-
Structural Insights into Membrane-targeting by the Flagellar
Calcium-binding Protein (FCaBP), a Myristoylated and Palmitoylated
Calcium Sensor in T. cruzi*
Jennifer N. Wingard,1# Jane Ladner,1 Murugendra Vanarotti,2
Andrew Fisher,2 Howard
Robinson,3 Kathryn T. Buchanan,4 David M. Engman,4 and James B.
Ames2|| 1Center for Advanced Research in Biotechnology, University
of Maryland, National Institute of
Standards and Technology, Rockville, MD 20850, 2Department of
Chemistry, University of California, Davis, CA 95616,
3Biology Department, Brookhaven National Laboratory, Upton, NY
11973-5000, 4Departments of Pathology and Microbiology-Immunology,
Feinberg School of Medicine,
Northwestern University, Chicago, IL 60611.
Running title: Crystal structure of FCaBP.
#Current address: Department of Molecular Physiology &
Biological Physics, University of Virginia, Charlottesville, VA
22908. ||To whom correspondence should be addressed: Department of
Chemistry, One Shields Avenue, University of California, Davis, CA
95616, Tel (530) 752-6358, FAX (530) 752-8995, E-mail:
[email protected]
http://www.jbc.org/cgi/doi/10.1074/jbc.M803178200The latest
version is at JBC Papers in Press. Published on June 17, 2008 as
Manuscript M803178200
Copyright 2008 by The American Society for Biochemistry and
Molecular Biology, Inc.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.orghttp://www.jbc.org/cgi/doi/10.1074/jbc.M803178200
-
Crystal Structure of FCaBP 2
The flagellar calcium-binding protein (FCaBP) of the protozoan
Trypanosoma cruzi is targeted to the flagellar membrane where it
regulates flagellar function and assembly. As a first step toward
understanding the Ca2+-induced conformational changes important for
membrane-targeting, we report here the x-ray crystal structure of
FCaBP in the Ca2+-free state determined at 2.2 Å resolution. The
first 17 residues from the N-terminus appear unstructured and
solvent exposed. Residues implicated in membrane-targeting (K19,
K22 and K25) are flanked by an exposed N-terminal helix (residues
26 – 37), forming a patch of positive charge on the protein surface
that may interact electrostatically with flagellar membrane
targets. The four EF-hands in FCaBP each adopt a “closed
conformation” similar to that seen in Ca2+-free calmodulin. The
overall fold of FCaBP is closest to that of grancalcin and other
members of the penta EF-hand superfamily. Unlike the dimeric penta
EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The
unstructured N-terminal region of FCaBP suggests that its
covalently attached myristoyl group at the N-terminus may be
solvent exposed, in contrast to the highly sequestered myristoyl
group seen in recoverin and GCAP1. NMR analysis demonstrates that
the myristoyl group attached to FCaBP is indeed solvent exposed in
both the Ca2+-free and Ca2+-bound states, and myristoylation has no
effect on protein structure and folding stability. We propose that
exposed acyl groups at the N-terminus may anchor FCaBP to the
flagellar membrane and that Ca2+-induced conformational changes may
control its binding to membrane-bound protein targets.
Flagellar calcium-binding protein (FCaBP) is a 24-kDa highly
immunogenic protein found in the flagellum of the protozoan
parasite Trypanosoma cruzi (1). FCaBP contains four EF-hand
calcium-binding motifs
(2,3) (Fig. 1), the third and fourth (EF-3 and EF-4) of which
bind calcium (4). The protein is modified at the N terminus by
covalent attachment of myristate at Gly2 and palmitate at Cys4,
both of which are required for association with the inner leaflet
of the flagellar membrane (5). Calcium is required for stable
flagellar localization as well, since FCaBP can be washed out of
detergent-permeabilized trypanosomes if calcium chelators are
included in the wash solutions. The N-terminal acylation and
calcium-dependent membrane localization of FCaBP suggested that the
protein may possess a functional calcium-acyl switch, similar to
the Ca2+-myristoyl switch observed previously for recoverin (6,7)
and other members of the neuronal calcium sensor (NCS) family (8).
Acyl switch proteins undergo calcium-dependent membrane association
by virtue of calcium-regulated extrusion or sequestration of a
myristate moiety that mediates membrane binding (9). However, an
FCaBP mutant unable to bind calcium still maintains its flagellar
localization, suggesting that FCaBP may not cycle on and off the
membrane like some calcium acyl switch proteins (4).
The best studied calcium myristoyl switch protein is recoverin,
a calcium-binding protein in retinal rod cells that inhibits
rhodopsin kinase (RK) only at high Ca2+ levels (10,11) and
regulates the recovery phase of phototransduction (12,13). In the
resting dark-state, recoverin binds two calcium ions and associates
with retinal rod outer segment (ROS) membranes through its exposed
myristoyl group (6,7). Photoexcitation of the rod cell results in a
lowering of cytosolic calcium (14), causing recoverin to lose its
bound calcium and adopt a conformation in which the myristoyl group
becomes sequestered within a hydrophobic cleft in the protein (9).
Ca2+-free recoverin then dissociates from RK at the membrane,
allowing RK to phosphorylate light-excited rhodopsin and promote
receptor inactivation.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 3
The calcium-myristoyl switch mechanism allows calcium regulation
of two proteins (RK and rhodopsin) that do not themselves bind
calcium. One major difference between FCaBP and recoverin is the
presence of palmitate in FCaBP, which may or may not participate in
a potential switch mechanism.
A variety of FCaBP-like proteins are found in other
trypanosomes: Trypanosoma rangeli, Trypanosoma lewisi, and
Trypanosoma brucei (Fig. 1). A flagellar calmodulin has also been
characterized in T. brucei (15). These Ca2+-binding proteins all
localize to the flagellum, a unique organelle that has many
functions, including motility, chemotaxis, and cell signaling. In
addition to the traditional "9 + 2" microtubule structure of the
axoneme, there is a structure known as the paraflagellar rod that
runs alongside the axoneme. The axoneme, paraflagellar rod, and
flagelloplasm are encased by the flagellar membrane. It has been
shown by freeze-fracture analysis that the flagellar membrane
contains a higher concentration of sterols than does the pellicular
(cell body) membrane (16), and the flagellum of T. brucei is highly
enriched in lipid rafts. Flagellar membrane stability and function
is also controlled by the recruitment of FCaBP to the membrane
surface, where it is believed to interact with a variety of
membrane-bound protein targets (4). We report here the x-ray
crystal structure of Ca2+-free FCaBP as a first step toward
understanding the Ca2+-induced conformational changes that control
membrane-targeting. This is only the second atomic-resolution
structure of a Ca2+-acyl switch protein in the Ca2+-free state. The
structure of Ca2+-free recoverin first showed its covalently
attached myristoyl group to be highly sequestered and buried deep
inside the protein (17). Also, the recent crystal structure of
Ca2+-bound GCAP1 indicates a sequestered myristoyl group (18). By
contrast, the structure of Ca2+-free FCaBP in this study suggests a
solvent exposed N-terminus, making its covalently attached acyl
groups accessible to interact with membrane targets.
We propose that the exposed acyl groups and N-terminal
positively charged residues (K19, K22, and K25) may promote the
binding of FCaBP to the flagellar membrane and that Ca2+-induced
protein conformational changes may control its binding to
membrane-bound protein targets. EXPERIMENTAL PROCEDURES Protein
expression and purification. To prepare recombinant unmyristoylated
FCaBP (and Se-methionine labeled FCaBP), FCaBP tagged with a
C-terminal (His)6 tract was expressed in Escherichia coli strain
BL21(DE3) carrying a derivative of the pET23d vector (Novagen) as
previously described (4) grown in M9 minimal medium supplemented
with or without Se-methionine, according to well-established
procedures (19-21).
Recombinant unmyristoylated FCaBP for the native crystal was
expressed as above in LB media and was initially purified via
Ni-affinity chromatography. Peak fractions were pooled and diluted
three-fold with buffer containing 20 mM Tris pH 7.5, 1 mM EDTA, 1
mM DTT and applied to a Q-HP HiTrap column (Amersham) at 5 ml/min.
FCaBP was eluted with a 200 ml linear KCl gradient to a final
concentration of 250 mM KCl. Peak fractions were pooled and
purified FCaBP was dialyzed and concentrated to 10 mg/ml in 20 mM
Tris, pH 7.5, 1 mM DTT in a centrifugal filter device with a 10,000
molecular weight cutoff (Amicon).
Recombinant myristoylated FCaBP was generated by co-expressing
FCaBP and N-myristoyl CoA transferase (pBB131-NMT) in BL21(DE3)
cells grown on M9 medium supplemented with myristic acid (10 mg/L).
Myristoylated and non-myristoylated FCaBP was purified from the
soluble fraction of bacterial cell lysates using Ni2+-chelate
affinity chromatography on a nitrilotriacetate-resin (Qiagen),
according the manufacturer’s instructions. Peak fractions were then
applied to an anion-exchange column (Hi-Trap DEAE-FF, Pharmacia)
equilibrated in buffer A (1 mM
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 4
EDTA, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 7.4) and eluted
with a linear salt gradient (0 to 0.2 M KCl) at flow rate of 5 ml
min-1 over the course of 150 min. Peak fractions were concentrated
to 5 ml and subjected to size-exclusion chromatography (Sephacryl
S-100, Pharmacia) in buffer B (1 mM dithiothreitol, 2 mM CaCl2, 50
mM HEPES, pH 7.4). Final purity was greater than 98%, as judged by
SDS-PAGE. Protein crystallization. Two different native crystals
were grown by the hanging drop method (22). Crystal form #1 was
obtained by combining 900 !l of original well solution (3.2 M
ammonium sulfate and 0.1 M MES pH 6.0) with 100 !l of OptiSalts
(Qiagen) optimization solution (0.75 M magnesium chloride and 0.1 M
sodium acetate pH 4.6). Crystal form #2 was obtained using a well
solution containing solely 3.2 M ammonium sulfate and 0.1 M MES pH
6.0. The selenomethionine protein crystal was grown in a sitting
drop with the well solution containing 3.2 M ammonium sulfate, 0.1M
MES pH 5.7. The drop was made with 6 microliters of protein (20
mg/mL) and 3 microliters of well solution. All crystallizations
were at room temperature. X-ray crystallography. Diffraction data
for the native structure (from crystal #1) were collected using a
Rigaku Micro Max 007 rotating anode generator (Rigaku/MSC, The
Woodlands, TX). The crystal was cooled to 100 K with a Cryocool low
temperature probe (Cryo Industries of America) and was
cryoprotected by pulling the loop mounted crystal through immersion
oil. The diffraction data were processed with CrystalClear/d*Trek
(23). Statistics are shown in Table 1 (crystal #1). SAD (single
wavelength anomalous dispersion) data for the selenomethionine
protein were collected at Brookhaven National Laboratory on
beamline X29 using the wavelength 0.9790 Å and processed with
HKL2000 (24). When the anomalous data were processed in space group
P212121, 15,413 measurements were rejected. With space group P1,
only 442 reflections were rejected
and the 33 Å axis was identified as the screw axis. When
processed in space group P21, 504 reflections were rejected. The
anomalous signal was weak but usable to 2.6 Å. The software
program, SHELXC/D/E (25,26) was used to find eight selenium sites
and examine the hand of the sites. A reasonable value for the
Matthews coefficient (1.8) indicated that there were two molecules
in the asymmetric unit for space group P21 or one for P212121. The
selenium sites were used in SOLVE/RESOLVE (27,28) to produce an
electron density map, and the map was examined in COOT (29) and
looked good. Using the iterative script (www.solve.lanl.gov) which
includes cycles of density modification and automated model
building by RESOLVE and molecular refinement by REFMAC5 (30), 271
of the 422 residues were built and 104 side chains were placed. The
model was completed by iteratively building in COOT and refining in
REFMAC5. The model was transferred to the native data and refined
in both space group P21 and P212121. The model and the statistics
were essentially the same. There was no compelling reason to use
P21 for the native data so the final statistics for the P212121
space group are shown in Table 2. The final model includes residues
17-208. The side-chains of 11 residues were modeled with double
conformations and C66 was modeled as s-oxy cysteine. The sequence
has four methionines, but the selenium sites seen were 122M, 145M,
and a double conformation for 135M. The loop region including
residues 102-113 has minimal density but the path is clear. In a
Ramachandran plot, 97.8% of the residues fall into the preferred
regions and 2.2% fall into the allowed regions as analyzed by COOT.
NMR spectroscopy. Samples for NMR analysis consisted of 15N-labeled
FCaBP with and without a covalently attached, 13C-labeled myristoyl
group. FCaBP protein for NMR studies (1.0 mM) was dissolved in 0.3
ml of a 95% H2O/5% {2H}H2O solution containing 10 mM Tris-HCl (pH
7.4) and 2 mM EDTA (Ca2+-free) or 2 mM CaCl2 (Ca2+-bound). All
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.orghttp://www.solve.lanl.gov/
-
Crystal Structure of FCaBP 5
NMR experiments were performed at 25 C on a Bruker DRX-600
spectrometer equipped with a four-channel interface and
triple-resonance probe with triple-axis pulsed field gradients; and
DRX-600 spectrometer equipped with an Ultrashield Bruker magnet, a
three-channel interface, and cryo-probe with Z-axis pulsed field
gradients. The 15N-1H HSQC spectra (see Fig. 4) were recorded on a
sample of 15N-labeled FCaBP (in 95% H2O, 5% 2H2O). The number of
complex points and acquisition times were: 256, 180 ms (15N (F1));
and, 512, 64 ms (1H (F2)). The 13C-1H HMQC spectra (Fig. 5) and
13C(F1)-edited, 13C(F3)-filtered NOESY-HMQC spectra were recorded
on a sample of unlabeled FCaBP protein attached to 13C-labeled
myristate (31) as well as 13C-labeled FCaBP bound to unlabeled
myristate (data not shown). Intermolecular NOESY experiments were
performed as described previously (32). 15N{1H}-NOE data were
measured using 2D [15N,1H]-HSQC based experiments as described
previously (33). Saturation was carried out with a series of 120º
1H pulses separated by 5 ms delays applied during the interscan
delay (3 s).
RESULTS AND DISCUSSION Protein crystallization. Recombinant
FCaBP crystallized in the orthorhombic space group, P212121 with
one molecule of FCaBP in the asymmetric unit. It is seen to be
monomeric in the crystal, as measured in solution by NMR and
dynamic light scattering analysis. The polypeptide structure
derived from diffraction data on crystal #1 (in the presence of
saturating Mg2+) and crystal #2 (apo-form) appear virtually
identical except for very minor differences in the EF2 loop
structure (residues, 102-113), suggesting that Mg2+ has almost no
effect on the overall structure. In each crystal form, the first 17
residues from the N-terminus appear disordered. Amino acid
sequencing of the protein from dissolved crystals showed that all
17 N-terminal residues remained intact in both
crystal forms and therefore were not cleaved by proteolysis
during crystallization. Three-dimensional structure. The x-ray
crystal structure of Ca2+-free FCaBP was solved at 2.2
Å-resolution, with a final R-factor of 16.95% and R-free of 21.97%
(Fig. 2). The entire polypeptide chain has been traced except for
the first 17 residues from the N-terminus. High temperature factors
for the N-terminal residues suggest they may be dynamically
disordered. NMR relaxation studies and heteronuclear 15N NOE
analysis on FCaBP also confirms that the first 17 N-terminal
residues are indeed unstructured (data not shown) and the
N-terminal myristoyl group is solvent exposed (see below). The
overall main chain structure for residues 17-208 of FCaBP
determined by x-ray crystallography was consistent with and nearly
identical to a much lower resolution NMR solution structure (not
shown). The protein structure visible in the crystal has a globular
fold with overall dimensions of 32 Å x 23 Å x 21 Å (Fig. 2). The
first structured residue in the crystal begins at A18 that marks
the start of an exposed, 5-residue conformation (residues, 18-23),
resembling a hook that forms a right angle with an adjacent "-helix
(residues, 24-37). Following this N-terminal helix is a compact
arrangement of four EF-hands: EF1 (residues 49-77, green); EF2
(residues 98-126, red); EF3 (residues 131-159, cyan); and EF4
(residues 168-196, yellow). Overall, FCaBP contains a total of
eight "-helices and four #-strands: "1 (residues 24-37), "2
(residues 44-57), "3 (residues 67-76), "4 (residues 87-105), "5
(residues 116-139), "6 (149-162), "7 (residues 169-176), "8
(residues 186-201), #1 (residues 64-66), #2 (residues 113-115), #3
(residues 146-148) and #4 (residues 183-185) (Fig. 1). The four
EF-hands of FCaBP associate into two pairs through a
characteristic, short, two-stranded #-sheet arrangement: EF1
(green) pairs with EF2 (red), while EF3 (cyan) pairs with EF4
(yellow). The two pairs of EF-hands are
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 6
connected by a long, central helix ("5) formed by merging the
exiting helix of EF2 into the entering helix of EF3. This topology
causes three long, central helices ("4, "5 and "8) to interact
closely as a twisted, vertical bundle. As a consequence, EF2 and
EF4 make close contact with one another. This globular arrangement
of EF-hands is most similar to that seen for the first four
EF-hands in the penta-EF-hand proteins, grancalcin (37) and domain
IV of calpain (38). However, unlike penta-EF-hand proteins, FCaBP
lacks a fifth EF-hand and is not dimeric. EF-hands and
Ca2+-binding. The individual metal-free EF-hands in FCaBP each
consist of a helix-turn-helix structure similar to the “closed
conformation” of EF-hands seen in previous Ca2+-free structures of
calmodulin (39,40), troponin C (41) and grancalcin (37). The
interhelical angles for the “closed” EF-hands in FCaBP are 119º
(EF1), 128º (EF2), 122º (EF3) and 120º (EF4). The binding of Ca2+
to each EF-hand in calmodulin causes a marked decrease in
interhelical angle that promotes formation of an open conformation,
leading to the exposure of many hydrophobic residues that interact
with protein targets (42,43). Functional Ca2+ binding to EF3 and
EF4 in FCaBP (4) may cause a similar Ca2+-induced exposure of
hydrophobic residues and might explain the observed Ca2+-induced
binding of FCaBP to protein phosphatase-2A (D. Engman, unpublished
results) and various other protein targets (4). Indeed,
Ca2+-dependent membrane binding by FCaBP might arise in part by its
Ca2+-induced binding to protein targets localized on the flagellar
membrane surface. The 12-residue Ca2+-binding loop of EF2 in FCaBP
appears loosely structured in crystal form #1 and adopts an
unusual, distorted conformation in crystal form #2, which may
explain why EF2 is not able to bind Ca2+ (4). The instability
and/or structural distortion of the EF2 loop may be due in part to
the presence of G109 at position-3 in the binding loop, which lacks
an acidic side-chain required for Ca2+ chelation at this key
position.
The lack of Ca2+ binding at EF2 may also explain why Ca2+ is not
able to bind to EF1, as the helices of EF1 and EF2 interlock to
form a four-helix bundle, causing both EF-hands to be cooperatively
locked in the Ca2+-free closed conformation. In addition, C66 at
position-9 in the EF1 loop is not able to form a hydrogen-bond with
E69, which may destabilize the binding-loop structure and prevent
Ca2+ binding at EF1. By contrast, EF3 and EF4 both adopt a more
favorable conformation for binding Ca2+, consistent with functional
Ca2+-binding measured at these sites (4). In the Ca2+-binding loop
of EF3, side-chain oxygen atoms from residues D140, S142, N144 and
E151 are held in a cage-like arrangement surrounding a bound H2O
molecule (Fig. 2A) that resembles the binding of Ca2+ with the
familiar pentagonal bipyramid geometry (3). For EF4, side-chain
oxygen atoms from residues D177, N179, T181 and E188 are also
pre-arranged with a similar geometry that surrounds a bound H2O. In
addition, the main-chain nitrogen of the sixth residue in the
Ca2+-binding loops of EF3 (M145) and EF4 (G182) both form hydrogen
bonds with aspartate at the one-position, forming a loop
conformation characteristic of Ca2+-bound calmodulin and troponin
C. Therefore, the Ca2+-binding loops in EF3 and EF4 both adopt a
favorable, pre-formed local conformation that should promote rapid
and functional Ca2+ binding. Surface Properties of FCaBP. A
space-filling representation of FCaBP reveals multiple charged
residues in FCaBP unevenly distributed on one side of the protein
surface (Fig. 3A) with an exposed hydrophobic patch on the opposite
side (Fig. 3B). Most striking is the arrangement of N-terminal Arg
and Lys residues (K19, K22, K25, K28, R33, and R35) whose
side-chains form a cluster of positive charge on the protein
surface (highlighted blue in Fig. 3A). Some of these N-terminal
basic residues (K19, K22, and K25) have been implicated previously
in membrane binding (D. Engman, unpublished results). We propose
that the exposed patch of N-terminal
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 7
Arg and Lys residues may promote electrostatic interactions with
negatively charged head groups on the surface of the flagellar
membrane. A similar electrostatic membrane interaction has also
been proposed for the myristoyl switch proteins, recoverin (44) and
the Src protein (45). The additional electrostatic attraction helps
augment membrane anchoring by the N-terminal myristoyl group and
can increase the membrane binding affinity by more than 10-fold
(45,46). The protein surface on the opposite face (Fig. 3B)
contains an exposed patch of hydrophobic residues near the
C-terminus (A170, A171, L172, L176, A191, W192, A195, V196, L198,
A200 and highlighted yellow in Fig. 3B). The exposed hydrophobic
residues are primarily located on the helices of EF4 ("7 and "8).
Ca2+-induced conformational changes expected in EF4 therefore might
alter the arrangement and environment of these hydrophobic
residues. We propose that the exposed hydrophobic patch in Fig. 3B
may represent a binding site for the interaction with
membrane-bound protein targets.
NMR analysis of myristoylation. The absence of detectable
electron density from the first 17 residues of FCaBP suggested that
the N-terminal region and myristoyl group may by structurally
disordered. To more directly examine the structural role of the
myristoyl group, we recorded two-dimensional (15N-1H HSQC) NMR
spectra of both myristoylated and unmyristoylated FCaBP,
demonstrating that both spectra are nearly identical (Fig. 4).
Hence, the presence of the myristoyl group does not appear to
affect the overall structure of Ca2+-free FCaBP, in contrast to the
large structural changes in Ca2+-free recoverin that result from
N-terminal myristoylation (17,31). The similarity in the spectra of
FCaBP and myr-FCaBP suggests that the fatty acyl chain may be
exposed to solvent and, hence, not interacting significantly with
the protein.
NMR experiments were also performed on FCaBP containing a
13C-labeled myristoyl
group to directly probe the structural environment and
disposition of the N-terminal myristoyl group. Previously,
two-dimensional (1H-13C HMQC) and three-dimensional (13C-filtered
NOESY-HMQC) NMR experiments on samples of recoverin that contained
a 13C-labeled myristoyl group were used to selectively probe the
chemical environment around the amino-terminal myristoyl group
(31,34). These studies revealed that the covalently-attached fatty
acyl chain in recoverin is sequestered in a hydrophobic pocket in
the Ca2+-free protein and that binding of Ca2+ leads to
conformational changes that extrude the N-myristoyl group into
solvent.
Similar NMR experiments were performed on samples of FCaBP that
contained a 13C-labeled myristoyl group (Fig. 5). Because the HMQC
experiment selectively probes protons that are covalently attached
to 13C, only the methylene and methyl proton resonances of the
fatty acyl chain appear in these spectra. Weak extraneous peaks
near 0.9, 1.4 and 1.7 ppm (1H dimension) are due to natural
abundance 13C signals from the protein. The spectrum of the
N-myristoyl group in Ca2+-free FCaBP (Fig. 5B) looks quite similar
to the spectrum of free myristic acid in solution (Fig. 5A) and the
resonance frequencies of corresponding peaks are nearly identical.
Assignments of the myristoyl group resonances for Ca2+-free FCaBP
(Fig. 5B) were therefore derived from assignments of those for free
myristic acid, which were determined previously (31). The
similarity in the spectrum of the myristoyl group in Ca2+-free
FCaBP to that of free myristic acid in solution indicates that, in
Ca2+-free FCaBP, the fatty acyl chain is solvent-exposed, in
contrast to Ca2+-free recoverin where the myristoyl chain is deeply
buried inside the core of the protein (9).
To further test whether the myristoyl group of FCaBP is
solvent-exposed, three-dimensional (13C/F1)-edited and
(13C/F3)-filtered NOESY experiments (31) were performed on
unlabeled FCaBP protein
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 8
containing a 13C-labeled myristate. These spectra selectively
probed atoms of residues in the protein that lie within 5 Å of the
labeled CH3-group of the myristoyl chain. Nuclear Overhauser effect
(NOE) dipolar interactions between the myristate methyl group and
the protein could not be detected (data not shown). The lack of
observable NOEs in this experiment suggests that the methyl group
of the fatty acyl chain is more than 5 Å away from atoms in the
protein, providing additional support for the conclusion that the
N-myristoyl group of FCaBP is solvent-exposed and does not interact
intimately with the protein.
The addition of Ca2+ to myr-FCaBP caused no discernable change
in the NMR spectrum of the myristoyl group (Fig. 5C). In addition,
NOE interactions between the myristate and protein could not be
detected (data not shown) in three-dimensional (13C/F1)-edited and
(13C/F3)-filtered NOESY spectra of Ca2+-bound 13C-myr-FCaBP,
suggesting that the methyl group of the fatty acyl chain does not
interact closely with the Ca2+-bound protein. Hence, the myristoyl
group of Ca2+-bound FCaBP appears solvent-exposed and does not
undergo a calcium-induced change in environment, in contrast to
what has been observed previously in recoverin (31). Structural
relationship with PEF proteins. The N-terminal myristoylation and
four EF-hands in FCaBP would suggest that it might be structurally
similar to recoverin (9,47) and related Ca2+-myristoyl switch
proteins (48,49). However, the overall three-dimensional structures
of FCaBP and recoverin are surprisingly quite different and
unrelated. Instead, the overall main chain fold of Ca2+-free FCaBP
is most similar to that of apo-grancalcin (37) and related proteins
that belong to the penta EF-hand (PEF) family (50). Interestingly,
FCaBP bears very little sequence homology to the PEF proteins (<
20% identity). The root-mean-square deviation (RMSD) is 3.5 Å when
comparing the main chain atoms of the four EF-hands of FCaBP with
those of the first four EF-hands of
grancalcin (Fig. 6). The structural similarity is even more
striking when comparing the main chain atoms from EF1, EF3 and EF4
(RMSD = 2.9 Å), whereas the structure of EF2 is much more divergent
between the two (RMSD = 4.5 Å). The second helix of EF2 and first
helix of EF3 in FCaBP are merged into a long, 23-residue helix ("5)
that is flanked in an anti-parallel fashion by helices, "4 and "8,
forming a twisted bundle. A similar helical bundle and overall
topology is also seen in grancalcin and seems to be a structural
hallmark of all PEF proteins. The similarity of the main chain
conformations between FCaBP and PEF proteins suggests that their
functions might be related. Indeed, grancalcin and PEF proteins
exhibit Ca2+-induced membrane binding (51-53) and interact with
target proteins such as L-plastin (54), annexins (55) and integrins
(56) implicated in cytoskeletal dynamics. FCaBP also binds to
membranes and has been shown recently to interact with several
candidate target proteins (4). Future studies are needed to
determine whether any of the FCaBP target proteins might be related
to L-plastin or other PEF protein targets that regulate
cytoskeletal adhesion and migration. An important structural
difference between FCaBP and PEF proteins is that FCaBP is
monomeric in solution and does not have a fifth EF-hand like the
dimeric PEF proteins. The fifth EF-hand of FCaBP may have been
deleted during evolution perhaps to allow for the exposure of
hydrophobic residues in EF4 (Fig. 3B) that otherwise would be
inaccessible if the fifth EF-hand were present. Another difference
is the presence of the lipophilic N-terminal extension (LNT) region
in PEFs that have variable Gly-Pro-Ala sequences implicated in
membrane targeting (57). By contrast, FCaBP has conserved basic
residues (K19, K22, K25) in the N-terminal region that we suggest
might be important for membrane-binding in addition to N-terminal
myristoylation and palmitoylation sites (Figs. 1 and 7). We propose
that the four EF-hands of FCaBPs are evolutionarily related to the
first four EF-hands of PEF proteins, and that
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 9
FCaBPs may represent a sub-branch of the PEF family that lack a
fifth EF-hand and have N-terminal myristoylation and palmitoylation
membrane-targeting motifs instead. It will be interesting to look
for other examples of truncated PEF proteins that are structurally
related to FCaBP and perhaps serve as myristoylated,
membrane-targeting calcium sensors. Membrane-targeting mechanism.
The crystal structure of FCaBP (Fig. 2) has implications for its
Ca2+-induced membrane-targeting mechanism (Fig. 7). The solvent
exposed myristoylation (and presumably palmitoylation (5)) sites at
the N-terminus suggest that the covalently attached fatty acyl
groups serve as membrane anchors that insert inside the lipid
bilayer. In addition, exposed basic residues in the N-terminal
region (Fig. 3A) are suggested to provide additional binding energy
by interacting electrostatically with negatively charged headgroups
on the membrane surface. A similar combination of electrostatic
effects and myristoylation was seen previously for membrane binding
by the myristoyl-electrostatic switch of Src and MARCKS protein
(45). Our NMR analyses (Figs. 4-5) indicate that the fatty acyl
group is exposed regardless of Ca2+ concentration, suggesting that
both the Ca2+-free and Ca2+-bound forms of FCaBP may be anchored to
the flagellar membrane (Fig. 7). But how then is membrane binding
by FCaBP regulated by calcium? We propose that the Ca2+ sensitive
localization of FCaBP at the flagellar membrane could be controlled
by Ca2+-dependent binding of FCaBP to various target proteins.
Previous studies have identified candidate target proteins that
differentially bind to either the Ca2+-free or Ca2+-bound forms of
FCaBP (4). The binding of Ca2+ to calmodulin and other EF-hand
proteins generally leads to the exposure of hydrophobic residues
that promotes their binding to protein targets (42,43). A similar
Ca2+-dependent exposure of hydrophobic residues in EF4 (Fig. 3B)
might promote association of Ca2+-bound FCaBP with a
membrane-bound target protein and preferentially localize FCaBP
at the flagellar membrane at high Ca2+ levels (Fig. 7B).
Alternatively, the Ca2+-free state of FCaBP might preferentially
bind to a cytosolic target protein that would localize FCaBP in the
cytosol at low Ca2+ levels (Fig. 7A). The binding of a cytosolic
target protein to Ca2+-free FCaBP might help to sequester the
N-terminal fatty acyl groups inside a protein environment (perhaps
involving exposed hydrophobic residues in EF4 (Fig. 3B)) like that
seen for Ca2+-free recoverin (17) or GCAP1 (18). Sequestration of
the myristoyl group would stabilize cytosolic FCaBP and therefore
promote its membrane dissociation only at low Ca2+ levels. Lastly,
reversible cleavage of the labile palmitoyl group attached to FCaBP
(by a thioester linkage) may further modulate its membrane
anchoring capacity. The enzyme catalyzed cleavage of palmitate may
also be regulated by Ca2+. In short, both the Ca2+-free and
Ca2+-bound FCaBP have exposed myristoyl groups (Fig. 5) that we
propose anchor the protein to the flagellar membrane. Thus, FCaBP
does NOT exhibit Ca2+-induced extrusion of the myristoyl group to
control membrane localization like what is seen for recoverin (6)
and related Ca2+-myristoyl switch proteins (58). Instead, we
propose that Ca2+-induced protein conformational changes in FCaBP
(4) may modulate its interaction with target proteins that control
membrane localization as depicted in Fig. 7. Future structural
studies on Ca2+-bound FCaBP bound to various target proteins are
needed to further test and refine the proposed membrane-targeting
mechanism.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 10
REFERENCES 1. Engman, D. M., Krause, K. H., Blumin, J. H., Kim,
K. S., Kirchhoff, L. V., and
Donelson, J. E. (1989) J. Biol. Chem. 264, 18627-18631 2.
Moncrief, N. D., Kretsinger, R.H., and Goodman, M. (1990) J. Mol.
Evol. 30, 522-562. 3. Ikura, M. (1996) Trends Biochem. Sci. 21,
14-17. 4. Buchanan, K. T., Ames, J. B., Asfaw, S. H., Wingard, J.
N., Olson, C. L., Campana, P.
T., Araujo, A. P., and Engman, D. M. (2005) J Biol Chem 280,
40104-40111 5. Godsel, L. M., and Engman, D. M. (1999) EMBO J. 18,
2057-2065. 6. Zozulya, S., and Stryer, L. (1992) Proc. Natl. Acad.
Sci. USA 89, 11569-11573 7. Dizhoor, A. M., Chen, C. K.,
Olshevskaya, E., Sinelnikova, V. V., Phillipov, P., and
Hurley, J. B. (1993) Science 259, 829-832 8. Burgoyne, R. D.
(2004) Biochim. Biophys. Acta. 1742, 59-68 9. Ames, J. B., Ishima,
R., Tanaka, T., Gordon, J. I., Stryer, L., and Ikura, M. (1997)
Nature
389, 198-202. 10. Kawamura, S. (1993) Nature 362, 855-857. 11.
Klenchin, V. A., Calvert, P. D., and Bownds, M. D. (1995) J. Biol.
Chem. 270, 16147-
16152. 12. Erickson, M. A., Lagnado, L., Zozulya, S., Neubert,
T. A., Stryer, L., and Baylor, D. A.
(1998) Proc. Natl. Acad. Sci. USA 95, 6474-6479 13. Gray-Keller,
M. P., Polans, A. S., Palczewski, K., and Detwiler, P. B. (1993)
Neuron 10,
523-531. 14. Koch, K. W., and Stryer, L. (1988) Nature 334,
64-66 15. Ridgley, E., Webster, P., Patton, C., and Ruben, L.
(2000) Mol. Biochem. Parasitol. 109,
195-201 16. Tetley, L. (1986) Acta. Trop. 43, 307-317 17.
Tanaka, T., Ames, J. B., Harvey, T. S., Stryer, L., and Ikura, M.
(1995) Nature 376, 444-
447. 18. Stephen, R., Bereta, G., Golczak, M., Palczewski, K.,
and Sousa, M. C. (2007) Structure
15, 1392-1402 19. Muchmore, D. C., McIntosh, L. P., Russell, C.
B., Anderson, D. E., and Dahlquist, F. W.
(1989) Methods Enzymol. 177, 44-86. 20. McIntosh, L. P., and
Dahlquist, F. W. (1990) Quart. Rev. Biophys. 23, 1-38. 21. Ames, J.
B., Tanaka, T., Stryer, L., and Ikura, M. (1994) Biochemistry 33,
10743-10753. 22. Jancarik, J., and Kim, S. H. (1991) J. Appl.
Cryst. 24, 409-411 23. Pflugrath, J. W. (1999) Acta. Crystallogr.
D55, 1718-1725 24. Otwinowski, Z., and Minor, W. (1997) Methods in
Enzymology 276, 307-326 25. Schneider, T. R., and Sheldrick, G. M.
(2002) Acta. Crystallogr. D58, 1772-1779 26. Sheldrick, G. M.
(2002) Kristallogr 217, 644-650 27. Terwilliger, T. C. (2003) Acta.
Crystallogr. D59, 38-44 28. Terwilliger, T. C. (2003) Acta.
Crystallogr. D59, 45-49 29. Emsley, P., and Cowtan, K. (2004) Acta.
Crystallogr. D60, 2126-2132 30. Murshudov, D. N., Vagin, A. A., and
Dodson, E. J. (1997) Acta. Crystallogr. D53, 140-
255 31. Ames, J. B., Tanaka, T., Ikura, M., and Stryer, L.
(1995) J. Biol. Chem. 270, 30909-
30913.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 11
32. Lee, W., Revington, M. J., Arrowsmith, C., and Kay, L. E.
(1994) FEBS Lett. 350, 87-90 33. Farrow, N. A., Muhandiram, R.,
Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G.,
Shoelson, S. E., Pawson, T., and Kay, L. E. (1994) Biochemistry
33, 5984-6003 34. Tanaka, T., Ames, J. B., Kainosho, M., Stryer,
L., and Ikura, M. (1998) J Biomol NMR
11, 135-152. 35. Nilges, M., Gronenborn, A. M., Brunger, A. T.,
and Clore, G. M. (1988) Protein Eng. 2,
27-38 36. Bagby, S., Harvey, T. S., Eagle, S. G., Inouye, S.,
and Ikura, M. (1994) Structure 2, 107-
122 37. Jia, J., Han, Q., Borregaard, N., Lollike, K., and
Cygler, M. (2000) J. Mol. Biol. 300,
1271-1281 38. Blanchard, H., Grochulski, P., Li, Y., Arthur, J.
S., Davies, P. L., Elce, J. S., and Cygler,
M. (1997) Nat. Struct. Mol. Biol. 4, 532-538 39. Schumacher, M.
A., Crum, M., and Miller, M. C. (2004) Structure 12, 849-860 40.
Zhang, M., Tanaka, T., and Ikura, M. (1995) Nat. Struct. Biol. 2,
758-767 41. Gagne, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and
Sykes, B. D. (1995) Nat. Struct.
Biol. 2, 784-789 42. Hoeflich, K. P., and Ikura, M. (2002) Cell
108, 739-742 43. Vetter, S. W., and Leclerc, E. (2003) Eur. J.
Biochem. 270, 404-414 44. Valentine, K., Mesleh, M., Ikura, M.,
Ames, J. B., and Opella, S. (2003) Biochemistry 42,
6333-6340 45. McLaughlin, S., and Aderem, A. (1995) Trends
Biochem. Sci. 20, 272-276 46. Resh, M. D. (1994) Cell 76, 411-413
47. Flaherty, K. M., Zozulya, S., Stryer, L., and McKay, D.B.
(1993) Cell 75, 709-716 48. Bourne, Y., Dannenberg, J., Pollmann,
V.V., Marchot, P., and Pongs, O. (2001) J. Biol.
Chem. 276, 11949-11955. 49. Vijay-Kumar, S., and Kumar, V. D.
(1999) Nature Struct. Biol. 6, 80-88. 50. Maki, M., Kitaura, Y.,
Satoh, H., Ohkouchi, S., and Shibata, H. (2002) Biochim.
Biophys.
Acta. 1600, 51-60 51. Teahan, C. G., Totty, N. F., and Segal, A.
W. (1992) Biochem. J. 286, 549-554 52. Meyers, M. B., Zamparelli,
C., Verzili, D., Dicker, A. P., Blanck, T. J., and Chiancone,
E. (1995) FEBS Lett. 357, 230-234 53. Mellgren, R. L. (1987)
FASEB J. 1, 110-115 54. Lollike, K., Johnsen, A. H., Durussel, I.,
Borregaard, N., and Cox, J. A. (2001) J. Biol.
Chem. 276, 17762-17769 55. Brownawell, A. M., and Creutz, C. E.
(1997) J. Biol. Chem. 272, 22182-22190 56. Huttenlocher, A.,
Palecek, S. P., Lu, Q., Zhang, W., Mellgren, R. L., Lauffenburger,
D.
A., Ginsberg, M. H., and Horwitz, A. F. (1997) J. Biol. Chem.
272, 32719-32722 57. Maki, M., Narayana, S. V., and Hitomi, K.
(1997) Biochem. J. 328, 718-720 58. Burgoyne, R. D., and Weiss, J.
L. (2001) Biochem. J. 353, 1-12 59. Wishart, D. S., Sykes, B. D.,
and Richards, F. M. (1992) Biochemistry 31, 1647-1651.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 12
FOOTNOTES *This work was supported by grants EY012347 and
NS045909 (J.B.A.), RR11973 (UC Davis NMR Facility) and AI46781
(D.M.E.) from the National Institutes of Health. The atomic
coordinates and structure factors (code 3CS1) have been deposited
in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org). 1The abbreviations used are: FCaBP,
flagellar calcium binding protein; HSQC, heteronuclear single
quantum coherence; HMQC, heteronuclear multiple quantum coherence;
NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; RMSD,
root-mean-squared deviation; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. ACKNOWLEDGMENTS We
thank Jeff de Ropp for help with NMR experiments, Dr. Frits
Abildgaard for providing NMR pulse-sequence programs, John Cieslak
for producing the E. coli strains coexpressing FCaBP and yeast
N-myristoyltransferase,and Frank Delaglio and Dan Garrett for
writing computer software for NMR data processing and analysis.
Certain commercial equipment, instruments, and materials are
identified in this paper in order to specify the experimental
procedure. Such identification does not imply recommendation or
endorsement by the National Institute of Standards and Technology,
nor does it imply that the material or equipment identified is
necessarily the best available for the purpose.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 13
Table 1. Data Statistics Table. native SeMet space group P212121
P21cell parameters (a,b,c) (Å) 32.91,37.67,141.33
37.71,32.91,140.88 cell parameters (!,",#) (°) 90.0,90.0,90.0
90.0,90.0,90.0 wavelength of data collection (Å) 1.541 0.979 no. of
measured intensities 53,305 52,133 no. of unique reflections 12,558
18,011 resolution of data (Å) 29.8-2.0 30.0-2.1 highest resolution
shell (Å) 2.07-2.00 2.18-2.10 Rsym (overall/high resolution shell)
0.070/0.308 0.059/0.261 completeness (%)(overall/high resolution
shell)
99.8/97.9 87.3/45.6
redundancy (overall/high resolution shell)
4.2/4.1 5.9/2.4
mean I/$ (overall/high resolution shell)
10.9/4.1 11.1/2.5
Table 2. Refinement Statistics Table resolution limits (Å)
20.0-2.0 number of reflections used/for Rfree 11,896/600 R-factor
(overall/high resolution shell) 0.211/0.251 Rfree (overall/high
resolution shell) 0.289/0.339 number of water molecules 65 rms
deviation bond length (Å) 0.019 rms deviation angle (°) 1.73
average B main chain/side chain/water (Å2) 30.3/31.7/32.4
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 14
FIGURE LEGENDS Figure 1. Primary and secondary structure
features of FCaBP. Alignment of the primary sequence of T. cruzi
FCaBP with that of T. rangeli and T. brucei. Secondary structural
elements indicated schematically were derived from the xray
structure and analysis of NMR data (3JHNH", chemical shift index
(59), and sequential NOE patterns). The four EF-hands (EF1, EF2,
EF3 and EF4) are highlighted green, salmon, cyan, and yellow,
respectively. Residues in the 12-residue Ca2+-binding loops are
underlined and chelating residues are highlighted bold. Invariant
basic residues on the protein surface and implicated in membrane
binding are colored blue. Figure 2. X-ray crystal structure of
FCaBP (PDB accession no., 3CS1). (A) Representative electron
density map of FCaBP, showing the Ca2+-binding loop of EF3. A bound
water molecule (red sphere) is present in the EF3 Ca2+-binding site
of apo FCaBP. (B) Ribbon diagrams depicting the main chain
structure of FCaBP viewed from the membrane-binding interface (top)
and rotated 180 (bottom). The EF-hands are colored as in Fig. 1.
Figure 3. Surface representation of FCaBP protein structure.
Space-filling models of FCaBP depicting (A) positively charged,
membrane-binding interface, and (B) exposed hydrophobic surface.
The orientations are similar to those in Fig. 2B. Exposed
hydrophobic, basic and acidic residues are highlighted in yellow,
blue and red, respectively. Figure 4. NMR spectral analysis of
myristoylated and unmyristoylated FCaBP. Two-dimensional (15N-1H
HSQC) NMR spectra of 15N-labeled unmyristoylated (black) and
myristoylated FCaBP (red) in the Ca2+-free state. Peaks
corresponding to the -NH2 groups of the side-chain amides of Gln
and Asn residues are connected by dotted lines. Sequence-specific
assignments are indicated. Figure 5. The N-terminal myristoyl group
of FCaBP appears to be solvent-exposed.
Two-dimensional (1H-13C HMQC) NMR spectra of free myristic acid
dissolved in chloroform (A) and the myristoyl group of Ca2+-free
(B) and Ca2+-bound (C) FCaBP. The myristoyl group was labeled with
carbon-13 and the protein was unlabeled. The peaks in the spectrum
represent protons attached to 13C-labeled fatty acyl chain. Figure
6. Structural comparison of FCaBP with penta EF-hand proteins. The
main chain atoms of the EF-hand regions of FCaBP (red, 3CS1.pdb)
and first 4 EF-hands of apo-grancalcin (blue, 1F4Q.pdb) are
superimposed (3.5 Å RMSD). Figure 7. Model of membrane-target
recognition by FCaBP.
Schematic diagram of FCaBP binding to membrane and protein
targets at low (A) and high (B) Ca2+ levels. Myristoyl and
palmitoyl groups highlighted magenta; EF-hands colored as in Fig.
1; N-terminal Arg and Lys residues colored blue; and cytosolic and
membrane-bound target proteins shown in gray.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 15
1 10 20 30 40 50 T. cruzi
MGACGSKGSTSDKGLA---SDKDGKKAKDRKEAWERIRQAIPREKTAEAKQRRIELFKKF T.
rangeli
MGACGSKGS------A---GNKDGKSATDRKVAWERIRQVIPREKTAEANERRIDLFKKF T.
brucei MGCSGSKNASNPKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKQF
60 70 80 90 100 110 T. cruzi
DKNETGKLCYDEVHSGCLEVLKLDEFTPRVRDITKRAFDKARALGSKLENKGSEDFVEFL T.
rangeli
DKNDTGKLSYDEVYSGCIEVLKLDEFTPRVH-ITKRAFNKAKDKGSKLENKGSEDFVEFL T.
brucei DTNGTGKLGFREVLDGCYSILKLDEFTTHLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFL
120 130 140 150 160 170 T. cruzi
EFRLMLCYIYDFFELTVMFDEIDASGNMLVDEEELKRAVPKLEAWGAKVEDPAALFKELD T.
rangeli
EFRLMLCYLYDYFELTVMFDEIDTSGNMLLDAKEFEKAVPKLEQWGAKIEDPAEVFKELD T.
brucei EFRLMLCYIYDIFELTVIFDTMDKDGSLLLELHEFKEALPKLKEWGVDITDATTVFNEID
180 190 200 T. cruzi KNGTGSVTFDEFAAWASAVKLDADGDPDNVPESA 211 T.
rangeli RNGSGSVTFDEFAAWASARKLDVDGDPDNVPESA 205 T. brucei
TNGSGVVTFDEFSCWAVTKKLQVSGDPDDEENGANEGDGANAGDGVPAAEGSA 233
"1 "2
"3#1 "4 "5#2
"5 "6#3 "7"5
"8#4
1 10 20 30 40 50 T. cruzi
MGACGSKGSTSDKGLA---SDKDGKKAKDRKEAWERIRQAIPREKTAEAKQRRIELFKKF T.
rangeli
MGACGSKGS------A---GNKDGKSATDRKVAWERIRQVIPREKTAEANERRIDLFKKF T.
brucei MGCSGSKNASNPKDGAASKGGKDGKTTADRKVAWERIRCAIPRDKDAESKSRRIELFKQF
60 70 80 90 100 110 T. cruzi
DKNETGKLCYDEVHSGCLEVLKLDEFTPRVRDITKRAFDKARALGSKLENKGSEDFVEFL T.
rangeli
DKNDTGKLSYDEVYSGCIEVLKLDEFTPRVH-ITKRAFNKAKDKGSKLENKGSEDFVEFL T.
brucei DTNGTGKLGFREVLDGCYSILKLDEFTTHLPDIVQRAFDKAKDLGNKVKGVGEEDLVEFL
120 130 140 150 160 170 T. cruzi
EFRLMLCYIYDFFELTVMFDEIDASGNMLVDEEELKRAVPKLEAWGAKVEDPAALFKELD T.
rangeli
EFRLMLCYLYDYFELTVMFDEIDTSGNMLLDAKEFEKAVPKLEQWGAKIEDPAEVFKELD T.
brucei EFRLMLCYIYDIFELTVIFDTMDKDGSLLLELHEFKEALPKLKEWGVDITDATTVFNEID
180 190 200 T. cruzi KNGTGSVTFDEFAAWASAVKLDADGDPDNVPESA 211 T.
rangeli RNGSGSVTFDEFAAWASARKLDVDGDPDNVPESA 205 T. brucei
TNGSGVVTFDEFSCWAVTKKLQVSGDPDDEENGANEGDGANAGDGVPAAEGSA 233
"1 "2
"3#1 "4 "5#2
"5 "6#3 "7"5
"8#4
Fig. 1
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 16
Fig. 2A.
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 17
Fig. 2B
"1
K22
K25 R33
N CEF1
EF2EF4
EF3
"2
"3
"4
"5
"5
"6
"7
"8
#1
#2#3
#4
K19
"1
K22
K25 R33
N CEF1
EF2EF4
EF3
"2
"3
"4
"5
"5
"6
"7
"8
#1
#2#3
#4
K19
C
NK19
K22
K25R33
"1 "2"3
"4 "5
"5
#3
"6
#4
"7
"8
EF1
EF2
EF3EF4
C
NK19
K22
K25R33
"1 "2"3
"4 "5
"5
#3
"6
#4
"7
"8
EF1
EF2
EF3EF4
Fig. 2B
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 18
Fig. 3
W19
2
V166
L172
V196
L198
A191
A170
A100
A171
B
W19
2
V166
L172
V196
L198
A191
A170
A100
A171
W19
2
V166
L172
V196
L198
A191
A170
A100
A171
B
K19
R88
K22
K25
K28
R33
R35
K79
K59
K56
R49
K55
A
K19
R88
K22
K25
K28
R33
R35
K79
K59
K56
R49
K55
A
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 19
Fig. 4
7.08.09.010.0
102.0
106.0
110.0
114.0
118.0
122.0
126.0
130.0
G202
G102 G73
T84T43
G14
T91
V147 T62 G180
G182
G63
D81
T181
N179N60
S142S194
L80L78
S183 K174 Q36F113
E32T100S98
D168E41
F83T185
V77I38 R33V114E138K55K153E118
I126
L176V196
K79
E76 R35Y127
E150L121
W162 L75E45V184
L53
R49E52V134
A200N206
R99A171
I51D140
A155K47
F54R50
D201
D128
N144
E29
Q48
A161K178A37
K165L15
K197
L159A30C74E151
D112F152
E131I34
W192K42
F130
E111
A191 D12
D18
L101A46K59
V207
A195
A211
A16
W31L146
N23
V166 E167A164
A44
R40
L198K19
F173
G163
E115
15N
(ppm
)
1H (ppm)7.08.09.010.0
102.0
106.0
110.0
114.0
118.0
122.0
126.0
130.0
G202
G102 G73
T84T43
G14
T91
V147 T62 G180
G182
G63
D81
T181
N179N60
S142S194
L80L78
S183 K174 Q36F113
E32T100S98
D168E41
F83T185
V77I38 R33V114E138K55K153E118
I126
L176V196
K79
E76 R35Y127
E150L121
W162 L75E45V184
L53
R49E52V134
A200N206
R99A171
I51D140
A155K47
F54R50
D201
D128
N144
E29
Q48
A161K178A37
K165L15
K197
L159A30C74E151
D112F152
E131I34
W192K42
F130
E111
A191 D12
D18
L101A46K59
V207
A195
A211
A16
W31L146
N23
V166 E167A164
A44
R40
L198K19
F173
G163
E115
15N
(ppm
)
1H (ppm)
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 20
2.0 1.5 1.0 0.5 2.0 1.5 1.0 0.5 2.0 1.5 1.0 0.5
20.0
16.0
24.0
28.0
32.0
36.0
C14
C13
C3
C4C - C5 11
C12C2
A C14
C13
C3
C - C4 11
C12C2
C14
C13
C3
C - C4 11
C12
C2
B C
1H (ppm)
13C
(ppm
)
2.0 1.5 1.0 0.5 2.0 1.5 1.0 0.5 2.0 1.5 1.0 0.5
20.0
16.0
24.0
28.0
32.0
36.0
C14
C13
C3
C4C - C5 11
C12C2
A C14
C13
C3
C - C4 11
C12C2
C14
C13
C3
C - C4 11
C12
C2
B C
1H (ppm)
13C
(ppm
)
Fig. 5
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 21
Fig. 6
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org
-
Crystal Structure of FCaBP 22
+ + +G2 C4
+ + +
EF2
EF3
EF4
+ + +G2 C4
+ + +
Ca2+membrane
Ca Ca
+ +
+
C4G2
EF1
Target Protein
"1
A. Low Ca2+ B. High Ca2+
Target Protein
+ + +G2 C4
+ + +
EF2
EF3
EF4
+ + +G2 C4
+ + +
Ca2+membrane
Ca Ca
+ +
+
C4G2
+ +
++
+ +
C4G2
EF1
Target Protein
"1
A. Low Ca2+ B. High Ca2+
Target Protein
Fig. 7
at Galter Health Sciences Library on July 14, 2008
www.jbc.orgDownloaded from
http://www.jbc.org