:
M
21

.
The buffer used was 1 m
M CaCl
2
,40mM potassium
phosphate pH 6.4.
Chemical cross-linking of CR I–II
Reactions were carried out in 20 mL volumes modified from
the method described by Staros et al. [46]. six micrograms
of protein and 1 m
M CaCl
2
were initially prepared in 10-mL.
A stock solution of 10 mL 100 m
M NaCl, 100 mM Tris
pH 8.0, with 20 m
M N-hydroxysuccinimide and 8 mM 1-
ethyl-3-(3-dimethylaminopropyl)-1-napthalene-sulfonic
acid (EDC), was then added. After 60 min in the dark at
room temperature, the reactions were terminated by the
addition of 4 Â SDS sample buffer and heating at 95 8C for
5 min. The reaction mixtures were separated by Tris/tricine/
SDS/PAGE [40], together with a low-range molecular
weight marker (Promega), on 10% acrylamide gels and
stained with Coomassie blue. Bovine S100B, bovine
a-lactalbumin and horse skeletal myoglobin (all from
Sigma) were used as control protein samples. Myoglobin
and a-lactalbumin produce single bands of monomeric
weight and S100B provides a positive control with a yield of
cross-linked dimer of < 30%.

RESULTS
Definition of CR I–II structure
The CR I –II and Calb I–II sequences have high
sequence identity, Fig. 1, and many other residues display
conservative changes. The region between residues 54 and
79 of CR I – II, encompassing the linker, helix C and start of
the second calcium-binding loop, has least identity to Calb
I–II. In this region, identity persists only for hydrophobic
residues of helix C that are expected to form part of the
hydrophobic core.
Standard protocols were followed to obtain spectra
suitable for backbone assignment of calcium-bound CR
I–II (full sample details, acquired NMR experiments and
assignments at pH 6.7 and 7.7 are available from BMRB
entry 4749). The
AUTOASSIGN program [37] was used to
obtain automatic assignments. By running the program with
several sets of parameters, we were able to obtain a
consistent auto-assignment for residues T17-I36, E70-I83
and T94-N97 that were subsequently confirmed manually.
Other assignments, most notably for helix B, were obtained
by manual assignment. The assignment was extended at
pH 6.7 for residues between L13 and F98, except for K39,
E40, K53, M58 and S61. Fig. 2 shows an assigned HSQC
spectrum of CR I– II at pH 7.7.
A summary of the secondary structure elements defined
by pH 7.7 NMR data is given in Fig. 3. Four helices are
defined by chemical shift index (CSI), medium-range NOEs
and measured
3

J
HNHa
values smaller than 5 Hz (A18–D31,
G38–K53, E66–K77 and A86–L92). Three short stretches
of extended structure (E15–T17, Y35– E37 and K82 –E84)
are suggested by CSI. However, the extended structure is
supported only by consistently very strong HN
i
,Ha
(i-1)
NOEs and
3
J
HNHa
values . 7 Hz for the latter two
segments. The short segments of extended structure found
Fig. 1. Pairwise alignment of CR I–II (1– 100) and Calb I–II
(1–93) sequences (both of rat origin). Additional residues at the
N terminus, that are the result of molecular biology manipulations, are
underlined. An EF-hand consensus sequence is placed between the
aligned EF-hand I and EF-hand II sequences [3]. The helices are shown
as boxes with conserved hydrophobic (h) and other amino acids (.), the
calcium-binding ligands are designated by their coordinates: x, y, z,-y,
-x and -z [60]. Residues discussed in the text are given in bold (G34,
K60 and G81 of CR I –II and G29, G66, R68, G71 and G74 of Calb
I–II). The SeqWeb alignment is shown between the aligned EF-hand
sequences: identical residues (|), homologous residues (:) and similar
residues (.) are marked, as scored by the program. The helices defined
by Klaus et al. for Calb I– II are boxed [31] and the secondary structure
elements are labeled (helices A–D; helices pre-A and D

0
found in Calb
I–II; the linker between EF-hand motifs; Cal I and Cal II denote the two
calcium-binding loops). For comparison, the position of helices A–D is
also boxed for the CR I –II sequences, as determined by the NMR data
described in this paper (see Fig. 3). Five asterisks in the linker at the
start of EF-hand II of the Calb I–II sequence denote a five-residue long
gap compared to the CR I–II sequence.
q FEBS 2001 Characterization of calretinin domain I– II (Eur. J. Biochem. 268) 6231
in the calcium-binding loops interact in a b-sheet-like
manner. This interaction between residues Y35–E37 and
K82–E84 is supported by interstrand NOEs (I36HN–
E84HA and I36HN–I83HN). The linker could not be
assigned at pH 6.7. The CSI scores at pH 6.7 are added to
Fig. 3 as this data could not be confirmed unambiguously at
pH 7.7.
Our secondary structure assignment is consistent with
known capping motifs. T13 and Q16 provides an N-terminal
helix capping motif in ShK toxin, which plays a role in helix
stabilization [47]. CR I–II (T17 and Q20) and Calb I–II
(T12 and Q16) contain analogous residues at the N-terminus
of helix A. The residues around G54 suggest that it forms
the C-capping motif of helix B [48], reinforcing the NMR
data that helix B of CR I –II is three residues shorter than
that of Calb I–II. The residues around G65 are also con-
sistent with an N-capping motif and agree with the Calb I –II
assignment, although N63 could provide an alternative
capping site [48]. A C-capping motif for the L92P93 pair of
residues, as suggested by Prieto and Serrano [49], agrees
with our secondary structure determination of CR I–II and

is also found in Calb I–II.
Fig. 4 shows the result of limited trypsinolysis of CR
I–II. Using the conditions used previously to cleave CR, we
found that CR I–II was fully digested in < 5 min, similar to
that reported previously for CR [39]. The resulting tryptic
fragments remained relatively resistant to further cleavage
(. 30 min) in the presence of calcium. MALDI-TOF and
N-terminal amino acid sequencing confirmed that the upper
band is CR(1–60) and the lower band is CR(61–100).
Preferential cleavage of CR I–II at K60 is consistent with
the secondary structure presented in Fig. 3 and the previous
results for CR [39]. All other potential trypsin cleavage sites
are found either in helical regions (K26, K50, R52, K53,
K67, K69, K74) or in the calcium-binding loops (K39, K77,
K82).
Both EF-hands of CR I–II bind calcium
The 12-residue long calcium-binding loops of EF-hands are
readily identified from their primary sequence. Significant
deviations from the consensus sequence usually lead to a
low-affinity calcium-binding site. Here we provide experi-
mental evidence that both EF-hands of CR I–II bind
calcium. CR fragments corresponding to the first (residues
1–60) and second (residues 61–100) EF-hands were
expressed as GST fusion proteins. We found thrombin
cleaved CR fragments difficult to purify and blot. Therefore,
45
Ca-overlay experiments were performed on the fusion
products together with CR and GST as controls, Fig. 5.
Ponceau red staining of the electroblot indicated that some
cleavage of the fusion proteins occurred and a lower band,

most probably GST, was observed. We observed that CR,
but not GST (also the lower bands of lanes 2 and 3), binds
45
Ca under the conditions used. GST –CR(1–60) and GST–
CR(61–100) provide
45
Ca-overlay bands of similar inten-
sity when blotted in similar quantities. This is consistent
with both EF-hand sequences containing a single calcium-
binding site.
NMR data also provides evidence suggesting that both
EF-hands of CR I–II bind calcium. The calcium-bound
Fig. 2.
1
H,
15
N HSQC of
15
N-labeled CR I –II with assignments. Of
note are residues G34 and G81, the ‘position 6’ glycine residues.
Residues I36, E37, I83, E84 and M85 have
1
H and
15
N chemical shifts
characteristic of extended conformation (confirmed by other assign-
ments). Other resonances were assigned at pH 6.7. These assignments
are missing from this figure as they fall in the crowded part of the
spectrum and their assignments could not be transferred unambigu-
ously, even though some of these peaks are still observed in this HSQC.

A pH titration indicated that the majority of resonances did not undergo
pH-induced changes in chemical shift . 0.1 p.p.m. (
1
H) or
. 0.5 p.p.m. (
15
N). Lines connect the unassigned glutamine and
asparagine side-chain resonances.
Fig. 3. Summary of the secondary structure assignments extracted
from NMR data at pH 7.7. Medium-range NOEs are given as lines
connecting assignments between HA and i 1 3ori1 4 HN resonances
(marked as NOE i 1 3 and NOE i 1 4, derived from a NOESY-HSQC
experiment). CSI parameters, scored as 1 1, 0 or 2 1, are calculated
from the backbone assignment according to Wishart et al. [61]. Note:
the CSI data for the linker region was determined from NMR data at
pH 6.7.
3
J
HNHa
values (marked as 3J) are calculated from a HNHA
spectrum using a
MATLAB routine.
Fig. 4. Limited trypsinolysis of CR I–II. Incubation times are given
above the lanes. The two proteolytic fragments correspond to CR(1– 60)
and CR(61–100) for the upper and lower bands, respectively.
6232 M. Palczewska et al. (Eur. J. Biochem. 268) q FEBS 2001
NMR assignments of conserved glycine amide protons, at
position six of the 12-residue long calcium-binding loops of
EF-hand proteins, have chemical shifts . 10 p.p.m. for
filled loops and chemical shifts of < 8 p.p.m. for empty

loops [50]. The chemical shifts of CR I–II, Calb I – II and
several reference EF-hand proteins are given in Fig. 6. The
chemical shifts agree with biochemical data that only certain
calcium-binding sites of the reference proteins are filled
[31,50–56].
CR I–II is monomeric
15
N NMR relaxation experiments can be used to determine
global correlation times of proteins that are related to the
size of the studied molecule. Using the isotropic form of
the Lipari–Szabo analysis [57], a global correlation time
of 6.7 ^ 0.5 ns was determined for calcium-bound
15
N-labeled CR I–II at pH 5.4 and 310 K (Fig. 7A). Also,
the diffusion rate of an unlabeled CR I–II sample was
evaluated under different conditions on an arbitrary log
10
scale at pH 8.1 and 278 K (Fig. 7B). Both sets of NMR data
for CR I –II clearly fit with a monomeric mass (12 kDa)
rather than a dimeric mass (24 kDa) when compared to
reference proteins (Fig. 7A,B). The NMR data indicate that
CR I–II exists as a monomer even at protein concentrations
as high as 1.5 m
M.
Fig. 5. CR(1 –60) and CR(61–100) bind calcium. Proteins were
separated by 10% Tris/tricine/SDS/PAGE and electroblotted to
nitrocellulose as described in Experimental procedures. (A) Ponceau
red stain of blotted proteins. Lane 1, GST; lane 2, GST– CR(1–60); lane
3, GST–CR(61–100); lane 4, CR. The fusion proteins in lanes 2 and 3
appear to be partly digested; the lower band corresponds to GST. (B)

45
Ca-overlay of the blot shown in part A indicating that CR and GST
fusion products of CR fragments bind
45
Ca.
Fig. 6. ‘Glycine 6’ of the calcium-binding loop
is a sensitive indicator of calcium loading. Filled
bars denote EF-hand loops that bind calcium and
empty bars those that do not, as determined by
biochemical or crystal structure characterization
[31,50–56]. (A) Frequenin (G33, G78, G114,
G162) [51,52]. (B) GCAP-2 (G38, G72, G110,
G163) [53,54]. (C) Calb I–II (G29, G71) [31]. (D)
Nereis diversicolor sarcoplasmic Ca
21
-binding
protein (G21, G55, N109, G143) [55,56]. (E)
Calerythrin (G22, G73, G117, G151) [50]. (F) CR
I–II (G34, G81).
Fig. 7. CR I–II is monomeric. (A) Plot of global
correlation time determined from NMR relaxation
data for three reference proteins vs. their known
molecular weights (X). The reference proteins are
ubiquitin (Martek Biosciences Corporation,
Columbia), Spo0F [62] and the dimeric HP-RNase
[63]. The experimentally determined correlation
time of CR I–II is plotted against its predicted
monomeric mass (B) and the predicted dimeric
mass (A). (B) Plot of relative diffusion coefficients
for several reference proteins, as in (A) vs. known

molecular weight (X). CR I –II experimental
DOSY data is plotted against its predicted
monomeric mass (B) and predicted dimeric mass
(A).
q FEBS 2001 Characterization of calretinin domain I– II (Eur. J. Biochem. 268) 6233
Analytical ultracentrifugation showed earlier that calcium-
bound Calb I –II is dimeric [31] and this method was used
under identical conditions to establish the extent of
dimerization of CR I–II. Analytical ultracentrifugation
revealed that $ 90% of 20 m
M CR I–II exists as a monomer
in the presence of 1 m
M CaCl
2
. Possible dimer, trimer and
tetramer species accounted for , 4% each sample.
In cross-linking experiments, a single band is observed
for CR I–II (lane 4, Fig. 8) at the molecular mass expected
for monomeric protein. A small amount of cross-linked CR
I–II, < 3% according to densitomeric analysis, is probably
the result of nonspecific cross-linking as the same amount of
cross-linked dimer species was detected for the monomeric
proteins a-lactalbumin and myoglobin. S100B, which forms
both covalent and noncovalent dimers, provides a yield of
30–40%. This is an acceptable yield for this method [46].
DISCUSSION
CR I–II EF-hand structure: comparison to Calb I–II
Only a partial backbone assignment of CR I – II was possible
at pH 7.7 because the intensity of some resonances are
diminished or unobserved in HSQC-based spectra (Fig. 2).

Moreover, several weak resonances do not provide sufficient
intensity in three-dimensional NMR experiments to enable
positive assignment. It is difficult to assign spectra of CR
I–II at lower pH due to the overlap of broad resonances.
When the pH was lowered to pH 6.7, it was possible to
assign most of the resonances between L13 and F98. Many
of the new pH 6.7 assignments resonated in the region
where residues in unstructured segments of protein are
usually found and their CSI scores confirmed this. The
N terminus of CR I –II could not be assigned even at pH 6.7.
However, NMR data for Ala2 and Gly3 at pH 6.7 suggests
that these residues are in an unordered state. The NMR data
of CR I–II is consistent with an unordered N terminus and
linker.
Klaus et al. described the N-terminal region as containing
a pre-A helix and noted that it is poorly formed [31]. This
region was not detected in the NMR spectra of CR I–II
(Fig. 3). There is also no sequence homology between CR
I–II and Calb I – II for this helix (Fig. 1). The C termini of
the CR I–II and Calb I–II domains contain a consensus
sequence (ENFLLXF, where X is a variable residue), which
is only partly observed in Fig. 1. This segment is present in
Calb and CR sequences of all species as well as the recently
described secretagogin sequence [58]. We do not observe
helix D
0
in CR I–II (Fig. 1) possibly because CR I–II is
three residues shorter than Calb I–II.
The secondary structures of CR I–II and Calb I–II are
almost identical within the EF-hand regions (see Fig. 1).

Helices A, C and D, together with the three-residue b-type
cross-strand interaction between the two calcium-binding
loops, are in identical positions. However, helix B of CR
I–II is three residues shorter than helix B in Calb I –II. A
number of identified helix capping motifs are consistent
with the determined secondary structure of CR I–II, includ-
ing the shorter helix B of CR I–II. Limited trypsinolysis of
CR I–II leads to fragmentation that is consistent with the
defined secondary structure: K60 is in the linker whereas the
other 10 potential cleavage sites are found in helical regions
or in the calcium-binding loops that scaffold the calcium-
binding ligands and are consequently protected. Helix B of
CR I–II is still longer than predicted by the EF-hand
consensus sequence, an unusual feature first described for
Calb I–II. The CR I–II sequence also has a five-residue
insertion in the linker compared to Calb I–II (see Fig. 1).
Therefore, the linker of CR I–II is 12 residues long
compared to only four residues in Calb I–II.
CR I–II binds two calcium cations
Based on a comparison with the consensus sequence, both
EF-hands of CR I–II are predicted to bind calcium. On the
other hand, the residues at positions 66, 68 and 74 (G66, R68
and G74) of Calb I–II lack the side-chain oxygens expected
to provide calcium-binding ligands. The absence of
calcium-binding to the EF-hand II motif of Calb has been
shown in the individual polypeptide, Calb I–II, Calb
mutants and full-length Calb using a number of experi-
mental techniques in several laboratories [26–29]. The
analogous residues of CR I– II (D76, N78 and E84), as well
as the whole second loop sequence, have suitable ligands

with side-chain oxygens where the consensus sequence
requires them for calcium binding. The second EF-hand of
CR I–II has a potential high calcium affinity.
The principal limited proteolysis products of CR I–II
conveniently correspond to individual EF-hand motifs
(residues 1–60 and 61– 100). We found that the PAGE
separated limited trypsinolysis products of CR I–II bound
45
Ca. However, GST fusion products of the same fragments
were produced in order to clarify our data due to the
poor blotting efficiency of the small CR fragments. Both
CR(1–60) and CR(61–100) bind
45
Ca as GST fusion
products. Individual synthetic Calb EF-hands I, III, IVand V
bind calcium [29]. This data of A
˚
kerfeldt et al. [29] agrees
with the large body of independent data collected on Calb
and truncated Calbs by Kumar’s group. This latter work
revealed that EF-hands II and VI of Calb do not bind
calcium and that Calb binds a total number of four calcium
cations [26–28]. Calcium-dependent NMR data for
‘position 6’ glycine residues also support calcium-binding
to both EF-hands of CR I–II (Fig. 6) but only to EF-hand I
of Calb I– II. These glycine chemical shifts report the
formation of a strong interaction between the glycine
amide and a carboxylate side-chain. This has been shown
empirically to occur when calcium ions are bound to the site.
In summary, the

45
Ca-binding data for CR fragments and
Fig. 8. 10% Acrylamide gel run under Tris/tricine/SDS/PAGE
conditions and stained with Coomassie blue showing the products of
chemical cross-linking of 0.3 mg
:
mL
21
protein in the presence of
1m
M CaCl
2
. Lane 1, S100B; lane 2, a-lactalbumin; lane 3, myoglobin;
lane 4, CR I–II; molecular weight standards are indicated to the left.
6234 M. Palczewska et al. (Eur. J. Biochem. 268) q FEBS 2001
NMR data for CR I–II indicate that the stoichiometry of
calcium binding in CR I–II is 2 : 1.
Calcium-bound CR I–II is monomeric
We speculate that the Calb I –II homodimer structure
mimics the interaction between Calb I –II and other parts of
the Calb sequence [31], in accordance with the Linse model
where all six EF-hand motifs of Calb interact within a single
globular domain structure [59]. In contrast to Calb I–II, CR
I–II is monomeric under various conditions. We have
not observed an interaction between CR I–II and various
complement CR fragments using fluorescence, chemical
cross-linking and size exclusion chromatography
(M. Palczewska, P. Groves & J. Kuz
´
nicki, unpublished

data). Therefore, we believe that CR does not form a single
globular domain. Perhaps the different oligomeric states of
CR I–II and Calb I–II are an indication of different
organizations of EF-hands within full-length CR and Calb.
CR I–II and Calb I–II may have different structures
CR I–II and Calb I–II have different biochemical
properties, despite their similar secondary structures. The
four-residue linker of Calb I –II could restrict the relative
positions of helices B and C to a greater degree than the
12-residue linker of CR I–II. The low-resolution NMR
structure of calcium-bound Calb I–II suggests that the
second calcium-binding loop may contain a distorted con-
formation [31]. We show that the second calcium-binding
loop of CR I–II binds calcium and is therefore expected to
adopt a characteristic conformation found in other EF-hand
protein structures. We predict that an overlay of future
high-resolution CR I–II and Calb I–II structures will reveal
that the largest structural differences lie in the orientation
of helix C, together with the adjacent linker and second
calcium-binding loop structures. There is a region of poor
sequence identity localized to residues 54–79 of CR I–II
that supports this prediction (Fig. 1). High resolution
structures of both apo- and calcium-bound domains are
essential to understand fully the differences in CR I–II and
Calb I–II structure and properties.
Are the functions of CR and Calb distinct?
A large body of physiological data supports a calcium-buffer
role for both Calb and CR. However, biochemical (and some
physiological) data suggest sensor roles, as do the inter-
action of Calb with at least two other proteins [20,21]. The

ongoing high-resolution structure projects on both CR I–II
and Calb I–II will provide a clearer answer as to the nature
of the calcium-induced structural changes, differences in
structure and clues to the function of the full-length proteins.
The present work establishes that the first domains within
CR and Calb have distinct structural features and bio-
chemical properties, and potentially perform different
functions within the full-length proteins.
ACKNOWLEDGEMENTS
We are greatly indebted to Werner Klaus (F. Hoffmann-LaRoche AG,
Basel) for discussions about the different oligomeric states of Calb I–II,
the subject of his earlier studies, and CR I–II. Francis Mueller and
Eric Kusznir (F. Hoffmann-LaRoche AG, Basel) determined the
analytical ultracentrifugation data on CR I–II under the same
conditions as used earlier for Calb I –II. Sa
´
ndor Ke
´
ki (Chemistry
Department, Debrecen) determined MALDI-TOF data for the limited
proteolysis products of CR I–II. We thank Walter Chazin (Vanderbilt
University, Nashville) for critical evaluation of the manuscript and
Barbara Zarzycka (Warsaw) for her technical assistance. This work was
supported by an International Center for Genetic Engineering and
Biotechnology (ICGEB) grant to J. K. [CRP/Pol97-01(t1)] and G. B.
[CRP/Hun97–01(t1)]. P. G. is supported by a grant from the State
Committee for Scientific Research, KBN no. 6 P04B 01015. G. B.
thanks the support of the Hungarian National Fund, OTKA T-029089.
The Bruker DRX 500 spectrometer used in these studies was purchased
from the following grants: PHARE-ACCORD H-9112-0198, OMFB

MEC 93-0098 and OTKA A084.
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