Modulation of Flash-Induced Photosystem II Fluorescence by Events Occurring a...
APO-S100B
1. proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS
Analysis of the structure of human
apo-S100B at low temperature indicates a
unimodal conformational distribution is
adopted by calcium-free S100 proteins
Shahid Malik,1
Matthew Revington,1
Steven P. Smith,2
and Gary S. Shaw1
*
1 Department of Biochemistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada
2 Department of Biochemistry, Queen’s University, Kingston, Ontario, K7L 3N6, Canada
INTRODUCTION
The S100 protein family makes up one of the largest EF-hand
groups of proteins, comprising at least 25 members in
humans.1,2 These low molecular weight (21–24 kDa) proteins
are generally expressed in a cell-specific manner having a variety
of biological roles including the regulation of cytoskeletal protein
assembly,3–7 control of protein phosphorylation,8–13 and modu-
lation of the activities of enzymes such as aldolase14,15 and
phosphoglucomutase.16 To date over 90 potential protein targets
have been identified for the S100 proteins,1 which include the
annexins (A1, A2, A5, A6, A11),17–21 the ubiquitination protein
CacyBP,22,23 F-actin,24 p53,25–27 and tau.28 Most of these
interactions are controlled through calcium binding and a subse-
quent conformational change of the S100 protein, revealing a
hydrophobic surface that recognizes the target protein sequence.
In general, this hydrophobic binding surface can also be probed
through binding of the calcium-bound S100 protein to phenyl
sepharose or anilinonaphthalene-8-sulfonic acid (ANS). These
probes have proven to be a connecting trademark for the S100
proteins, placing them within the calcium ‘‘sensor’’ group of EF-
hand proteins that also includes the muscle protein troponin-C
and the multifunctional protein calmodulin.29,30 Further interest
in the S100 members has evolved because some of these proteins,
such as S100B and S100A8/S100A9 show elevated expression lev-
els in Alzheimer’s disease31,32 and rheumatoid arthritis,33,34
respectively, suggesting they may have roles in these diseases.
S100B is one of the best-characterized members of the S100
protein family. As with all other S100 proteins, except S100G
(calbindin D9k), S100B can form homodimers, comprising two
noncovalently associated 91-residue subunits. Further, yeast two-
hybrid and optical biosensor experiments have indicated that
Abbreviations: nOe, nuclear overhauser effect; RDC, residual dipolar couplings; rmsd, root
mean square deviation.
Grant sponsor: Canadian Institutes of Health Research (GSS).
*Correspondence to: Gary S. Shaw, Department of Biochemistry, The University of Western
Ontario, London, Ontario, N6A 5C1, Canada. E-mail: gshaw1@uwo.ca.
Received 30 November 2007; Revised 8 February 2008; Accepted 19 February 2008
Published online 2 April 2008 in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/prot.22037
ABSTRACT
S100B is one of the best-characterized members of the
calcium-signaling S100 protein family. Most S100
proteins are dimeric, with each monomer containing
two EF-hand calcium-binding sites (EF1, EF2). S100B
and other S100 proteins respond to calcium increases
in the cell by coordinating calcium and undergoing a
conformational change that allows them to interact
with a variety of cellular targets. Although several
three dimensional structures of S100 proteins are
available in the calcium-free (apo-) state it has been
observed that these structures appear to adopt a wide
range of conformations in the EF2 site with respect to
the positioning of helix III, the helix that undergoes
the most dramatic calcium-induced conformational
change. In this work, we have determined the struc-
ture of human apo-S100B at 108C to examine
whether temperature might be responsible for these
structural differences. Further, we have used this
data, and other available apo-S100 structures, to
show that despite the range of interhelical angles
adopted in the apo-S100 structures, normal Gaussian
distributions about the mean angles found in the
structure of human apo-S100B are observed. This
finding, only obvious from the analysis of all avail-
able apo-S100 proteins, provides direct structural evi-
dence that helix III is a loosely packed helix. This is
likely a necessary functional property of the S100 pro-
teins that facilitates the calcium-induced conforma-
tional change of helix III. In contrast, the calcium-
bound structures of the S100 proteins show signifi-
cantly smaller variability in the interhelical angles.
This shows that calcium binding to the S100 proteins
causes not only a conformational change but results
in a tighter distribution of helices within the EF2 cal-
cium binding site required for target protein interac-
tions.
Proteins 2008; 73:28–42.
VVC 2008 Wiley-Liss, Inc.
Key words: calcium-binding proteins; structure; nmr.
28 PROTEINS VVC 2008 WILEY-LISS, INC.
2. S100B can form heterodimers in the cell with S100A1,
S100A6, and S100A11.35,36 Each S100B monomer com-
prises a globular domain containing two EF-hand cal-
cium-binding motifs. Sequence comparison of the S100
proteins reveals the presence of a basic N-terminal
pseudo (C) calcium-binding site, comprising 14 residues
(site I) between helices I and II (EF1), and an acidic 12-
residue canonical calcium-binding site in the C-terminus
(site II) flanked by helices III and IV (EF2). The region
joining the two calcium-binding sites (linker), and the
extreme N- and C-terminal regions of the S100 family
members are more divergent in their sequences.37 Three-
dimensional structures of S100A1038 and S100A1139
show that residues within these latter three regions are
important for the interactions with the annexin proteins
that are thought to be important in membrane vesicula-
tion processes.
Three-dimensional structures of several S100 proteins
in the calcium-free (apo) and calcium-bound states,
including S100B,40–43 S100A644–46 and S100A11,39,47
shows that they undergo a significant conformational
change on binding calcium, exposing several hydrophobic
residues that are required for interacting with specific bi-
ological target molecules. For example, the conforma-
tional change in S100A11 indicates a 408 reorientation of
helix III occurs on calcium binding leading to a more
open or exposed conformation. In other S100 proteins,
such as S100B and S100A6, the calcium-induced confor-
mational change ranges between $608 and $908. These
observations might indicate that the calcium-induced
structural change is different for each S100 protein. Alter-
natively, the structures could reflect a wide range of con-
formational space that can be sampled by helix III in ei-
ther the apo- or calcium-bound structures. For example,
the unstable nature of helix III in apo-S100B is consistent
with amide protons in this helix that exchange up to
three orders of magnitude faster than those in helices I,
II, or IV.48 In principle, a distinction between different
orientations for helix III (and therefore different confor-
mational changes) or multiple orientations that sample a
broad range of conformations should be possible through
a detailed analysis of existing or new S100 structures. To
date, however, this has not been done.
In this work, we have determined the water-refined,
three-dimensional structure of the calcium-free human
S100B dimer at low temperature (108C) to define
whether major temperature-induced structural changes
occur compared to other S100 structures determined at
higher temperatures. We have also used chemical shift in-
formation and residual dipolar coupling experiments to
determine if more subtle temperature-induced structural
changes occur in apo-S100B, and in particular helix III.
Using this data, and the structure of human apo-S100B,
we have assessed the interhelical interactions and super-
positions for the 11 calcium-free S100 protein structures
currently available. We find that the apo-S100 proteins
exhibit normal Gaussian distributions expected for a sin-
gle conformational distribution. These results provide
evidence that the structures of apo-S100 proteins reflect
their ability to survey a wide range of conformational
space rather than adopting a specific conformation. The
structure of human apo-S100B is close to the prototypi-
cal structure in this population.
MATERIAL AND METHODS
Sample preparation
Uniformly 15
N- and 13
C-labeled recombinant human
S100B was expressed in Escherichia coli strain N99, and
purified to homogeneity as previously described.49 All
NMR experiments utilized 1–2 mM human S100B sam-
ples dissolved in 90% H2O/10% D2O containing 50 mM
KCl at pH 7.19. DSS was used as an internal standard. A
single 15
N/13
C-labeled sample was used for all triple reso-
nance NMR experiments.
NMR spectroscopy
All NMR spectroscopy utilized Varian 600 and 800
MHz spectrometers with pulse field gradient triple reso-
nance probes. Data for the backbone sequential assign-
ments was collected at 108C and included HNCA,50
HNCACB,51 CBCA(CO)NH,52 HNCO, and 1
H-15
N
HSQC experiments. Side chain assignments were made
from C(CO)NH, HC(CO)NH,53 HCCH-COSY54 (90%
D2O/10% H2O), HBCBCGCDHD,55 and 1
H-13
C HSQC
experiments. Interproton distances were measured from
15
N-NOESY (mixing time 150 ms), 13
C-NOESY (mixing
time 100 ms, in 90% D2O/10% H2O) and F1-filtered, F3-
edited NOESY56 experiments (mixing time 100 ms) were
collected at 800 MHz at the Canadian National High
Field NMR Centre (NANUC). Slow-exchanging amide
protons were identified via a series of 1
H-15
N HSQC
experiments taken at time intervals (5 min, 5 h, 10 h,
and 16 h) after dissolving the protein in D2O. 1
H-15
N re-
sidual dipolar couplings were measured from a series of
IPAP-HSQC experiments57–59 using a 0.7 mM sample
of 15
N-labeled apo-S100B in 12 mg/mL Pf1 phage with
50 mM KCl at pH 7.19. Spectra were acquired at 35, 25,
15, 10, and 58C. An identical sample lacking phage was
used to measure isotropic 1
H-15
N splittings at the same
temperatures. All NMR spectra were processed using
NMRPipe60 and analyzed using NMRView61 software.
Structure calculations
Three-dimensional structures of apo-S100B were calcu-
lated using the simulated annealing protocol in the pro-
gram CNS62 with the use of noncrystallographic symme-
try. A total of 2504 nOes, including 1328 intraresidue,
556 sequential, 370 short-range, 160 long-range, and 90
intermolecular distances were used. After the initial
Conformational Distribution for Apo-S100 Proteins
PROTEINS 29
3. structure calculation was completed to define the second-
ary structure of the protein, 124 hydrogen bond distance
restraints and 336 dihedral restraints were included in
the calculations. Interproton distances for all the proton
pairs, except intermolecular nOes, were calibrated using
maximum and minimum nOe intensities for known
DNHa distances.63 For all intermolecular nOes, an upper
distance of 5 A˚ was used. Dihedral angle restraints were
determined using the TALOS protocol64 based on chemi-
cal shift data from Ha, Ca, Cb, and CO resonances of
apo-S100B. Angular restraints were only selected when 9/
10 dihedral matches fell within an allowed region of the
Ramachandran plot. The resulting w and / angles were
restricted to two times the error from the TALOS output
for the structure calculation with a minimum error of
Æ208. Hydrogen bonds were identified from slowly
exchanging NH resonances at the 16 h time point from
the incubation of apo-S100B in D2O and where tempera-
ture coefficients > 24.5 ppb/K could be measured. For
each hydrogen bond distance restraints for NHÀÀO (1.8–
2.3 A˚ ) and NÀÀO (2.3–3.3 A˚ ) were used. The final water
refinement utilized the RECOORD method65 for CNS.
Structures were viewed and analyzed using MOLMOL.66
VADAR67 (University of Alberta) was used to calculate
the accessible surface area for all proteins described. All
interhelical angles were calculated using the Vector Ge-
ometry Mapping method.29,68 In cases where NMR
structures were used, calculations were done for multiple
structures and the average Æ standard deviation reported.
Pairwise comparisons and rmsd calculations for all apo-
S100 structures were done using the program Chimera.69
The atomic coordinates for the 20 lowest energy struc-
tures of human apo-S100B have been deposited in the
RCSB under accession number 2PRU.
RESULTS AND DISCUSSION
The backbone and side chain resonance assignments
for apo-S100B at 108C and 358C were determined by the
acquisition and analysis of standard two- and three-
dimensional NMR experiments. In total, assignments of
1
H, 13
C, and 15
N resonances for 90 of the 91 residues in
apo-S100B were completed. The aromatic side-chain res-
onances for all seven phenylalanine, one tyrosine, and
four histidine residues in each apo-S100B subunit were
accomplished and were critically important for the struc-
ture determination of the protein since a large number
of intra- and intermolecular nOe correlations were
observed to these residues.
Temperature dependence of apo-S100B
The majority of three-dimensional structures for apo-
S100 proteins have been completed using NMR spectros-
copy at temperatures above 258C. For example, data used
for the structures of apo-S100A6,37,44 S100B40 and
S100P70 was acquired at 25–388C. In contrast, the only
two X-ray structures of calcium-free S100 proteins, apo-
S100A371 and apo-S100A646 had data collected at
21738C, although crystals were grown near ambient
temperatures. To determine the influence of temperature
on the apo-S100B structure a series of 1
H-15
N HSQC
spectra were collected at temperatures ranging between
10–358C. As shown in Figure 1 the majority of the amide
correlations shift downfield in both 1
H and 15
N dimen-
sions as the temperature is lowered. In general, most of
the resonances exhibited some line broadening at the
lower temperatures, which is consistent with a longer
correlation time due to an increased viscosity/tempera-
ture ratio. Further, analysis of the temperature-dependent
changes in the cross peak position showed that each res-
onance shifted in a linear fashion. The largest change in
amide proton chemical shift was $0.2 ppm as the tem-
perature was lowered. Similar temperature dependent
changes in 1
H chemical shift have been noted for the EF-
hand protein troponin-C72 where a large structural
change has not been noted. In addition, the changes in
chemical shift with decreased temperature for apo-S100B
are much less than the 1
H chemical shift changes on cal-
cium-binding or interaction with other proteins73,74
where a large structural reorganization of the protein
occurs. The temperature coefficients for each amide reso-
nance (2DdHN/T) are shown in Figure 2. The data shows
Figure 1
1
H-15
N HSQC spectra for human apo-S100B shown as a function of
temperature. The spectra are superimposed and plotted using filled contours for
108C and a single contour levels at 25 and 358C. Assignments for most of the
isolated correlations are indicated near the 108C spectrum. Arrows are used to
show the direction of movement for some of the most affected resonances over
the 358C to 108C temperature range.
S. Malik et al.
30 PROTEINS
4. the average temperature coefficient is 23.2 ppb/K and
most of the residues in apo-S100B are found within one
standard deviation (Æ2.3 ppb/K) of this value. Since
there are no regions of the sequence that exhibit anoma-
lous coefficients this further indicates that large tempera-
ture-induced structural changes in apo-S100B do not
occur as the temperature is lowered. It has been shown
for bovine pancreatic trypsin inhibitor and lysozyme that
temperature coefficients more positive than 24.5 ppb/K
indicate an amide proton that is hydrogen bonded.75
This appears to hold true for many residues within the
helical regions of apo-S100B. However, several amide
protons lie in the lower portion of [Fig 2(A)], below the
24.5 ppb/K threshold, indicating poorer hydrogen bond-
ing. Many of these are located in unstructured regions,
including those that lie either at the extreme C-terminus
of the protein (E91) or within calcium-binding sites I
(G19, G22, D23, K24, S30) and II (D61, G64, E67, F70).
It is interesting to note that two amide resonances that
shift greater than one standard deviation from the mean
lie at the observed S100 dimerization interface involving
helix I (L3, L10). While a loss of intramolecular hydrogen
bonding could cause this effect, this result may arise
from a minor temperature dependent alteration of the
helix I-helix I0
interface. An additional significant change
was noted at the N-terminus of helix III (I47, Q50), a
region that contains some of the fastest exchanging am-
ide protons in apo-S100B.48
To assess whether any re-orientation of helices in apo-
S100B occurred between 358C and 108C, we measured
HN residual dipolar couplings using two-dimensional
IPAP 1
H-15
N HSQC experiments at both temperatures
[Fig. 2(B)]. Residual dipolar couplings provide orienta-
tional information about the NÀÀH bond vector in a
partially oriented sample in a magnetic field. The figure
shows the expected clustering of negative couplings for
helices I and IV since these helices are nearly coplanar as
shown in previously determined S100 structures.40 Over-
all, at the two temperatures studied most residual dipolar
couplings changed by less than 1 Hz, which is close to
the measurement error for the experiments. This is in
contrast to much larger changes observed for the ribonu-
clease S-peptide, where a significant temperature-induced
structural alteration occurs. In apo-S100B, the residual
dipolar coupling experiments suggest that little reorienta-
tion of any of the helices occurred over the 10–358C
temperature range. Alternatively, it is possible that these
experiments report only the average position of the heli-
ces in apo-S100B and would not be sensitive to helix
movement or helices that occupy a range of conforma-
tions. To determine this latter case, a more detailed ex-
amination of the apo-S100B structure at lower tempera-
ture and its comparison with other apo-S100 structures
was completed.
Description of the structure of
human apo-S100B
The three-dimensional structure of human apo-S100B
was determined at 108C using a total of 2504 nOes, 336
dihedral restraints, and 124 hydrogen bonds. Hydrogen
bonds and dihedral restraints were only used in a-helical
regions clearly defined from nOe patterns. The amide
protons involved in hydrogen bonding were identified
from their slow exchange with D2O and measured tem-
perature coefficients > 24.5 ppb/K. The 20 lowest energy
structures obtained from CNS calculations were used for
further refinement using a full nonbonded potential to-
gether with explicit solvent molecules. While use of this
approach made little difference to the overall structure,
Figure 2
A: 1
H temperature coefficients for the backbone amide (HN) resonances of
human apo-S100B. The coefficients were determined from the slope of a plot of
HN chemical shift vs. temperature between 308 K and 283 K and plotted. The
average 1
H temperature coefficient was 23.2 ppb/K (dashed line). Temperature
coefficients larger than 24.5 ppb/K (solid line) are consistent with hydrogen
bonding. Residues that exhibited temperature coefficients more negative than
24.5 ppb/K, are not expected to be hydrogen bonded and are labeled for clarity.
B: HN residual dipolar coupling constants for human apo-S100B measured at
358C (*) and 108C (n). Only residues that exhibited clearly resolved HN
correlations were measured. Bars above each figure are shown to represent the
positions of the four a-helices, determined in the current work.
Conformational Distribution for Apo-S100 Proteins
PROTEINS 31
5. improvements were noted in the average nOe and angu-
lar restraint violations, and their corresponding energies.
In addition, the number of close contacts dropped by
nearly 70% in the water-refined approach, a similar result
to that noted for other systems.65 The ensemble of the
20 lowest-energy structures of human apo-S100B is
shown in Figure 3 and structural statistics are listed in
Table I. Structures were chosen based on their low ener-
gies and contained no distance violations greater than
0.5 A˚ and no angle violations greater than 58. The family
of structures shows two S100B monomers, each with
four well-defined helices (Fig. 3: I–IV, I0
–IV0
). The sym-
metric relationship between the monomers is evident
from a twofold rotational axis passing through the dimer
interface approximately perpendicular to helices I and I0
,
and parallel to helices IV and IV0
. The NMR spectra were
consistent with this symmetric nature as one resonance
was observed for most peaks. Some peaks, such as L3
exhibited multiple peaks (see Fig. 1) because of the pres-
ence of formyl- and desformyl methionine forms of the
protein that have been previously characterized.76 In all
cases, these peaks shifted nearly identically during tem-
perature studies (see Fig. 1) indicating the differential N-
terminal modification has little affect on dimerization.
The rmsd of all helices in the dimer relative to the mean
structure was 0.55 Æ 0.07 A˚ for the backbone and 1.03
Æ 0.09 A˚ for all heavy atoms.
Each S100B monomer is formed by two helix-loop-he-
lix EF-hand calcium-binding motifs joined by a linker
region [Fig. 3(A)]. The N-terminal EF-hand consists of
helix I (E2-Y17), calcium binding loop I (S18-K28), and
helix II (K29-E39). The C-terminal EF-hand comprises
helix III (Q50-D60), calcium-binding loop II (D61-D69),
and helix IV (F70-T81). Each of the four helices is well
defined in all 20 low-energy structures, with the rmsd
values 0.33 Æ 0.08 A˚ for helix I, 0.23 Æ 0.07 A˚ for helix
Figure 3
Three-dimensional structure of human apo-S100B determined at 108C. A: Ribbon diagram of apo-S100B showing helices I and I0
(blue), II and II0
(yellow), III and III0
(pink), and IV and IV0
(red). B: Superposition (N, Ca, C0
) of the family of 20 low-energy structures of apo-S100B obtained after water-refinement shown in the same
orientation as (A).
Table I
Structural Statistics of Apo-S100B
Restraints for RECOORD refined structure calculation
Total NOEs 2504
Intraresidual NOEs 1328
Sequential NOEs 556
Long-range NOEs 530
Intermolecular 90
Dihedral angles /; w (TALOS) 336
Hydrogen bonds 124
Energies (kcal/mol)
ETotal 28066.5 Æ 224.8
ENOE 0.81 Æ 0.10
Echid 0.83 Æ 0.50
EL-J 2739.6 Æ 35.2
Structure quality
r.m.s.d. from experimental restraints
Angular (8) 0.1798 Æ 0.0357
Distance (Š) 0.0173 Æ 0.0015
r.m.s.d. from idealized geometry
Bond (Š) 0.0051 Æ 0.0004
Bond angles (8) 1.3380 Æ 0.0637
Improper torsion (8) 1.7632 Æ 0.1050
Ramachandran plot statistics
Residues in favored regionsa
94.9%
r.m.s.d. to mean structure (Š)b
Backbone atoms 0.55 Æ 0.07
Heavy atoms 1.03 Æ 0.09
Bad contacts (avg. per structure) 2.4
a
Reflects residues in both most favored and additionally favored regions.
b
Precision is calculated for residues found in helices I (3–16), II (29–39), III
(50–60), and IV (70–80) in apo-S100B.
S. Malik et al.
32 PROTEINS
6. II, 0.14 Æ 0.05 A˚ for helix III, and 0.17 Æ 0.03 A˚ for he-
lix IV. For the calcium-binding loop regions, long-range
nOes were found for residues L27 with C68 and D69, K28
with E67 and C68, and K29 with C68 indicating the two
calcium-binding loops were in close proximity. Evidence
from nOe data also showed that extensive hydrophobic
interactions occurred between the four helices. These
interactions include contacts between V13 and F14 (helix
I) with L35 (helix II), S30, L32, and I36 (helix II) with
V56, M57, and L60 (helix III), V52, V56 (helix III) with
F76, M79 and V80 (helix IV), L10 (helix I) with residue
F73 (helix IV), and L32, L35, I36 (helix II) with F73 and
V80 (helix IV). Analysis of the family of structures indi-
cated that 94.9% of all the residues were in the allowed
regions of the Ramachandran plot.
The dimerization of apo-S100B occurs through an anti-
parallel alignment of helices I-I0
and IV-IV0
, and the per-
pendicular association of these pairs of helices to form an
X-type bundle. Residues that define the dimer interface in
human apo-S100B were identified from a 13
C F1-filtered,
F3-edited NOESY spectrum using 13
C, 15
N-labelled and
unlabelled proteins in a 50:50 ratio in solution. Unambig-
uous intermolecular nOes from this data showed interac-
tions between A6 (helix I) and A60
, A90
and L100
(helix I0
),
and L3 (helix I) with V130
(helix I0
). The perpendicular
association of helices I and IV0
was evident from unambig-
uous intermolecular nOes between L3 and M7 (helix I)
with V770
and T810
(helix IV0
), respectively. The orienta-
tion of helices IV and IV0
was determined by nOes between
M74 (helix IV) with M740
(helix IV0
) and, F70 (helix IV)
with T820
and F870
(helix IV0
). Further hydrophobic inter-
actions at the dimer interface were observed between the
N-terminus of helix I (L3), and the N-terminus of the
linker region in the partner monomer (L400
).
The three-dimensional structures of rat40 and bovine43
apo-S100B have been previously determined using NMR
spectroscopy. A comparison of these structures reveals a
backbone rmsd of 2.00 A˚ (residues 2–84) between the two
structures indicating there are some significant differences
between these two proteins. This arises mostly from dissimi-
larities in the orientation of helix III and calcium-binding
loop II. While it is possible sequence variation could con-
tribute to this difference, this seems unlikely given that rat
and bovine sequences share 95.6% identity (87 of 91 resi-
dues). Further, these proteins have identical helix III
sequences and a single minor change in calcium-binding
loop II (E62 in rat; S62 in bovine). The current structure of
human apo-S100B, which shares 97.8% and 96.7% identi-
ties with rat and bovine proteins, respectively, allows some
of the differences between the rat and bovine structures to
be resolved. Using the helices of these apo-S100 proteins for
comparison, there is a backbone rmsd of 1.76 A˚ between
human and rat apo-S100B that increases to 2.29 A˚ between
the human and bovine proteins. These inequities largely
result from differences in the positions of helices III and IV
where an rmsd of 1.24 A˚ exists between human and rat pro-
teins but increases to 2.32 A˚ between human and bovine
proteins. Only a single minor, conservative residue change
occurs between human and bovine proteins at position 80
(I80 in bovine; V80 in human) in helix IV indicating the
sequence difference probably does not account for the larger
variance in helix positions between these proteins. Thus,
when comparing the a-helices it is clear that human apo-
S100B is more similar to the rat S100B structure than the
bovine structure. The similarity between the orientation of
a-helices in human and rat apo-S100B proteins becomes
more apparent when interhelical angles are considered
(described later).
The human apo-S100B structure (see Fig. 3) indicates
that calcium-binding loops I (G19-E32) and II (D61-
E72) are less well defined (backbone rmsd 2.27 and
1.18 A˚ , respectively) than the helices. A similar observa-
tion has been made for bovine apo-S100B43 although
calcium-binding loop I is the better defined of the two
loops in that protein. However, in rat apo-S100B,40 the
backbone rmsds for calcium-binding loops I (0.22 A˚ )
and II (0.15 A˚ ) are similar to those found in the a-heli-
ces suggesting the loops adopt a tight structure with lim-
ited flexibility. In human apo-S100B, a network of nOes
similar to those observed for helices I-IV was not evident
in calcium-binding loops I and II, contributing to its
poorer definition. Further, amide exchange experiments
show that most of the amide protons within calcium-
binding loops I and II have protection factors up to six
orders of magnitude lower than amide protons in the a-
helices.48 This indicates that a poorer series of hydrogen
bonds exists in the calcium-binding loops of human apo-
S100B and that most of the amide protons are exposed
to solvent. In addition, 15
N relaxation experiments77
have shown that the order parameters (S2
) for residues
in calcium-binding loops I and II average 0.80, perhaps
indicating a greater degree of flexibility within the loops
than the helices (S2
5 0.84–0.87). It is interesting that
15
N relaxation experiments with another EF-hand cal-
cium-binding protein troponin-C,78,79 have indicated
calcium-binding sites I and II in the N-terminal domain
have lower order parameters also, characteristic of greater
flexibility. Thus, the poor definition of the calcium-bind-
ing loops in human apo-S100B is most consistent with
flexibility of the backbone within these regions. This
interpretation is in agreement with crystallographic stud-
ies of apo-S100A371 and apo-S100A646 where thermal
factors about two-times those found in the helices have
been observed for site I in apo-S100A6 and site II in
apo-S100A3, corresponding to backbone atomic displace-
ments near 0.6 A˚ .
The apo-S100 proteins show variations in
interhelical angles
All S100 proteins, with the exception of S100A10,
undergo a calcium-induced conformational change that
Conformational Distribution for Apo-S100 Proteins
PROTEINS 33
7. allow for their interaction with a variety of target pro-
teins. In general, this change involves a reorientation of
helix III and repacking of helix II that results in the ex-
posure of a broad, hydrophobic surface. To assess the
details of this conformational change and to determine
whether specific S100 proteins adapt to calcium binding
in different manners, knowledge of the structures of
many S100 protein family members is required in the
apo- and calcium-bound states. We have used the struc-
ture of human apo-S100B to assess the detailed structure
of all S100 proteins in the calcium-free state to under-
stand the first part of the calcium-binding response.
The structure of human apo-S100B determined at
108C was compared with NMR and X-ray crystallo-
graphic structures of S100A1, S100A3, S100A4, S100A6,
S100A11, S100A13, bovine and rat S100B, and S100P
using a variety of criteria, including interhelical distances
and angles, helix rotation and buried surface area. The
helix–helix relationship in this broad range of S100 struc-
tures was examined using the vector geometry mapping
(VGM) method, which provides important information
about the tip angle (y) between helices, the helix projec-
tion angle (/) and the helix role (x), and gives the best
overall picture of the spatial arrangement of helices.29,68
In our assessment, first shown for apo-calmodulin, we
chose helices adjacent to the calcium-binding loops such
that the incoming helix ended at the hydrophobic residue
immediately preceding the calcium-binding loop. In apo-
S100B this corresponded to Y17 and L60 in helices I and
III. The exiting helix started two residues before the
bidentate glutamate-coordinating residue that terminates
the calcium-binding loop (E31 and E72 in helices II and
IV of human S100B). In all cases, a careful examination
was made of the helix selections by shifting the helix des-
ignation by one or two residues to insure the y and /
were not grossly affected. It was observed that some heli-
ces, especially helices I and III, were very sensitive to this
selection due to the presence of some helix twist or
bend.
The interhelical angles y and / for human apo-S100B
and 10 other apo-S100 structures are listed in Table II
and shown graphically in Figure 4. The y and / angles
between helices I and II for the pseudo EF-hand (EF1) in
human apo-S100B are 72 Æ 48 and 98 Æ 88, respectively.
These are in excellent agreement with the average angles
(63 Æ 108, 99 Æ 78) obtained when all other apo-S100
structures are considered, indicating that helices I and II
adopt near identical orientations amongst all available
apo-S100 protein structures. This is shown clearly in
[Fig. 4(A)], where there is a clustering of the exiting he-
lix II for each structure. In addition, the pairwise super-
positions for helices I and II in these structures (55 com-
binations) was binned and fit with a Gaussian distribu-
tion having a midpoint of 1.46 Æ 0.56 A˚ [Fig. 5(A)].
This data passed multiple tests for a normal distribution.
The excellent agreement of the midpoint for the Gaussian
fit, the median (1.46) and positive normal distribution
tests was strong evidence for a single structural popula-
tion. This would indicate that the distribution of angles
for helices I and II in the apo-S100 structures is most
consistent with a single conformational family. Further,
the range of angles for EF1 in the apo-S100 proteins (Ta-
ble II) is considerably tighter than that observed for heli-
ces I and II in all EF-hand protein structures (1158).80
A comparison of helices I and II in EF1 from the S100
family to apo-calmodulin shows that calmodulin has a
significantly smaller tip angle (y) of 46 Æ 28, and larger
horizontal angle (/) of 123 Æ 38, both of which lie out-
side the ranges observed for any single S100 protein. As
shown in Figure 4, the position of the C-terminus of he-
lix II is closer to the N-terminus of helix I for apo-cal-
modulin than for any of the S100 proteins, resulting in y
and / angles for apo-calmodulin that differ by about
2178 and 248, respectively, when compared with the av-
erage apo-S100 structure. Human apo-S100B and the rest
of the S100 proteins thus have a more open conforma-
tion for EF1 than apo-calmodulin, a result reaffirmed
when considering interhelical distances between helices I
and II of EF1. The separation of these helices in the S100
proteins is approximately 3.6 A˚ greater than those in
apo-calmodulin.
The tip and horizontal angles for EF2 of human apo-
S100B are y 5 25 Æ 28 and / 5 2146 Æ 88, respectively
(Table II). These are well within the range observed for
all apo-S100 proteins (y 5 25 Æ 88, / 5 2142 Æ 468).
In general the exiting helix IV in the apo-S100B proteins
is angled in an opposite direction compared with apo-
calmodulin (see Fig. 4), which has a positive / angle
most similar to S100P. Unlike EF1, the larger deviations
in the y and / angles for EF2 indicate that potential dif-
ferences might exist between the structures due to
sequence differences, helix–helix interactions or both.
Alternatively, the differences in the structures could
reflect a lack of definition in the NMR structures due to
limited numbers of nOes between helices III and IV. This
does not appear to be the case since most of the apo-
S100 structures have tight ranges of y and / angles
( 208) indicating the nOe distance information used in
these structures was sufficient. In addition, variation is
noted for the two x-ray structures that are available
(S100A3 and S100A6) that have / angles that differ by
>208, although this difference is clearly smaller than that
exhibited by the apo-S100 structures determined by
NMR spectroscopy.
An examination of the y and / angles for the apo-
S100 structures shows that, with the exception of S100P,
all the apo-S100 structures are found within 88 of the av-
erage y angle with approximately equal numbers on ei-
ther side of the average. This indicates that the opening
angles between helices III and IV are similar as noted by
the mostly parallel arrangement of the helices for the
apo-S100 proteins [Fig. 4(D)]. On the other hand, the
S. Malik et al.
34 PROTEINS
9. differences in the positions of helix IV with respect to
helix III mostly arise from the large variation in / angle.
For example, some structures (S100A13, S100A1) have /
angles about 608 more positive and others (S100A11,
S100P) have / angles about 608 more negative than
found in human apo-S100B. The VGM plot in Figure 4
fixes helix III along the z-axis and gives the impression
that the N-termini of helix IV, closest to the calcium-
binding loop of EF2 takes on a range of positions with
respect to helix III. Superposition of the apo-S100 pro-
teins, however, shows that it is the C-terminus of helix
III that occupies a variety of locations.
Despite the similarity in y angles observed for the pro-
teins it is tempting to suggest that the range of / angles
observed for the EF2 calcium-binding site might arise
from different structural subfamilies in the apo-S100 pro-
teins. In turn, the different interhelical relationships
between helices III and IV might be important for the
extent that these helices change conformation upon cal-
cium binding. Several factors indicate this is not the case.
For example, statistical analysis of the pairwise superposi-
tions of helices III and IV in EF2 for human apo-S100B
and all other apo-S100 proteins shows a Gaussian distri-
bution [Fig. 5(B)] that is satisfied at the 95% confidence
level and passes several normality tests (with the excep-
tion of S100P). Further, the median of this superposition
(1.85 A˚ ) falls near the midpoint for a Gaussian distribu-
tion of the helices (1.88 Æ 0.73 A˚ ). This result is similar
to that observed for helices I, II and IV (1.72 Æ 0.48 A˚ )
or all four helices (1.84 Æ 0.63 A˚ ) shown in Figure 5.
Using the pairwise rmsd comparisons (see Fig. 5) as a
guide in combination with the VGM analysis (see Fig. 4),
it is apparent the large structural differences arise due to
the displacement of the N-termini of helix IV with
respect to the C-termini of helix III. For example, human
apo-S100B and S100A1 have a relatively small rmsd
Figure 4
Helix orientation for human apo-S100B and other S100 proteins obtained using Vector Geometry Mapping.29,68 The figure shows helices I and II (A, B) from calcium-
binding site EF1 and helices III and IV (C, D) from calcium-binding site EF2. In both cases the incoming helix of each structure is aligned along the z-axis with respect
to the N-terminal helix of EF1 in calmodulin. The exiting helices (II in EF1 and IV in EF2) are shown for the calcium-free proteins calmodulin (grey), human S100B
(red), bovine S100B (brown), rat S100B (maroon), S100A1 (cyan), S100A3 (green), S100A4 (magenta), human S100A6 (blue), rabbit S100A6 (light blue), S100A11
(dark purple), S100A13 (orange), and S100P (yellow). The plots show the helices from calcium-binding site EF1 as viewed down the 1z axis (A, C) and rotated $908
about y and $458 about z (B, D). The directions of the helices are denoted by N?C either along the z-axis or the exiting helix (B, D).
S. Malik et al.
36 PROTEINS
10. when helices III and IV are superimposed (1.30 A˚ ) yet
the N-termini of helix IV are separated by about 6 A˚ .
This is evident in [Fig. 4(C,D)] where the C-termini of
helix IV from human apo-S100B (red) and apo-S100A1
(cyan) are close together, but their N-termini are more
divergent.
The comparison of the interhelical y and / angles of
the apo-S100B structures shows that human apo-S100B
lies very close to the midpoint of tip and horizontal
angles for both EF1 and EF2 calcium-binding sites. Anal-
ysis of the angular ranges and pairwise backbone rmsd
indicates that all apo-S100 structures to date, with the
exception of S100P (y angle for EF2), fall within a single
Gaussian distribution although the ranges of the horizon-
tal angle (/) for EF2 is nearly sevenfold larger for EF2
than for EF1.
Helix III shows the broadest range
of accessible surface areas in the
S100 proteins
The VGM results indicate there is a significant range
of conformations that helices III and IV adopt with
respect to each other within the S100 family. In order to
pinpoint a rationale for this, the accessible surface area
for human apo-S100B, presented here, and other cal-
cium-free S100 proteins was analyzed. The fraction acces-
sible surface area for human apo-S100B reveals a large
number of residues in helix I (L3, A6, M7, A9, L10, I11,
V13, and F14) and helix IV (F70, F73, M74, F76, V77,
A78, V80, and T81) that have >80% of their side chains
buried (see Fig. 6). Most of these residues form the
hydrophobic dimer interface in apo-S100B derived from
the near perpendicular arrangement of helices I and I0
Figure 5
Comparison of apo-S100 structures showing the distribution of structures as a function of backbone rmsd between helices. The figure shows (A) helices I and II, (B)
helices III and IV, (C) helices I, II, and IV, and (D) sum of all helices for human apo-S100B and the other ten apo-S100 structures described in Table II. The helices used
were L3-Y17 (helix I), K29-E39 (helix II), Q50-L60 (helix III), and F70-V80 (helix IV) in human apo-S100B and the corresponding regions in other S100 proteins based
on alignment using T-Coffee.81 For NMR structures, the most representative structure as listed in the PDB file was used. In each case the rmsd values between all
possible pairs of structures (55 comparisons) were tabulated and binned (0.2 A˚ bins). The number of occurrences was plotted against the bin center. Each graph shows the
best-fit Gaussian curve to the binned rmsd data centered at (A) 1.46 Æ 0.56 A˚, (B) 1.88 Æ 0.73 A˚, (C) 1.72 Æ 0.48 A˚, and (D) 1.84 Æ 0.63 A˚. The structures for apo-
S100A13 and apo-S100P were removed from datasets (A) and (B–D), respectively based on ANOVA statistics.
Conformational Distribution for Apo-S100 Proteins
PROTEINS 37
11. with IV and IV’. For example, L3 in helix I interacts with
L10 and V13 in helix I0
. In helix IV, residue F73 interacts
with L3 of helix I, and L10 and F14 of helix I0
. Compari-
son with other apo-S100 structures reveals this pattern of
buried hydrophobic residues is nearly perfectly preserved
[Fig. 6(A,D)]. An examination of the S100 sequences
along with this analysis indicates the dimerization motif
for these S100 proteins is LXX[A/C/S][M/L/I/V]XX[M/L/
I/V][I/V]X[V/I/T]F (residues 3-14; helix I) and FXE[F/
Y][V/I/L/M]X[L/F][V/L/I][A/G/S]X[V/L/I][T/A] (residues
70–81; helix IV). The excellent agreement between the
buried surface area and sequence conservation of the apo-
S100 structures is in accord with 15
N relaxation77 and am-
ide proton exchange experiments48 for apo-S100B. In par-
Figure 6
Comparison of side chain fractional accessible surface area (FASA) for residues in human apo-S100B and other apo-S100 structures. Residues in (A) helix I, (B) helix II,
(C) helix III and (IV) helix IV are plotted as a function of fraction accessible surface area by each side chain. The numbering scheme used is that of human apo-S100B
and other proteins were aligned based on sequence using T-Coffee.86 The proteins shown are human S100B (n), bovine S100B (~), rat S100B (!), S100A1 (^),
S100A3 (l), S100A4 (h), human S100A6 (!), rabbit S100A6 (~), S100A11 (^), S100A13 (3) and S100P (*). All fractional accessible surface areas were calculated
from available PDB coordinates using the program VADAR.67 For NMR structures, the most representative structure as listed in the PDB file was used.
S. Malik et al.
38 PROTEINS
12. ticular, helices I and IV had protection factors that were
similar to free energies for the unfolding of apo-S100B
indicating the dimer interface, maintained by the helix I
and IV motifs is a major contributor towards the stability
of all S100 proteins.
Extending this analysis to helix II shows the hydropho-
bic portions of residues K29, L32, L35, and I36 are all
buried >80% in human apo-S100B. As with helices I
and IV these positions in helix II are highly conserved in
the S100 sequences. The large variation in accessible sur-
face area at the N-terminus of helix II is also accompa-
nied by decreased protection from amide exchange.48 In
helix III, the shortest of the helices in apo-S100B, a large
variation in the accessible surface area exists between
structures. Only two residues (V56, L60) show a consist-
ent pattern of burial between human apo-S100B and the
other S100 proteins and these are both accompanied by
high conservation at these positions. Other residues are
less well conserved and show poorer patterns of accessi-
ble surface area. For example, V52 in the apo-S100B
structures is nearly completely buried while residues in
S100A6 (E52) and S100A3 (D54) are more exposed con-
sistent with their significant differences in side chain po-
larity. Notably, position 57 in helix III, where methionine
is highly conserved, shows an inconsistent pattern. In
human apo-S100B it shows about 30% accessible surface
area, midway between the observed range for this residue
(2%–62%), despite the conserved nature of the methio-
nine residue in all sequences but S100A13 and S100P.
Further differences are seen at the second position in the
calcium-binding loop occupied by N62 in human apo-
S100B, where it is exposed but nearly completely buried
in S100A11 where a leucine residue occupies this posi-
tion. The large variations in buried surface area through-
out helix III and especially for conserved residues (V53,
M57) are most consistent with this helix being loosely
packed. This conclusion is borne out by amide hydrogen
exchange rates for residues in helix III that are 2–3 orders
of magnitude faster than helices I, II, and IV.48 In addi-
tion, although amide exchange rates have not been meas-
ured for rat or bovine apo-S100B, 15
N relaxation experi-
ments indicate helix III in rat apo-S100B has the lowest
order parameters of the four helices.77 A similar obser-
vation has been made in the N-terminal domain of tro-
ponin-C78,79 where helix C, one of the helices that
undergoes a conformational change upon calcium bind-
ing, has the lowest order parameters of the helices in that
domain.
Function relevance for the S100 proteins
An analysis of the tip and horizontal angles for the
EF1 and EF2 calcium-binding sites in human apo-S100B
and several other apo-S100 proteins reveals a range of
orientations are adopted by EF2 with a mean near that
determined for human apo-S100B. It was also shown
that helix III is clearly the most loosely packed of the
four helices in all structures of apo-S100 proteins. This
data indicates that helix III is likely a flexible helix able
to sample a range of orientations with respect to helix
IV. Is this important for the function of the S100 pro-
teins? As shown in Figure 7, the broad range of tip and
horizontal angles displayed in the apo-S100 structures is
not exhibited in the calcium-bound forms. As with the
apo-proteins the structure of the calcium-bound form of
human S100B has tip and horizontal angles for EF2 (818,
100.18) that are representative of the average angles of
67.5 Æ 9.5 and 100.3 Æ 8.88 for the calcium-bound
structures. For human S100B, this indicates that the pro-
tein undergoes changes of about 558 and 1148 in the tip
and horizontal angles respectively in response to calcium
binding. This opening of the horizontal angle is 2.5–3.0
times larger than observed for calmodulin82 or
troponin-C.83,84 This structural change is responsible
for the shallow binding surface noted in the calcium-
bound forms of the S100 proteins compared to a nar-
rower cleft found in calmodulin and troponin-C.85,86 It
is interesting that the calcium-bound S100 structures
compared (4 NMR, 6 X-ray structures) display a much
narrower range of horizontal angles (100.3 Æ 8.88) clearly
indicating a more homogeneous group of structures
compared to their apo forms (Table II). This would indi-
cate that calcium binding might act to restrict the freedom
of helix III, presumably through an ordering of the EF2 cal-
cium-binding site. For S100B, amide exchange experiments
Figure 7
Helix reorientation for the EF2 calcium-binding site in S100 proteins. The
relative distribution for each S100 protein is plotted versus the horizontal angle
(/) calculated using Vector Geometry Mapping.29,68 NMR structures are
represented as a Gaussian curve centered at the mean and having a width at
half-height corresponding to Æ the standard deviation. X-ray structures are
shown as vertical sticks. The structures shown are; human S100B (red), bovine
S100B (orange), rat S100B (maroon), S100A1 (green), S100A3 (ochre), S100A4
(blue), human S100A6 (sky blue), rabbit S100A6 (dark green), S100A11
(magenta), S100A12 (pink) S100A13 (rose), and S100P (black).
Conformational Distribution for Apo-S100 Proteins
PROTEINS 39
13. have shown that the amide rates of exchange in this cal-
cium-binding site are slowed by 1–2 orders of magni-
tude. However, amide exchange rates for helix III remain
the fastest of the helices in S100B indicating this helix is
still exposed.48 Still, Figure 7 provides evidence that cal-
cium binding to the S100 proteins enables EF2 to move
from a broad range of conformations to a tighter more
compressed distribution.
CONCLUSIONS
Based on amide chemical shift changes and residual
dipolar changes the three-dimensional structure of
human apo-S100B does not appear to undergo signifi-
cant structural changes between 35 and 108C. A compari-
son of the three dimensional structure of human apo-
S100B with those of rat and bovine apo-S100B indicates
the human form has similar helix orientations and preci-
sion as the rat form of the protein but has calcium-bind-
ing loops more similar to those obtained for the bovine
structure. Using human apo-S100B as a template the
interhelical angles for EF-hands EF1 and EF2 were com-
pared with other apo-S100 protein structures. The
arrangements of EF1 (y 5 72 Æ 4, / 5 98 Æ 8) and
EF2 (y 5 25 Æ 2, / 5 2146 Æ 8) were found to be
near the mean for all apo-S100 protein structures deter-
mined to date. Although a broad distribution of the EF2
helical angles exist for the apo-S100 proteins, we have
shown that these structures assume a normal Gaussian
distribution about this conformation indicative of a sin-
gle structural population. Calcium binding alters the con-
formation of the EF2 helices (III, IV) but more signifi-
cantly leads to a tighter less variable arrangement of the
helices.
ACKNOWLEDGMENTS
The authors would like to thanks Kathryn Barber
(UWO) for her technical support. We are grateful to
Lewis Kay (University of Toronto) for providing pulse
sequences, Frank Delaglio for NMRPipe and DYNAMO,
and Bruce Johnson for NMRView. We would like to
thank the Canadian National High Field NMR Centre
(NANUC) for their assistance and use of the facilities.
Operation of NANUC is funded by the Canadian Insti-
tutes of Health Research, the Natural Science and Engi-
neering Research Council of Canada and the University
of Alberta.
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42 PROTEINS
