A Novel S100 Target Conformation Is Revealed by the Solution
Structure of the Ca2+-S100B-TRTK-12 Complex*
Kimberly A.
McClintock and
Gary S.
Shaw
From the Department of Biochemistry and McLaughlin Macromolecular
Structure Facility, the University of Western Ontario, London,
Ontario N6A 5C1, Canada
Received for publication, October 17, 2002, and in revised form, December 9, 2002
 |
ABSTRACT |
The Alzheimer-linked neural protein S100B is a
signaling molecule shown to control the assembly of intermediate
filament proteins in a calcium-sensitive manner. Upon binding calcium,
a conformational change occurs in S100B exposing a hydrophobic surface
for target protein interactions. The synthetic peptide TRTK-12
(TRTKIDWNKILS), derived from random bacteriophage library screening,
bears sequence similarity to several intermediate filament proteins and
has the highest calcium-dependent affinity of any target
molecule for S100B to date (Kd <1
µM). In this work, the three-dimensional structure of the
Ca2+-S100B-TRTK-12 complex has been determined by NMR
spectroscopy. The structure reveals an extended, contiguous hydrophobic
surface is formed on Ca2+-S100B for target interaction. The
TRTK-12 peptide adopts a coiled structure that fits into a portion of
this surface, anchored at Trp7, and interacts with multiple
hydrophobic contacts in helices III and IV of Ca2+-S100B.
This interaction is strikingly different from the
-helical structures found for other S100 target peptides. By using the TRTK-12
interaction as a guide, in combination with other available S100 target
structures, a recognition site on helix I is identified that may act in
concert with the TRTK-12-binding site from helices III and IV. This
would provide a larger, more complex site to interact with full-length
target proteins and would account for the promiscuity observed for
S100B target protein interactions.
 |
INTRODUCTION |
The S100 proteins are low molecular weight (10-12 kDa) members of
the EF-hand family of calcium-binding proteins. Many of these proteins,
including several of the S100s, the muscle contractile protein troponin
C, and the ubiquitous protein calmodulin act as signaling molecules by
converting an influx of cellular calcium into a biological response.
Calcium binding to these EF-hand proteins triggers a conformational
change and allows the protein to interact with an appropriate target
molecule. The S100 proteins are unique among this family because,
unlike troponin C or calmodulin, they exist in solution as homo- or
heterodimers. Each S100 monomer contains two helix-loop-helix
calcium-binding motifs as follows: a basic N-terminal pseudo EF-hand
comprising 14 residues (site I), and a canonical and acidic C-terminal
EF-hand of 12 residues (site II). A central linker region joining the
two EF-hands along with the extreme N and C termini of these proteins
exhibit the most sequence divergence among family members and are
therefore believed to provide specificity for target protein
interactions. This feature, along with the dimeric state of the S100
proteins, likely indicates these calcium-signaling proteins have the
distinctive ability to interact with more than one target molecule at a time.
S100B,1 a homodimer of
91-residue S100
monomers, is found primarily in glial cells and has
been implicated in neurological diseases including Alzheimer's disease
and Down's syndrome (1-3). More than 20 calcium-sensitive in
vitro binding partners have been identified for S100B (4)
including several cellular architecture proteins such as tubulin (5)
and GFAP (6), where S100B can inhibit polymerization of these
oligomeric molecules. Furthermore, S100B inhibits the phosphorylation
of multiple kinase substrates including the Alzheimer protein tau (7,
8) and neuromodulin (GAP-43) (9) through a calcium-sensitive
interaction with the protein substrates. Consistent with the
calcium-induced conformational change mechanism, a comparison of
three-dimensional structures of apo- and calcium-bound S100B reveals
that binding of calcium leads to the exposure of a hydrophobic
surface(s) for protein-protein interactions (10).
The 12-residue peptide TRTK-12, derived from random bacteriophage
library experiments, has been used in previous studies as a model for
S100B-target protein interactions. Experiments utilizing this peptide
have indicated that peptides containing the consensus sequence
(R/K)(L/I)(XWXXIL) bind specifically to S100B in
a calcium-sensitive manner (11). Furthermore, this consensus sequence
is conservatively found in the cytoskeletal proteins tubulin, desmin,
vimentin, and GFAP, shown previously to interact with
Ca2+-S100B (12). The TRTK-12 peptide competes with S100B
target proteins including CapZ-
and GFAP for Ca2+-S100B
binding (6, 11) and has the highest affinity (Kd ~260 nM) (13) of any known S100B target. Fluorescence
studies (13) have shown that Trp7 of TRTK-12 is a key
residue for the calcium-sensitive interaction with S100B becoming
buried at the protein-peptide interface. In addition, deletion of
residues 85-91 from S100B leads to a >2000-fold decrease in affinity
for TRTK-12 indicating the C-terminal helix is an important site of interaction.
To date, only three structures are available for S100-target peptide
complexes. In each case the target peptide adopts a 2.5 turn
-helical structure that interacts via two distinct modes with the
S100 protein. The structures of human S100A10 and S100A11 in complex
with peptides from the binding regions of annexin II and I,
respectively, are nearly identical (14, 15) with the annexin peptides
bridging the two S100 monomers through contacts in the linker region
and C terminus of one monomer and the N terminus of the other monomer.
Surprisingly, this similarity of interaction occurs despite little
sequence similarity between the annexin peptides. In contrast, the
interaction of rat S100B with a 23-residue peptide from the tumor
suppressor protein p53 shows each S100
monomer binds to a single
peptide through interactions with helix III and a portion of helix IV
(16). This orients the
-helical p53 peptide about 90° from that
found for the annexin peptides with respect to their S100 partners.
These structural variations of the S100A10, S100A11, and S100B
complexes indicate that recognition differences in the protein and the
target must exist for S100 proteins. In an effort to clarify these
interactions, we present the three-dimensional solution structure of
Ca2+-S100B in complex with the TRTK-12 peptide. The amino
acid sequence of TRTK-12 does not correspond to a natural sequence for
any known S100B target. However, its unusually high affinity for S100B
may indicate that it contains structural determinants that remain to be
uncovered for recognition of an S100 binding partner. The binding
interaction of TRTK-12 is unexpected with the peptide adopting an
extended and reversed orientation compared with the p53 interaction.
Furthermore, there is no interaction with helix I of S100B as found in
the S100A11/S100A10 annexin structures thus providing a novel third
mode of recognition for an S100-target protein complex.
 |
EXPERIMENTAL PROCEDURES |
Sample Preparation--
Human S100B protein was expressed in
Escherichia coli (strain N99) and purified as described
previously (17). Uniformly 15N-labeled or
15N/13C-labeled S100B was prepared using M9
minimal media containing 1 g/liter 99% 15NH4Cl
or 1 g/liter 99% 15NH4Cl and 2 g/liter
[13C]glucose. Uniformly
2H/15N-labeled S100B was prepared in M9 minimal
media containing 1 g/liter 99% 15NH4Cl and
99% D2O. Unlabeled TRTK-12 peptide
(Ac-TRTKIDWNKILS-NH2) and
[13C]isoleucine/[13C]acetyl-labeled peptide
(*Ac-TRTK*IDWNK*ILS-NH2) were synthesized by the
Queen's Peptide Synthesis Lab (Queen's University, Kingston, Canada).
Purity was confirmed using reversed phase-high pressure liquid
chromatography and mass spectrometry. Typically, NMR samples contained
1 mM S100
monomer, 1.2 mM TRTK-12 peptide,
35 mM KCl, 4 mM CaCl2, and 5 mM dithiothreitol in 90% H2O, 10%
D2O (v/v), pH 7.05. For a single sample, a mixed
13C/12C S100B dimer was prepared by incubating
20 mg each of 13C-labeled S100B and unlabeled S100B (total
S100B concentration) in D2O at 37 °C for 150 h.
Evolution of the mixed dimer over time was monitored by mass
spectrometry. The final concentration of 13C/12C S100B dimer in the NMR sample was
~1.5 mM, and the sample was prepared in 100%
D2O. Experimental conditions were otherwise identical to
those reported above.
NMR Spectroscopy--
NMR experiments were performed at 35 °C
on Varian 500-, 600-, and 800-MHz spectrometers with pulsed field
gradient probes. Backbone resonances for Ca2+-S100B in the
complex were sequentially assigned using HNCACB (18), CBCA(CO)NH (19),
and HNCO (20) experiments. Side chain assignments were made using
C(CO)NH (21), H(CCO)NH (21), 15N-edited TOCSY, and
HCCH-TOCSY (22) experiments. Some aromatic ring protons were assigned
using (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments (23). TRTK-12
1H assignments were determined from homonuclear
two-dimensional NOESY and two-dimensional TOCSY experiments with wet
and watergate water suppression, respectively, using uniformly
15N,2H-labeled S100B and unlabeled TRTK-12.
Side chain 13C resonances were obtained from a natural
abundance 13C-HSQC. 13C and 1H
assignments for the two isoleucine residues (Ile5 and
Ile10) were confirmed using specifically labeled TRTK-12.
Spectra were processed and analyzed on a Silicon Graphics work station
using NMRPipe, NMRDraw (24), and Pipp and Stapp (25) programs.
Structure Determination--
Approximate interproton distances
in S100B were determined using 15N-filtered NOESY
(26)(
mix = 150 ms) and simultaneous
13C/15N-separated three-dimensional NOESY-HSQC
(27)(
mix = 100 ms) experiments. NOEs derived from the
15N-filtered NOESY were calibrated based on known
dHN
and sequential d
HN distances. The
13C/15N-separated three-dimensional NOESY-HSQC
was calibrated to geminal protons or protons on adjacent carbon atoms.
In cases where direct calibration was not possible, the maximum
distance of 6.0 Å was used.
and
angles for
Ca2+-S100B and the bound TRTK-12 peptide were incorporated
where greater than 9 of 10 predictions fell within the same region of
the Ramachandran plot based on the TALOS program (28). Minimum standard
deviations of ±20° and ±30° were used for
and
,
respectively.
angle restraints were confirmed with
3JNH-H
coupling constants derived
from an HNHA experiment (29). Hydrogen bond distance
restraints of rNH-O = 1.4-2.7 Å and
rN-O = 2.4-3.7 Å were implemented based on the
identification of helical regions in initial structures. NOEs between
the two S100
monomers were unambiguously assigned using a mixed
13C/12C S100B dimer and a 13C
F1-edited F3-filtered NOESY-HMQC experiment
(30) which detects only NOEs between a proton attached to
13C and a proton attached to12C. These NOEs
were calibrated in the same fashion as cross-peaks from the
13C/15N-separated three-dimensional
NOESY-HSQC.
Secondary structure of the TRTK-12 peptide was determined using a
two-dimensional wetNOESY experiment and 15N,2H
S100B protein. Dihedral restraints for the peptide were included using
the same stipulations as for Ca2+-S100B. Intermolecular
NOEs between S100B and TRTK-12 were unambiguously identified using the
same 13C F1-edited F3-filtered
NOESY-HMQC experiment (30) described above with
13C/15N-labeled S100B protein and unlabeled
TRTK-12 as well as a simultaneously 13C/15N-separated three-dimensional NOESY-HSQC
using 15N-labeled S100B and specifically labeled
TRTK-12.
Initial structures were calculated using the hybrid distance geometry
and simulated annealing protocol in the Crystallography and NMR System
program, version 1.1. The two monomeric subunits of S100B were
constrained to be identical using the non-crystallographic symmetry
definition and a force constant of 10 kcal/mol. The same constraint was
applied for the two molecules of TRTK-12.
Circular Dichroism Spectroscopy--
Experiments were performed
at 25 °C on a Jasco J-810 spectropolarimeter. For the TRTK-12,
Ca2+-S100B, and Ca2+-S100B-TRTK-12 samples,
spectra from three scans (200-250 nM) in a 1-mm path
length cuvette were averaged, and the buffer background was subtracted.
Protein samples were prepared in 50 mM KCl, 10 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM CaCl2 to final protein concentrations of 100 µM for S100
and 100 µM for TRTK-12 and 100 µM S100
; 100 µM TRTK-12 for the complex.
 |
RESULTS |
Binding of the TRTK-12 peptide has been monitored previously by
NMR and fluorescence spectroscopy and found to have a
Kd = 260 nM (13). In this work,
formation of the complex was measured using a 15N-labeled
S100B sample and monitoring the change in resonances as a function of
added TRTK-12 peptide. Complex formation was complete at a ratio of 2:1
TRTK-12:S100B indicating that two TRTK-12 peptides bind to each S100B
dimer protein. The resulting 1H-15N HSQC
spectrum of this complex showed a single set of resonances in the NMR
spectra for most residues (Fig. 1)
indicating the arrangement of the Ca2+-S100B-TRTK-12
complex dimer is symmetric. Exceptions were noted for residues
Ser1, Leu3, Lys5,
Ser41, and His42 which have duplicate peaks
resulting from the presence of formyl and desformyl N-terminal
methionine S100B species as observed previously for apo- and
Ca2+-S100B (31). Several residues in the N-terminal
calcium-binding loop have weak (Ser18, Glu21,
His25, Lys26, Leu27, and
Lys28) or absent (Gly22 and Asp23)
amide correlations in the 1H-15N-HSQC likely
due to exchange with the H2O solvent. Gly66
shows a substantial downfield 1H shift characteristic of
calcium binding to the C-terminal calcium loop. A comparison of this
spectrum with that for Ca2+-S100B alone indicated that
several residues including Ile47, Val53,
Ala78, Ala83, and Cys84 had shifted
upon TRTK-12 binding.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1.
1H-15N HSQC spectrum
(500 MHz) of Ca2+-S100B bound to TRTK-12. Backbone
amide resonances are indicated. All backbone amides were identified
with the exception of Gly22 and Asp23.
Correlations of side chain amide groups are indicated by
horizontal lines. Duplicate peaks are observed for residues
at the extreme N terminus (S1, L3, and K5) and in
the linker region (S41 and H42) due to the
presence of both formyl and desformyl N-terminal methionine S100B
forms.
|
|
To identify the specific interactions between TRTK-12 and human
Ca2+-S100B, the solution structure of the complex (24 kDa)
was determined using 2072 experimental distance restraints in
combination with 536 dihedral and 136 hydrogen bond restraints. This
generated a family of 17 well defined
structures (Fig. 2) based on the superposition of the 8 helices
in the dimer (root mean square deviation 0.63 ± 0.07)
(Table I). The TRTK-12 peptide was well
defined between residues 4 and 12 forming a coil structure that folds
back on itself. As observed in the rat S100B-p53 structure (16), each S100B monomer binds one TRTK-12 molecule.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Structure of the
Ca2+-S100B-TRTK-12 complex. The N and C termini are
labeled for one S100 monomer and TRTK-12, and helices are indicated
for S100 . A, stereo view of the backbone superposition of
the final ensemble of 17 NMR derived structures of the complex. S100
monomers are shown in magenta and blue, and the
TRTK-12 peptide molecules are shown in light blue. B,
ribbon structure of the complex. Each monomer consists of four
-helices (helix I, blue; II, magenta; III,
green; and IV, yellow) and two anti-parallel
-strands (orange, blue). TRTK-12 is shown in
dark blue.
|
|
Structure of Ca2+-S100B in the Complex--
Overall,
the structure of human Ca2+-S100B in the TRTK-12 complex
retains many structural features of the calcium-bound form alone (32).
The dimer interface is characterized by antiparallel interactions
between helices I and I' (interhelical angle (
) = 164 ± 4°) and helices IV and IV' (
= 172 ± 4°) forming an
X-type four helix bundle (Fig. 2). Each S100
monomer consists of an N-terminal pseudo EF-hand motif (site I) comprising helix I
(Leu3-Tyr17), calcium binding loop I
(Ser18-Glu31), and helix II
(Ser30-Glu39) and a C-terminal canonical
EF-hand (site II) comprising helix III
(Gln50-Leu60), calcium binding loop II
(Asp61-Glu72), and helix IV
(Phe70-Cys84). Calcium binding loops I and II
are associated through a short antiparallel
-sheet
(Lys26-Lys28 and
Glu67-Asp69). The arrangement of helices I and
II in site I (
= 129 ± 3°) is similar to that of
Ca2+-S100B (
= 138 ± 4°) and calbindin
D9k (
= 130 ± 2°), indicating that TRTK-12
binding to Ca2+-S100B results in little conformational
change to this region. In contrast, significant changes are identified
in site II. The helix III-IV interhelical angle (
= 104 ± 4°) is dramatically opened by more than 90° than found in
apo-S100B (
=
166 ± 1°). Furthermore, the arrangement
of helices III-IV shows distinct differences from human
Ca2+-S100B (
= 148 ± 4°) being more similar
to the III-IV arrangement found in the S100A11-annexin I complex
(
= 103°) and more "open" compared with either the
S100B-p53 (
= 110 ± 1°) or S100A10-annexin II (
= 117°) complexes. These observations suggest that the relative orientations of helices III and IV may be a governing factor for target specificity.
Structure of TRTK-12 in the Complex--
Our previous studies (33)
have shown that the TRTK-12 peptide is unstructured in the absence of
Ca2+-S100B. Consistent with this, the circular dichroism
spectrum of TRTK-12 (Fig. 3) showed an
ellipticity minimum at <200 nm indicative of a random coiled
structure. Upon binding, TRTK-12 assumes a coiled conformation that
folds back upon itself (Fig. 2). The structure determination of TRTK-12
was aided by using a peptide having uniform 13C-labeling at
Ile5 and Ile10 and the N-terminal acetyl
CH3 positions thus providing 13C markers
throughout the peptide length. The coiled structure of TRTK-12 was
supported by definitive NOEs between the side chains of residues
Trp7-Leu11, and peptide spectra were
characterized by an absence of
-helical NOEs and
-proton (and
C for Ile5 and Ile10) chemical shifts
not consistent with
-helical structure. These findings are supported
by CD spectra of Ca2+-S100B (Fig. 3) that show ellipticity
minima at 208 and 222 nm typical of an
-helical protein. Upon
addition of 1 eq of TRTK-12 per S100
monomer (Fig. 3). little
difference in this spectrum occurs indicating the TRTK-12 peptide does
not assume an
-helical conformation. Furthermore, the
208/
222 ratio for the complex appears
nearly identical to that of Ca2+-S100B, indicating little
change in helix-helix interactions occurs.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Far-UV circular dichroism spectra of
Ca2+-S100B in the presence of the TRTK-12 peptide. The
spectra show TRTK-12 ( ), Ca2+-S100B ( ), and
Ca2+-S100B in the presence of TRTK-12 ( ). The complex
was formed using a 1:2 ratio of Ca2+-S100B (50 µM) and TRTK-12 (100 µM) consistent with
the binding of one TRTK-12 peptide for each S100 monomer.
|
|
Ca2+-S100B-TRTK-12 Interactions--
The interface
between Ca2+-S100B and TRTK-12 is defined by several
residues in helix III (Val52, Lys55,
Val56, Thr59, and Leu60) and helix
IV (Phe76, Ala80, Ala83, and
Cys84). In particular, 13C-labeled
Ile5 and Ile10 revealed definitive interactions
to residues Thr82 in helix IV and Val52 and
Val56 in helix III, respectively (Fig.
4). Furthermore, residues
Lys9, Ile10, and Leu11 in the C
terminus of TRTK-12 have multiple contacts in helix III, and
Thr1, Thr3, and Lys4 are in close
proximity to Ala83 in helix IV. These interactions position
the N terminus of the TRTK-12 peptide near helix IV and the C terminus
near helix III resulting in a peptide orientation opposite to that
observed for the S100B-p53 complex. Although 13C labeling
of the N-terminal acetyl group in TRTK-12 was used, no observable
peptide-protein cross-peaks resulting from this label were observed in
13C-edited NOE spectra, indicating the extreme N terminus
of TRTK-12 is exposed to solvent and does not interact with residues in
helix I as the annexin peptides do in the S100A10 and S100A11
structures. A large hydrophobic cavity exists on Ca2+-S100B
where TRTK-12 is bound (Fig. 5). This
observation is consistent with previous results showing that calcium
binding to human S100B results in exposure of a hydrophobic surface
(10). Trp7 of the peptide is the anchoring residue nestled
in a hydrophobic pocket including residues Val56,
Thr59, Phe76, and Val80 of S100B.
This environment results in the observed blue shift and an increase in
fluorescence intensity for Trp7 upon protein binding (33).
Comparison of the exposed surface area between Ca2+-S100B
alone and in complex with TRTK-12 shows the side chains of
Val56, Thr59, and Ala83 decrease
their surface exposure by 86, 96, and 99%, respectively, upon TRTK-12
binding. Similarly, residues Trp7 and Ile10 of
TRTK-12 have more than 84 and 90%, respectively, of their surface area
buried in the complex. Recent fluorescence studies of the S100B-TRTK-12
interaction have indicated that deletion of the C-terminal 7 residues
in S100B results in a drastic decrease in TRTK-12 binding affinity
(13). This was attributed to a loss in
-helical structure near
Ala83 in the truncated S100B protein. Presumably this would
considerably alter the arrangement of residues Val80,
Ala83, and Cys84 resulting in a significantly
reduced affinity.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
Ca2+-S100B-TRTK-12
interactions. A, strip plots extracted from
mix = 100-ms 13C/15N-edited
NOESY-HSQC spectrum of Ca2+-S100B bound to
[13C]Ile-labeled TRTK-12 peptide (Ile5
and Ile10, 1st 3 panels) and mix = 100-ms 13C F1-edited, F3-filtered
NOESY-HMQC experiment of 13C-labeled Ca2+-S100B
(Ala83 and Lys55, last 2 panels)
bound to unlabeled TRTK-12. Each strip represents the 13C
plane and 1H chemical shift of the indicated residue.
Asterisks indicate NOEs involving TRTK-12 residues.
B, structural details of the binding interface of the
Ca2+-S100B-TRTK-12 complex. The TRTK-12 peptide
(blue) shows residues Thr1, Lys4,
Ile5, Lys9, and Ile10 (*), where
NOE contacts were observed to residues Val52,
Lys55, Val56, Thr82, and
Ala83 in Ca2+-S100B (magenta) as
shown in A.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Electrostatic potential surface showing the
TRTK-12-binding site on Ca2+-S100B. Negative potential
is indicated in blue, and positive potential is shown in
red for Ca2+-S100B. A, location of
the two TRTK-12 molecules (green) on opposite sides of
Ca2+-S100B. The TRTK-12 peptide fits into the hydrophobic
cleft generated by helices III and IV of each monomer. Note that on
either side of the TRTK-12-binding site a significant uncharged region
exists. B, environment of the anchoring Trp7
residue from TRTK-12 (green) showing interactions with S100B
residues (blue) in helix III (Val56 and
Thr59) and IV (Phe76 and
Val80).
|
|
 |
DISCUSSION |
Examination of the structure of Ca2+-S100B-TRTK-12 and
comparison to the existing structures of S100A10-annexin II,
S100A11-annexin I, and S100B-p53 allows a distinction to be made
between target recognition by these S100 proteins. Furthermore, the
TRTK-12 sequence was identified from random bacteriophage peptide
selection and therefore may contain unique binding features in
comparison to the natural sequences for the annexin and p53 peptides.
In the current structure, the TRTK-12 peptide lies in a hydrophobic
cleft formed between helices III and IV maximizing the interaction of the hydrophobic residues Ile5, Trp7,
Ile10, and Leu11 with residues in both helices
of S100B. Nearly all the interaction is hydrophobic in nature
reaffirming earlier work that the S100B-TRTK-12 interaction is tighter
at increased ionic strength (13). The interaction is distinct from that
of the rat S100B-p53 peptide (16), likely due to the
sequence differences of the interacting peptides. The target sequence
for p53 (SRHKKLMFKT) bears little similarity to TRTK-12 containing a
highly positively charged C terminus that orients near the highly
acidic N terminus of helix III (EEIKEEQE). In contrast, the charged
residues in TRTK-12 are more dispersed throughout its sequence and most
are occupied through potential hydrogen-bonding interactions.
Nevertheless, some similarities between the target peptide molecules
exist. The anchoring residues for the p53 peptide are
Leu383 and Phe385, spaced only one residue
apart, as are Ile5 and Trp7 in TRTK-12. The
distinguishing feature of these two residues in each peptide is their
positions with respect to S100B. In the current work Trp7
is positioned in an analogous region to Leu383 in p53,
whereas the side chain of Ile5 is located nearby that of
Phe385 in p53, albeit somewhat shifted due to a different
orientation. The reverse nature of these two interactions together with
the differential charge balance in the peptides likely results in the
reversal of orientation of TRTK-12 to p53 upon binding to S100B.
The regions of interaction of TRTK-12 and p53 differ significantly from
that of both annexin I and II with S100A11 and S100A10, respectively.
Both of the annexin peptides have interactions with several residues in
the N-terminal helix I of these S100 proteins (Fig.
6A). An examination of the
sequences and interactions reveals three key residues in the annexin
peptides (Val3, Glu5, and Leu7)
that have identical interactions with the N-terminal residues Glu5, Met8, Glu9, and
Met12 in S100A10 (Glu9, Ile12,
Glu13, and Ile16 in S100A11). This pattern in
the target peptide, where two hydrophobes are separated by three
residues, including a central acidic residue, is not found in either
the TRTK-12 or p53 peptides. Furthermore, the side chain carboxylate of
Glu9 in S100A10 (Glu13 in S100A11) has
conserved hydrogen bonds to the Thr2 and Val3
amides in annexin II (Met2 and Val3, in
S100A11). In S100B the position of Glu9 (Glu13)
is held by a hydrophobe (Val8) removing its side chain
hydrogen bonding ability. Interestingly, this position is among the
least conserved within the S100 protein family. This sequence
divergence in both the S100B and S100A10/S100A11 proteins and in the
peptide motif is likely responsible for the different target peptide
recognition sites between the proteins. Consistent with this, previous
experiments have shown that TRTK-12 shows no observable
interaction with S100A11 (33) when monitored by Trp7
fluorescence.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
Interactions of S100 proteins with annexin I,
annexin II, and TRTK-12. A, aligned sequences of helices I
(top) for S100B, S100A11, and S100A10 along with observed
interactions to their respective target peptides ( ). The annexin I
and II peptides were aligned based on the structurally conserved
interactions to the N-terminal residues of S100A11 and S10010,
respectively (green), and involving the identical residues
(Val, Glu, and Leu) in the annexin peptides (dark shading).
The conserved interaction involving Glu9 in S100A10
(Glu13 in S100A11) is unlikely in S100B where a valine
residue occupies this position (blue). B, the
aligned sequences of helix III and the C-terminal portion of the linker
are shown together along with the common interactions to
Trp7 (pink) in the TRTK-12 peptide and Trp11 in
the annexin I peptide ( ). Interactions found between S100B and
TRTK-12, but not in annexin I, are also shown (- - -). C,
proposed full-length target site for S100B. The accessible surface for
Ca2+-S100B was calculated based on the complex with TRTK-12
in the absence of the peptide. Residues where the side chain
interactions with the TRTK-12 peptide (green) and those
where a proposed site exists based on the S100A11-annexin I structure
(yellow) are shown based on the observations in
A. Residues that have common contacts to the bridging Trp
residue, as shown in B, are shown in red. A
comparison of the structures reveals that the hydrophobic surface used
by S100B for recognition of TRTK-12 (green) is largely
exposed in the S100A11-annexin I structure and vice versa.
|
|
The interaction of Trp7 in TRTK-12 likely contributes to
the tightest binding for any S100B target to date. It is also
interesting that replacement of Phe385 in the p53 peptide
with a Trp residue increases its affinity for rat S100B by about 5-fold
(34). In addition, important similarities exist between the
environments of Trp7 (TRTK-12) and Trp11
(annexin I) even though the orientation of the two target peptides is
different with respect to the protein helices in each complex. A
comparison of the anchoring Trp7 of TRTK-12 reveals side
chain interactions (<6.5 Å) with Ile47,
Val52, and Val56 in a majority of the NMR
structures. The analogous interactions, involving Gln52,
Val57, and Met61 exist for S100A11 (Fig.
6B). Despite this similarity, the differences of the peptide
orientations for TRTK-12 and annexin I result in a significant
hydrophobic surface for each protein that remains exposed, even in the
presence of the bound peptide (Fig. 5A and Fig.
6C). This may occur simply because the target peptides
presently used are too short or that the interaction can be modulated
through a second region of interaction. As a result, it has been
suggested recently (35) that S100 proteins may bind full-length targets using more than one site. The similarity of the locations for Trp7 (TRTK-12) and Trp11 (annexin I) would
provide a unique bridging residue to encompass a larger, contiguous
binding region that includes an
-helical annexin type interaction,
utilizing the VEL residues found in annexin I and II, and an extended
TRTK-12 type interaction based on hydrophobic interactions anchored by
Trp7 (Fig. 6C). It is interesting that the
recent structure of the calmodulin-calmodulin kinase kinase
peptide complex displays exactly these features, where the N-terminal
portion of the peptide forms an
-helical structure and the
C-terminal region is more extended (36), folding back on the helix in
addition to having important protein contacts. As in calmodulin, this
larger interaction site would also provide a rationale for the
promiscuity of S100B target interactions, including cellular
architecture proteins such as tubulin, vimentin, desmin, GFAP, and
caldesmon, which contain the TRTK-12 consensus motif (12). It remains
to be seen whether the natural sequences for the intermediate filament
proteins interact in a similar manner as TRTK-12 or via the
-helical
mode found for either annexin or p53 peptides.
Coordinates--
Chemical shift assignments for S100B and
TRTK-12 in the complex have been deposited in the BioMagResBank
(accession number 5377).
 |
ACKNOWLEDGEMENTS |
We thank Kathryn Barber for technical
support; Frank Delaglio and Dan Garrett (National Institutes of Health)
for NMRPipe and Pipp; Lewis Kay (University of Toronto) for all pulse
sequences; and The National High Field Nuclear Magnetic Resonance
Facility (NANUC, Edmonton, Alberta, Canada) for acquisition of
13C F1-edited F3-filtered
NOESY-HMQC experiments. Funding for 500 and 600 MHz NMR spectrometers
was made possible by grants from the Canada Foundation for Innovation,
the Medical Research Council of Canada, and the Academic Development
Fund of the University of Western Ontario and generous gifts from R. Samuel McLaughlin Foundation and London Life Insurance Company of Canada.
 |
FOOTNOTES |
*
This work was supported by operating and maintenance grants
from the Canadian Institute for Health Research (to G. S. S.) and
graduate studentships from the Natural Sciences and Engineering Research Council (to K. A. M.). This work was presented at the XXth
International Conference on Magnetic Resonance in Biological Systems,
August 25-30, 2002, Toronto, Ontario, Canada.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 1MQ1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Fax: 519-661-3175;
E-mail: gshaw1@uwo.ca.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210622200
 |
ABBREVIATIONS |
The abbreviations used are:
S100B, dimeric
S100
;
GFAP, glial fibrillary acidic protein;
TRTK-12, acetyl-TRTKIDWNKILS-NH2;
NOE, nuclear Overhauser effect;
MOPS, 4-morpholinepropanesulfonic acid.
 |
REFERENCES |
1.
|
Marshak, D. R.,
Pesce, S. A.,
Stanley, L. C.,
and Griffin, W. S. T.
(1991)
Neurobiol. Aging
13,
1-7
|
2.
|
Van Eldik, L. J.,
and Griffin, W. S. T.
(1994)
Biochim. Biophys. Acta
1223,
398-403[Medline]
[Order article via Infotrieve]
|
3.
|
Griffin, W. S. T.,
Stanley, L. C.,
Ling, C.,
White, L.,
MacLeod, V.,
Perrot, L. J.,
White, C. L.,
and Araoz, C.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
7611-7615[Abstract]
|
4.
|
Donato, R.
(1999)
Biochim. Biophys. Acta
1450,
191-231[Medline]
[Order article via Infotrieve]
|
5.
|
Donato, R.
(1988)
J. Biol. Chem.
263,
106-110[Abstract/Free Full Text]
|
6.
|
Bianchi, R.,
Garbuglia, M.,
Verzini, M.,
Giambanco, I.,
Ivanenkov, V. V.,
Dimlich, R. V. W.,
Jamieson, G. A., Jr.,
and Donato, R.
(1996)
Biochim. Biophys. Acta
1313,
258-267[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Baudier, J.,
Mochly-Rosen, D.,
Newton, A.,
Lee, S.-H.,
Koshland, D. E.,
and Cole, R. D.
(1987)
Biochemistry
26,
2886-2893[Medline]
[Order article via Infotrieve]
|
8.
|
Baudier, J.,
and Cole, R. D.
(1988)
J. Biol. Chem.
263,
5876-5883[Abstract/Free Full Text]
|
9.
|
Lin, L.-H.,
Van Eldik, L. J.,
Osheroff, N.,
and Norden, J. J.
(1994)
Mol. Brain Res.
25,
297-304[Medline]
[Order article via Infotrieve]
|
10.
|
Smith, S. P.,
and Shaw, G. S.
(1998)
Structure
6,
211-222[Medline]
[Order article via Infotrieve]
|
11.
|
Ivanenkov, V. V.,
Jamieson, G. A., Jr.,
Gruenstein, E.,
and Dimlich, R. V. W.
(1995)
J. Biol. Chem.
270,
14651-14658[Abstract/Free Full Text]
|
12.
|
McClintock, K. A.,
and Shaw, G. S.
(2000)
Protein Sci.
9,
2043-2046[Abstract]
|
13.
|
McClintock, K. A.,
Van Eldik, L. J.,
and Shaw, G. S.
(2002)
Biochemistry
41,
5421-5428[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Rety, S.,
Sopkova, J.,
Renouard, M.,
Osterloh, D.,
Gerke, V.,
Tabaries, S.,
Russo- Marie, F.,
and Lewit-Bentley, A.
(1999)
Nat. Struct. Biol.
6,
89-95[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Rety, S.,
Osterloh, D.,
Arie, J.-P.,
Tabaries, S.,
Seeman, J.,
Russo-Marie, F.,
Gerke, V.,
and Lewit-Bentley, A.
(2000)
Structure
8,
175-184[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Rustandi, R. R.,
Baldisseri, D. M.,
and Weber, D. J.
(2000)
Nat. Struct. Biol.
7,
570-574[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Smith, S. P.,
Barber, K. R.,
Dunn, S. D.,
and Shaw, G. S.
(1996)
Biochemistry
35,
8805-8814[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Wittekind, M.,
and Mueller, L.
(1993)
J. Magn. Reson. Ser. B.
101,
171-180
|
19.
|
Grzesiek, S.,
and Bax, A.
(1992)
J. Am. Chem. Soc.
114,
6291-6293
|
20.
|
Kay, L. E., Xu, G. Y.,
and Yamazaki, T.
(1994)
J. Magn. Reson. Ser. A
109,
129-133[CrossRef]
|
21.
|
Grzesiek, S.,
Anglister, J.,
and Bax, A.
(1993)
J. Mag. Reson.
101,
114-119[CrossRef]
|
22.
|
Kay, L. E., Xu, G.,
Singer, A. U.,
Muhandiram, D. R.,
and Forman-Kay, J. D.
(1993)
J. Magn. Reson. Ser. B
101,
333-337[CrossRef]
|
23.
|
Yamazaki, T.,
Forman-Kay, J. D.,
and Kay, L. E.
(1993)
J. Am. Chem. Soc.
115,
11054-11055
|
24.
|
Delaglio, F.,
Grzesiek, S.,
Vuister, G. W.,
Zhu, G.,
Pfeifer, J.,
and Bax, A.
(1995)
J. Biomol. NMR
263,
277-293
|
25.
|
Garrett, D. S.,
Powers, R.,
Gronenborn, A. M.,
and Clore, G., M.
(1991)
J. Magn. Reson.
95,
214-220
|
26.
|
Zhang, O.,
Kay, L. E.,
Olivier, J. P.,
and Foreman-Kay, J. D.
(1994)
J. Biomol. NMR
4,
845-858[Medline]
[Order article via Infotrieve]
|
27.
|
Pascal, S. M.,
Muhandiram, D. R.,
Yamazaki, T.,
Forman-Kay, J. D.,
and Kay, L. E.
(1994)
J. Mag. Reson. Ser. B
103,
197-201
|
28.
|
Cornilescu, G.,
Delaglio, F.,
and Bax, A.
(1999)
J. Biomol. NMR
13,
289-302[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Vuister, G. W.,
and Bax, A.
(1993)
J. Am. Chem. Soc.
115,
7772-7777
|
30.
|
Lee, W.,
Revington, M.,
Arrowsmith, C.,
and Kay, L. E.
(1994)
FEBS Lett.
350,
87-90[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Smith, S. P.,
Barber, K. R.,
and Shaw, G. S.
(1997)
Protein Sci.
6,
1110-1113[Abstract/Free Full Text]
|
32.
|
Smith, S. P.,
and Shaw, G. S.
(1997)
J. Biomol. NMR
10,
77-88[Medline]
[Order article via Infotrieve]
|
33.
|
Barber, K. R.,
McClintock, K. A.,
Jamieson, G. A., Jr.,
Dimlich, R. V.,
and Shaw, G. S.
(1999)
J. Biol. Chem.
274,
1502-1528[Abstract/Free Full Text]
|
34.
|
Rustandi, R. R.,
Drohat, A. C.,
Baldisseri, D. M.,
Wilder, P. T.,
and Weber, D. J.
(1998)
Biochemistry
37,
1951-1960[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Otterbein, L. R.,
Kordowska, J.,
Witte-Hoffmann, C.,
Wang, C.-L. A.,
and Dominguez, R.
(2002)
Structure
10,
557-567[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Kurokawa, H.,
Osawa, M.,
Kurihara, H.,
Katayama, N.,
Tokumitsu, H.,
Swindells, M. B.,
Kainosho, M.,
and Ikura, M.
(2001)
J. Mol. Biol.
312,
59-68[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Laskowski, R. A.,
MacArthur, M. W.,
Moss, D. S.,
and Thornton, J. M.
(1993)
J. Appl. Crystallogr
26,
283-290[CrossRef]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.