From the Architecture et Fonction des
Macromolécules Biologiques, CNRS UMR-6098, 13402, Marseille Cedex
20, France, the
Universitaet Hamburg, Zentrum für
Molekulare Neurobiologie, Institut für Neurale
Signalverarbeitung, D20249 Hamburg, Germany, and the ** Ingénierie
des Protéines, CNRS UMR-6560, Institut Fédératif de
Recherche Jean Roche, Université de la Méditerranée,
13916 Marseille Cedex 20, France
Received for publication, October 13, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Frequenin, a member of a large family of
myristoyl-switch calcium-binding proteins, functions as a calcium-ion
sensor to modulate synaptic activity and secretion. We show that human
frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in
similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4K Frequenin (Frq),1 or
neuronal calcium-sensor 1, is a member of a family of related
calcium-myristoyl-switch proteins that have been proposed to function
as calcium-ion sensors. Members of this family include recoverin, GCAP,
neurocalcin, visinin, and others (1). Recoverin and GCAP have been
implicated in the control of recovery and adaptation in visual signal
transduction. In vertebrate rod outer segments, GCAP apparently
inhibits guanylate cyclase when the cytosolic concentration of
Ca2+ is high in the dark, whereas recoverin may inhibit
rhodopsin kinase (2, 3). Lowering the cytosolic Ca2+ during
light illumination attenuates the inhibitory activities of GCAP and
recoverin, leading to an activation of these enzymes.
Frq, on the other hand, has attracted much attention because it may
function as a calcium-ion sensor to modulate synaptic activity and
secretion (4-7). Drosophila Frq has been implicated in the
facilitation of neurotransmitter release at neuromuscular junctions of
third instar larvae (Drosophila melanogaster). Drosophila mutants that overexpress Frq show a facilitated neurotransmitter release that dramatically depend on the frequency of stimulation (4).
Similarly, overexpression of a rat homolog of Frq in PC12 cells evokes
an increased release of growth hormone in response to agonists like ATP
(5). These results are consistent with the idea that Frq activity and
regulated secretion are coupled.
More recently, it has been shown that the yeast homolog of Frq
functions as a Ca2+-sensing subunit of the yeast
phosphatidylinositol (PtdIns)-4-OH kinase, PIK1, a key enzyme in the
phosphoinositide signaling system (6). In Saccharomyces
cerevisiae, PIK1 participates in the mating pheromone-signal
transduction cascade and regulates secretion at the Golgi. PIK1 is
essential in yeast cells for normal secretion, Golgi and vacuole
membrane dynamics, and endocytosis (7). Yeast PIK1ts
mutants exhibit severe protein trafficking defects and accumulate morphologically aberrant Golgi membranes (8). The aberrant Golgi
morphology is strikingly similar to that found in yeast cells lacking a
functional ARF1 GTPase (7). ARF1 has been implicated in multiple
membrane trafficking events including the recruitment of the mammalian
PIK1 homolog, PI4K Here we show that human Frq (HuFrq) colocalizes with ARF1 in COS-7
cells and occurs in similar cellular localizations as PI4K HuFrq Cloning, Expression, and Purification--
Human
poly(A)RNA was isolated from HEK293 cells using the Fast Track II Kit
(Invitrogen), and cDNA was synthesized with Superscript II Reverse
Transcriptase (Life Technologies, Inc.). The HuFrq-encoding cDNA
was amplified in a polymerase chain reaction (PCR) using PfuTurbo-Polymerase (Life Technologies, Inc.) with the first
strand cDNA as template and the primers
5'-ATACCATGGGGAAATCCAACAG-3' (sense) and 5'-CTATACCAGCCCGTCGTAGAGG-3'
(antisense). Primer sequences were derived from the HuFrq
nucleotide sequence (GenBankTM/EBI accession no. AF186409).
The restriction sites NcoI and NdeI were used
for subcloning into expression vector pET-16b (Promega) to generate
expression plasmid pET-HuFrq. All nucleotide sequences were verified by
automated sequencing.
The expression plasmid pET-HuFrq was transformed into Escherichia
coli strain BL21(DE3) (Novagen). Transformed cells were grown in
Luria-Bertani (LB) medium containing ampicillin (100 µg/ml) at
37 °C. HuFrq expression was induced overnight at an A600 of 0.8 with 0.5 mM
isopropyl-1-thio- Site-directed Mutagenesis--
Point mutations into
Frq cDNA were introduced by PCR using the following
mutation primers: 5'-GGCAGGATCGTGTTCTCCGAATTC-3' and 5'-TCGGAGAA
CACGATCCTGCCAT-3' for E81V (EF2); 5'-ACATCGCCAG AAACGAGATGCTG-3' and
5'-CTGGTTTCTGC CGATGTAGCCGTC-3' for T117A (EF3);
5'-GGGAAGCTAGCTCTTCAGGAGTTC-3' and 5'-CCTGAAGAGCTAGCTTCCCATCA-3' for
T165A (EF4). PCR fragments were cloned into pET16b and sequenced before use.
45Calcium Binding Assay--
Protein samples were
resolved by SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. The 45Ca2+ blotting was
performed as previously described (10); autoradiographic exposure time
was 2 days.
Immunofluorescence Microscopy--
The polyclonal anti-HuFrq
antibodies were raised in rabbit against purified HuFrq and purified by
affinity chromatography on HuFrq-Sepharose 4B according to the
manufacturer's instructions (Amersham Pharmacia Biotech); specificity
analyses performed by enzyme-linked immunosorbent assay showed full
recognition of the immunizing HuFrq but no recognition of either
recoverin or neurocalcin (11). COS-7 cells were cultured at 37 °C in
Dulbecco's modified Eagle's medium (Life Technologies) supplemented
with 10% (v/v) fetal calf serum (Life Technologies). They were grown
to 60% confluency on poly-L-lysine-coated
glass-coverslips. The cells were fixed with 4% (v/v) paraformaldehyde
in phosphate-buffered saline for 15 min at room temperature, washed
twice with phosphate-buffered saline and blocked with 1% (v/v) goat
serum and 1% (w/v) bovine serum albumin in phosphate-buffered saline.
Permeabilization used a blocking solution containing 0.3% (v/v) Triton
X-100. Successive incubations with primary and secondary antibodies
were carried out for 1 h at room temperature. Cells were washed in
phosphate-buffered saline, and coverslips were mounted with Fluoromount
GC (Southern Technologies). The cells were visualized and confocal
images acquired using a confocal laser scanning microscope (Leica TCS
NT). Primary antibodies were detected by species-specific cyanine
dye-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories): Cy3-conjugated antibodies were used for visualization of
anti-HuFrq and anti-PI4K Crystallization and Data Collection--
Crystals were obtained
at 4 °C using the vapor diffusion technique. Typically, 5 µl of
the HuFrq solution were mixed with 5 µl of a reservoir solution made
of 0.1 M sodium cacodylate, pH 6.5, 0.2 M NaAc,
30% polyethylene glycol Mr 8,000 (Crystal Screen I, solution 28, Hampton Research). Under rare circumstances, three different crystal forms grew from this solution: needle-like (form A), thin plates (form B), and thick plates (form C). Form A
crystals belong to the hexagonal space group P61/5 with
unit cell dimensions: a = b = 82.2 Å and c = 56 Å and contains one HuFrq molecule per
asymmetric unit. Both form B and C crystals belong to the monoclinic
space group P21 with unit cell dimensions: a = 53.8 Å, b = 55.5 Å,
c = 77.7 Å, Structure Determination and Refinement--
Initial phases for
form B crystals were obtained by molecular replacement using the
structure of neurocalcin (14) (PDB code 1BJF) as a search model with
the AMoRe package (15), giving a correlation coefficient of 36% and an
R-factor value of 48% in the 15 to 4 Å resolution range.
Rigid-body refinement, performed on each molecule with CNS (16) using
data between 20 and 3 Å, gave an R-factor of 49%. For 2%
of the reflections against which the model was not refined,
R-free was 48%. The model was refined to 1.9 Å resolution
using CNS, including bulk solvent and anisotropic B-factor corrections;
the resulting 2Fo-Fc and Fo-Fc electron density maps were used to
correct the model with the graphics program TURBO-FRODO (17). Solvent
molecules automatically added using CNS were carefully examined on the
graphics display. The final model comprises residues
Asn5-Val190 and
Asn5-Gly188, respectively, for the two
molecules in the asymmetric unit. High temperature factors and weak
electron density are associated with residues 1-7, 49-60, and
133-138. The average r.m.s.d. between the two HuFrq molecules is 0.6 Å for 182 C Chromosomal Localization--
A search in the HTGS data bank
with the HuFrq cDNA sequence revealed that the
HuFrq gene is located on chromosome 9. A comparison of the
publicly available chromosome 9 DNA sequence with the frequenin cDNA sequence showed that the exon-intron organization between Drosophila (4) and HuFrq genes has been
conserved. Both open reading frames are interrupted by the same
exon-intron borders and each are composed of same 8 exons (Fig.
1A). About 50 kilobases upstream of the first Frq exon are located several STS markers, e.g. DGS1924, A001W37, STSG22304, placing the Frq
gene at 9q34.11. To our knowledge, a human disease has not been
associated yet with this locus.
Molecular Identification and Immunocytochemical
Localization--
Previously, mammalian homologs of
Drosophila Frq have been cloned (22, 23). We have used this
information to clone HuFrq from human first strand cDNA (Fig.
1A). The predicted HuFrq sequence contains 190 amino acids
(Fig. 1B) with a theoretical monoisotopic mass of 21,865.91 and exhibits four EF hand motifs that represent potential
Ca2+-binding domains. In HuFrq, as in other members of the
family, the first of the four EF hand motifs is not likely to be a
functional Ca2+-binding site as it lacks two
Ca2+-coordinating amino acids. The HuFrq N terminus
contains the consensus sequence MGXXX(S/T)K for
myristoylation (24); hence it could be myristoylated like the rat
homolog (5). The HuFrq sequence is 100% homologous to those of rat (5)
and mouse (23) and it differs by a single amino acid from that of
Xenopus (25). Remarkably, the yeast (6) and HuFrq protein
sequences also show a high degree of conversation: 143 of 190 amino
acids (75%) are either identical or correspond to conservative
replacements (Fig. 1B).
In yeast, Frq has been shown to stimulate the activity of the
PtdIns-4-OH kinase PIK1 (6), an enzyme that is essential for normal
secretion, Golgi and vacuole membrane dynamics, and endocytosis (7).
Xenopus Frq rescues a yeast Frq deletion mutant, indicating
that Frq from higher eukaryotes is able to fulfill similar functions
like yeast Frq (25). Consistent with the functional role of Frq in
yeast are the phenotypes that have been described for
Drosophila mutants (4) and for mammalian cells
overexpressing Frq (5). In both cases, it appears that evoked secretion
is stimulated by Frq (26).
The activity of PI4K Calcium-binding Properties--
We mutated EF hands EF2, EF3, and
EF4 of HuFrq together or in pairwise combinations utilizing in
vitro mutagenesis. In all four cases, we mutated the amino acid
residues at the -X position as previously described for
Drosophila Frq (Fig. 1B) (4). Accordingly, we
generated four HuFrq mutants: E81V/T117A/T165A (Frq2,3,4),
E81V/T117A (Frq2,3), E81V/T165A (Frq2,4), and
T117A/T165A (Frq3,4). Bacterial lysates containing
approximately equal amounts of each HuFrq mutant were blotted onto
nitrocellulose. The blot was incubated with
45Ca2+ to investigate the
Ca2+-binding capacity of the HuFrq mutants in comparison to
wild-type HuFrq (Fig. 3). The results
showed that wild-type HuFrq yielded the highest
45Ca2+ signal. We noted for the recombinant
HuFrq mutants with pairwise mutations attenuated
45Ca2+-signals of comparably reduced intensity.
The pairwise HuFrq mutants each contained a single intact EF hand (EF2
in Frq3,4, EF3 in Frq2,4, and EF4 in
Frq2,3), yet they bound Ca2+ with high
affinity; this suggests that EF hands EF2, EF3, and EF4 not only are
functional in HuFrq but also are independent from each other.
Previously, it was shown that single mutations in yeast Frq1 EF hands
did not display a temperature-sensitive phenotype like the quadruple
mutant in the frq1-Its allele, consistent with our
observations. By contrast, the triple mutant Frq2,3,4 did
not bind 45Ca2+ to a significant extent; hence
in HuFrq, EF hand 1 does not constitute a high affinity
Ca2+-binding site in HuFrq, as predicted earlier from
sequence analysis.
Overall Structure--
The crystal structure of HuFrq was solved
by the molecular replacement method using neurocalcin (14) as a search
model and was refined to 1.9 Å resolution. The structure consists of
residues Asn5-Val190 with good
stereochemistry; clear electron density maps could be observed for all
structural elements (Fig. 4A).
As predicted, HuFrq shares the typical Structural Comparison with Homologous Proteins--
For residues
8-175, the HuFrq overall fold is similar to that of neurocalcin (14)
with an r.m.s.d. value of 1.3 Å for 164 C Novel Hydrophobic Crevice--
The dominant and striking feature
of the HuFrq structure is the large positional shift of the C-terminal
helix J compared with its position in the recoverin and neurocalcin
structures. Indeed, helix J has moved by 45° as a hinge-type
rigid-body motion, with the residue pair
Asp176-Pro177 acting as a pivot point, to adopt
an original position that is well ordered in our structure (Fig.
5, A and C). As a
result, the helix is tightly packed against loop
The major structural significance of the helix J repositioning for
ligand binding is obvious from the complete exposure of the hydrophobic
surface region. This new conformation dramatically alters the
solvent-accessible molecular surface of the molecule when compared with
those of neurocalcin or recoverin. In HuFrq, a large patch of
hydrophobic residues line the crevice and still are fully accessible to
the solvent (Fig. 5A), whereas they are sequestered into the
neurocalcin and recoverin protein cores (Fig. 5, B and
C). Actually, fragments of the polyethylene glycol used for
crystallization are found well ordered within the crevice, where they
could mimic an interacting ligand/membrane partner and shield the
crevice from the solvent. Presence of the detergent octyl-glucoside was
a requisite to solubilize and reduce aggregation of yeast Frq (29). In
HuFrq, the unique disordered surface loop, loop 133-137, along with
the C-terminal part of helix C that also presents high mobility, are
located at the periphery of the crevice and may also play a role upon
ligand/membrane recognition.
Is this The Calcium Myristoyl Switch--
HuFrq may share the molecular
mechanisms of calcium-myristoyl switch proteins as seen from the
solution structures of recoverin, where the myristoyl group is
sequestered in a deep hydrophobic box in the Ca2+-free
state (33) and ejected into the solvent to interact with a lipid
bilayer membrane in the Ca2+-bound state (34). HuFrq
possesses two discrete conformations at the hinge point
Lys7-Leu8 in the N-terminal region, which
therefore is a flexible arm. Residues Gly42 and
Gly96 are the two hinge points that distinguish between the
Ca2+-free and Ca2+-bound conformations in
recoverin, and these two residues are conserved in HuFrq. Of the five
helices that participate in the formation of the myristoyl box in
recoverin, four (helices B, C, E, and F), line the large crevice in
HuFrq. In addition, most of the hydrophobic residues that are recruited
to accommodate the myristoyl group in recoverin are conserved in HuFrq
and are solvent-exposed at the crevice surface. In HuFrq, residue
Gly96, which is one of the two hinge points in recoverin,
establishes van der Waals contacts with helix J, suggesting that a
conformational shift at this position might modify the position of this
helix. Would HuFrq use the same structural switch as seen in recoverin, the shape and/or size of its hydrophobic crevice would be dramatically altered, a modification that could reflect a regulatory mechanism. Further biochemical experiments must await production of myristoylated HuFrq to test this hypothesis, although recent studies suggest that the
presence of a N-myristoyl group in yeast Frq does not affect its
overall structure whether Ca2+ is present or not (29).
Rat Frq, as opposed to recoverin, neurocalcin, and hippocalcin, showed
a Ca2+-independent interaction with membranes (35),
suggesting that the presence of the hydrophobic crevice, so far a
feature unique to the Frq, may account for this difference in calcium
dependence. As well, yeast Frq binds to and activates PIK1 in a
Ca2+-independent manner (6), a feature also consistent with
our proposal of the hydrophobic crevice as a functionally important binding site. In contrast, the Ca2+-enhanced interaction of
yeast Frq with membranes (29) would suggest that in
Ca2+-free yeast Frq the shape of the hydrophobic crevice
might be altered.
In summary, we have cloned HuFrq from human cDNA and analyzed its
cellular localization in COS cells. HuFrq was overexpressed in E. coli, purified to homogeneity, and crystallized in the
Ca2+-bound state. This Ca2+-bound HuFrq
structure is a critical step toward identification of a physiological
protein partner using biological experiments. Further crystallographic
investigations will help identify the interactions involved in complex
formation and conformation.
, the mammalian homolog of the yeast kinase PIK1. In
addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 Å resolution. The
overall fold of frequenin resembles those of neurocalcin and the
photoreceptor, recoverin, of the same family, with two pairs of
calcium-binding EF hands and three bound calcium ions. Despite the
similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.
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, to the Golgi membrane (9).
. In
addition, in a further step toward understanding the cellular function
of Frq, we report the crystal structure of unmyristoylated Ca2+-bound human Frq (HuFrq) refined to 1.9 Å resolution.
This structure confirms that frequenins belong to the large family of
myristoyl-switch Ca2+-binding proteins and reveals the
architecture of the Ca2+-binding sites. Most importantly,
comparative analysis of the HuFrq structure with those of neurocalcin
and recoverin highlights a unique wide crevice and a solvent-exposed
carboxyl terminus that could be responsible for ligand recognition and
account for the broad substrate specificity among members of the family.
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-D-galactopyranoside and reached
average yields of 20 mg/liter. Cells were harvested by centrifugation,
resuspended in 20 ml of lysis buffer (50 mM HEPES, pH 7.4;
100 mM KCl; 1 mM EGTA; 1 mM
dithiothreitol; 1 mM MgCl2) per 1 liter medium,
and lysed in a French pressure cell. Protamine sulfate was added to a
final concentration of 0.1% for 10 min and then the lysate was cleared
by centrifugation (40,000 × g, 30 min, 4 °C). The
supernatant was filtered (0.45 µm), adjusted to 1 mM
CaCl2, and applied to a 15-ml phenyl-Sepharose CL4-B column (Amersham Pharmacia Biotech). The column was washed with buffer A (20 mM Tris/HCl, pH 7.9; 1 mM MgCl2; 1 mM dithiothreitol) containing 1 mM
CaCl2 until A280 was below 0.01, and
then the protein was eluted with buffer A containing 2 mM
EGTA. The eluate was applied to a 3-ml HiTrapQ column (Amersham
Pharmacia Biotech). The column was washed with buffer A containing 60 mM NaCl and 1 mM CaCl2, and eluted
with 120 mM NaCl in buffer A. The HuFrq-containing fractions were extensively dialyzed against water and concentrated to
10 mg/ml using microconcentrators (Pall Filtron). MALDI-TOF analysis of
the purified HuFrq used the linear mode and a 337-nm nitrogen laser
(Voyager-DETMRP BioSpectrometer work station, Perseptive Biosystems).
(Upstate Biotechnology) staining, whereas
Cy2- and Cy5-conjugated antibodies were used for anti-
-adaptin
(Sigma) and anti-ARF1 (Santa Cruz Biotechnology) staining, respectively.
= 107.6°, and a = 29.6 Å, b = 105.2 Å, c = 55.2 Å,
= 106.6°, respectively, and contain two HuFrq molecules per
asymmetric unit. Crystals selected for data collection were briefly
soaked into the reservoir solution supplemented with 10% (v/v)
ethylene glycol, flash-cooled at 100 K in the nitrogen gas stream and
stored in liquid nitrogen. No single crystal could be selected for form
C. Data for forms A and B were collected on beamline ID14-EH2 of ESRF
(Grenoble, France). Oscillation images were integrated with DENZO (12)
and scaled and merged with SCALA (13). Amplitude factors were generated with TRUNCATE (13). Form A crystals were found to be twinned and the
collected data could not be used.
atoms with the largest deviation (1.4 Å) for residue
Gln54. The stereochemistry of the model was analyzed with
PROCHECK (18); no residues were found in the disallowed regions of the Ramachandran plot. Data collection and refinement statistics are summarized in Table I. The coordinates
and structure factors of HuFrq have been deposited with the Protein
Data Bank (1G8I). Fig. 1B was generated by ALSCRIPT (19) and
Figs. 4-5 with SPOCK (20) and Raster3D (21).
Data collection and refinement
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Fig. 1.
Exon-intron map of the HuFrq
gene and sequence. A, black boxes correspond to
the open reading frame and broken lines to introns.
Because the available genomic DNA sequence data of the HuFrq
gene are not contiguous, exact sizes of introns are not known.
Likewise, the 5'- and 3'-ends of Hufrq mRNA have not
been mapped. Exons are drawn to scale as indicated by the
bar. Exons 1 and 7 may contain untranslated sequence as
indicated by dotted lines. The open reading frame in the
first exon starts at nucleotide 155870 of the genomic clone
AC006241. The STS marker D9S1924 is located at map position 9q34.11
corresponding to nucleotides 121075-121194. B, amino acid
sequence alignment of HuFrq with homologous proteins. Invariant
residues within HuFrq are highlighted in white with a
black background. The secondary structural elements are
shown in gray with the four EF hands labeled EF1 to EF4.
Ca2+-bound residues are indicated by gray dots
and residues that are solvent exposed in the crevice by black
dots. The N-terminal myristoylation site is underlined,
and the mutated residues are indicated with black
dots.
, the likely mammalian homolog of
yeast PIK1 (26), is recruited by the small GTPase ARF1 to the Golgi and
contributes to the regulation of Golgi membrane dynamics and
Golgi-dependent vesicle formation (9). Accordingly, in
immunocytochemical experiments we have compared the immunostaining patterns obtained with anti-PI4K
, anti-ARF1, and anti-HuFrq
antibodies, respectively; overlapping immunostaining reactions were
observed (Fig. 2). For comparison, we
also included in our investigations experiments with anti-
-adaptin
antibodies, a typical trans-Golgi network (TGN) marker.
Paraformaldehyde-fixed COS-7 cells were first incubated with primary
antibodies, e.g. polyclonal anti-HuFrq rabbit antibodies,
monoclonal anti-
-adaptin mouse antibodies, polyclonal anti-PI4K
rabbit antibodies, and polyclonal anti-ARF1 goat antibodies,
respectively. Then, we used secondary Cy2-, Cy3-, or Cy5-labeled
antibodies for immunocytofluorescent staining and localization of
-adaptin, HuFrq and PI4K
, and ARF1, respectively, using confocal
microscopy (Fig. 2). The anti-HuFrq antibodies revealed a pattern with
a crescent of staining on one side of the nucleus and some punctuate
staining within the cytoplasm (Fig. 2A). Staining could be
eliminated by preincubation of the primary antibody with the immunizing
HuFrq protein (not shown), indicating that the observed
immunofluorescence is generated by HuFrq-specific antibodies. A similar
staining pattern was obtained with
-adaptin (Fig. 2B),
which in double-labeling experiments colocalized with the
HuFrq-immunostaining pattern (Fig. 2C). Previously,
-adaptin has been shown to be localized in the TGN and the late
endosomes (27). The double-immunostaining patterns also indicate a
colocalization for
-adaptin and PI4K
(Fig. 2, D-F) in
agreement with a recent report (28). Finally, we coimmunostained the
COS-7 cells with anti-HuFrq and anti-ARF1 antibodies (Fig. 2,
G-I). Again, we observed a crescent of staining on one side
of the nucleus (presumably the TNG) and some punctuate immunostain
extending to the plasma membrane, which colocalized with the HuFrq
immunostain (Fig. 2I). The results indicate similar
subcellular distributions for HuFrq, ARF1, and PI4K
. The
colocalization is consistent with the proposal that frequenin proteins
modulate PtdIns-4-OH kinase activity both in yeast and in mammalian
cells and thus may have similar regulatory functions in secretion and
Golgi membrane dynamics.
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Fig. 2.
Immunocytofluorescent localization of HuFrq
in COS-7 cells. COS-7 cells were double stained with anti-HuFrq
(A) and anti- -adaptin antibodies (B).
Anti-HuFrq was detected by Cy3-conjugated goat anti-rabbit antibodies
and anti-
-adaptin by Cy2-conjugated goat anti-mouse antibodies.
C, confocal microscopy emphasizing the colocalization of
HuFrq and the Golgi marker
-adaptin (D-F). COS-7 cells
were double stained with anti-PIK4
(D) and
anti-
-adaptin antibodies (E). Anti-PIK4
was detected
by Cy3-conjugated goat anti-rabbit antibodies and anti-
-adaptin as
in B. F, confocal microscopy emphasizing the
colocalization of
-adaptin and PIK4
. G-I, COS-7 cells
were double stained with anti-HuFrq (G) and anti-ARF1
antibodies (H). Anti-HuFrq was detected as in A and
anti-ARF1 by Cy5-conjugated mouse anti-goat antibodies.
I, confocal microscopy emphasizing the colocalization of
HuFrq and ARF1. Scale bars in C, F, and I are representative
for all images shown at same magnification.
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Fig. 3.
Ca2+-binding sites of HuFrq.
45Ca2+-binding assay is shown. Bacterial
lysates of E. coli overexpressing HuFrq (wt) or
HuFrq mutants E81V/T117A/T165A (Frq2,3,4), E8IV/T117A
(Frq2,3), E81V/T165A (Frq2,4), and T117A/T165A
(Frq3,4) were resolved in 12% polyacrylamide, 0.1% SDS
gels. Gels were Coomassie Blue-stained (bottom) or blotted
in parallel to nitrocellulose for 45Ca2+
incubation (top) as described under "Experimental
Procedures." Lysates of E. coli transformed with an empty
expression vector were used for control.
-helical fold found in
homologous proteins, with overall dimensions of 35 × 60 × 40 Å. HuFrq contains 10 helices labeled A to J (Fig. 4B).
Consistent with the recently proposed NMR-derived model of yeast Frq
(29), the four EF hands, which all present the typical architectural
short
-strand, come in two pairs: an N-terminal pair (EF1, EF2) and
a C-terminal pair (EF3, EF4), which are connected by the hinge loop
93-97 and related by an approximate 2-fold axis. The hinge loop
conformation positions the four EF hands on one side of the molecule in
a tandem linear array. This structural arrangement is similar to the
ones seen in other members of the myristoyl-switch
Ca2+-binding proteins, e.g. neurocalcin,
recoverin, and GCAP (1). It is, however, different from the dumbbell
arrangement found in e.g. calmodulin and tropinin C (30).
The HuFrq structure contains three calcium ions bound to EF hands EF2,
EF3, and EF4. The Ca2+ coordination in HuFrq is virtually
identical to recoverin and neurocalcin. The side chains of residues x,
y, and z in the 12-residue loop each provide an oxygen atom, as does
the main chain carbonyl of residue y. Two additional oxygen atoms come
from the conserved glutamic side chain of residue z and a water
molecule.
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Fig. 4.
Quality of the map and overall fold of
HuFrq. A, stereo view of the 1.9 Å resolution omit
2Fo-Fc averaged electron density map, contoured at 1 , showing part
of helix J. The coordinates of this region were omitted and the protein
coordinates refined by simulated annealing before the phase
calculation. B, stereo ribbon diagram of HuFrq
with bound Na+ and Ca2+ ions. Secondary
structure elements are indicated as in Fig. 1. Labels Ca2,
Ca3, and Ca4 refer to the Ca2+ ions
bound to EF hands EF2, EF3, and EF4, respectively. Functionally
important side-chain residues in the vicinity of helix J are shown as
blue/orange bonds with red oxygen and
blue nitrogen atoms. Hydrogen bonds are shown as
dotted lines. C, stereo overlay of HuFrq
(yellow/cyan) and neurocalcin (orange/green)
oriented as in B. Helix J is highlighted in cyan
for HuFrq and green for neurocalcin.
atoms (Fig.
4C). Extended comparison with the structure of recoverin
(31) yields a r.m.s. deviation of 1.7 Å for only 123 C
atoms. These
values indicate that the HuFrq structure is more closely related to
that of neurocalcin. This reflects the high sequence identity existing
between the two proteins, which is 61% for the whole molecule,
compared with only 41% between HuFrq and recoverin. However, whereas
neurocalcin dimerizes in solution (14), no dimeric assembly could be
observed for HuFrq in concentrated solution or in the crystals (data
not shown); this suggests that the distinct oligomeric assembly of
these proteins may be related to their biological functions.
E-
F where it
establishes numerous polar and hydrophobic interactions, consistent
with an average mobility similar to that of other structural elements in the molecule. As a consequence of helix J motion, a large
hydrophobic crevice, with dimensions 30 × 15 × 15 Å, is
unmasked on the HuFrq face that is opposite to the four EF hands (Fig.
5A). The crevice is made of 46 residues provided by 8 helices, of which helices D, E, and F contribute to the floor of the
crevice, helices C, G on one side and helix J on the other side
contribute to the walls, and helices H and B contribute to the top and
bottom, respectively. Hence, a quarter of the HuFrq molecule
contributes to the structure of the crevice.
View larger version (49K):
[in a new window]
Fig. 5.
Structural differences between HuFrq and
homologous proteins. Molecular surface of HuFrq (A),
viewed down the large hydrophobic crevice (orange) and
oriented as in Fig. 2., neurocalcin (B) and recoverin
(C) (same orientation). Secondary structure elements are
labeled. The C-terminal helices, helix J in HuFrq (cyan) and
in neurocalcin (green) and helices J and K in recoverin
(green) are displayed.
-helix conformational shift specific to HuFrq? To our
knowledge, such a large positional shift in the C-terminal portion is
novel and unique to HuFrq compared with other members of the family and
exemplifies how a structurally related fold can exhibit a distinct
binding site. Within the crystal asymmetric unit, a sodium ion is bound
in the N-cap of helix J and connects residue Ala175 in the
I-
J loop that precedes helix J of one molecule to residues Thr17, Thr20, and Phe22 of the
second molecule via the carbonyl atoms; however, there is no evidence
for an active role of this ion in the
-helical shift. Instead,
sequence differences in the C-terminal ends of proteins of the family
may explain the conformational features specific to the HuFrq
structure. Indeed, seven residues are conserved in the C-terminal
region of HuFrq compared with other members: Ala182,
Asp187, Gly188, and Leu189, which
are exposed at the periphery of the crevice and may have a role for
ligand recognition, and Ser184, Tyr186, and
Asp187, which are located on the opposite face where they
tightly anchor helix J to the protein core (Fig. 4B). A
recent report on a yeast Frq model, designed from a combination of
partial NMR data and homology modeling based on the recoverin
structure, might suggest a similar location of helix J in the yeast
homolog (29). Hence, the fact that Drosophila Frq possesses
a glycine doublet in place of the conserved residue pair
Tyr186-Asp187 appears most surprising because
these substitutions would be expected to destabilize helix J. Finally,
a Kv channel-interacting protein has recently been identified as a
novel member of the calcium-binding protein family (32), and its
C-terminal sequence presents high similarity with the C-terminal region
of HuFrq. This suggests that a hydrophobic crevice similar to that
found in HuFrq may exist and be involved in the binding of the
cytoplasmic N termini of Kv4
-subunits.
![]() |
ACKNOWLEDGEMENT |
---|
We thank the European Synchroton Radiation Facility staff for technical support in data collection.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 444-B4, to O. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G8I) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 33-4-91-16-45-08; Fax: 33-4-91-16-45-36; E-mail: yves@afmb.cnrs-mrs.fr.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M009373200
1
Frq, frequenin; NCS-1, neuronal sensor 1; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR; TGN, trans-Golgi-network; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HEK, human embryonic kidney cells; r.m.s.d., root mean
square deviation; PIK1, yeast phosphatidylinositol-4-OH kinase; PI4K, mammalian homolog of phosphatidylinositol-4-OH kinase.
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