proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Analysis of the structure of humanapo-S100B at low temperature indicates aunimodal conformational distribution isadopted by calcium-free S100 proteinsShahid Malik,1 Matthew Revington,1 Steven P. Smith,2 and Gary S. Shaw1*

1Department of Biochemistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada

2Department 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.

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. Theseobservations 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 15N- and 13C-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 15N/13C-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 1H-15N

HSQC experiments. Side chain assignments were made

from C(CO)NH, HC(CO)NH,53 HCCH-COSY54 (90%

D2O/10% H2O), HBCBCGCDHD,55 and 1H-13C HSQC

experiments. Interproton distances were measured from15N-NOESY (mixing time 150 ms), 13C-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 1H-15N HSQC

experiments taken at time intervals (5 min, 5 h, 10 h,

and 16 h) after dissolving the protein in D2O.1H-15N re-

sidual dipolar couplings were measured from a series of

IPAP-HSQC experiments57–59 using a 0.7 mM sample

of 15N-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 1H-15N 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

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

�208. 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 NH��O (1.8–

2.3 A) and N��O (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 � 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 of1H, 13C, and 15N 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 1H-15N 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 1H and 15N 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 1H 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 1H 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 11H-15N 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

the average temperature coefficient is 23.2 ppb/K and

most of the residues in apo-S100B are found within one

standard deviation (�2.3 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 1H-15N HSQC experiments at both temperatures

[Fig. 2(B)]. Residual dipolar couplings provide orienta-

tional information about the N��H 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 10–358Ctemperature 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 ofhuman 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-helicalregions 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 2A: 1H 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 1H 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

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 � 0.07 A for the backbone and 1.03

� 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 � 0.08 A for helix I, 0.23 � 0.07 A for helix

Figure 3Three-dimensional structure of human apo-S100B determined at 108C. A: Ribbon diagram of apo-S100B showing helices I and I 0 (blue), II and II 0 (yellow), III and III 0

(pink), and IV and IV 0 (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 IStructural Statistics of Apo-S100B

Restraints for RECOORD refined structure calculationTotal NOEs 2504Intraresidual NOEs 1328Sequential NOEs 556Long-range NOEs 530Intermolecular 90Dihedral angles /; w (TALOS) 336Hydrogen bonds 124

Energies (kcal/mol)ETotal 28066.5 � 224.8ENOE 0.81 � 0.10Echid 0.83 � 0.50EL-J 2739.6 � 35.2

Structure qualityr.m.s.d. from experimental restraintsAngular (8) 0.1798 � 0.0357Distance (�) 0.0173 � 0.0015r.m.s.d. from idealized geometryBond (�) 0.0051 � 0.0004Bond angles (8) 1.3380 � 0.0637Improper torsion (8) 1.7632 � 0.1050

Ramachandran plot statisticsResidues in favored regionsa 94.9%

r.m.s.d. to mean structure (�)b

Backbone atoms 0.55 � 0.07Heavy atoms 1.03 � 0.09

Bad contacts (avg. per structure) 2.4

aReflects residues in both most favored and additionally favored regions.bPrecision 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

II, 0.14 � 0.05 A for helix III, and 0.17 � 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 13C F1-filtered,

F3-edited NOESY spectrum using 13C, 15N-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, 15N 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 that15N 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 ininterhelical angles

All S100 proteins, with the exception of S100A10,

undergo a calcium-induced conformational change that

Conformational Distribution for Apo-S100 Proteins

PROTEINS 33

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 � 48 and 98 � 88, respectively.These are in excellent agreement with the average angles

(63 � 108, 99 � 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 � 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 � 28, and larger

horizontal angle (/) of 123 � 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 yand / 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 � 28 and / 5 2146 � 88, respectively(Table II). These are well within the range observed for

all apo-S100 proteins (y 5 25 � 88, / 5 2142 � 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

TableII

EF-H

andAngles

ofApo-S100Proteinsa

Protein

EF1b

EF2

N-terminal

coordina

teof

seco

ndhe

lixy

(deg

ree)

/(deg

ree)

x(deg

ree)

N-terminal

coordinate

ofseco

ndhe

lixy

(deg

ree)

/(deg

ree)

x(deg

ree)

hS100B

c11.8�

0.7,23.3�

1.7,21.1�

0.7

72�

498

�8

129�

88.4�

1.1,11.5�

0.7,21.8�

0.5

25�

22146�

883

�8

bS100B

d10.3�

1.7,1.6�

1.5,25.4�

2.1

53�

2105�

493

�4

1.4�

1.3,10.8�

1.4,28.2�

1.3

10�

42124�

1045

�3

rS100B

e12.3�

0.3,24.0�

1.2,25.2�

0.7

58�

193

�3

129�

47.8�

0.6,10.4�

0.5,24.6�

0.5

15�

12127�

595

�4

S100A1f

13.1�

1.3,23.3�

2.4,24.7�

1.3

68�

391

�5

166�

63.3�

1.9,14.3�

0.5,20.3�

0.3

33�

1284

�4

73�

8S100A3g

11.2,2

0.6,24.2

55102

129

11.7,10.9,

23.0

262116

107

S100A4h

12.4�

1.2,22.2�

1.4,23.2�

0.7

68�

2105�

3114�

48.1�

2.8,11.9�

2.1,23.7�

1.4

19�

22151�

770

�10

# S100A

6i10.1�

0.7,0.4�

0.3,24.8�

0.5

56�

1103�

3113�

310.1�

0.8,11.0�

0.3,22.0�

0.4

34�

22161�

395

�3

S100A6j

10.361,2

0.181,

24.809

56105

121

11.4,11.4,

23.5

242138

111

S100A11

k9.9�

0.4,21.3�

0.7,24.3�

0.3

63�

2101�

1123�

88.7�

2.9,6.9�

1.3,22.2�

1.2

30�

4163�

1073

�20

S100Pl

10.8�

0.7,21.0�

0.7,25.9�

1.1

54�

3105�

4104�

29.0�

0.2,1.49

�0.7-4.7�

0.5

70�

3121�

386

�4

S10013

m13.5�

1.5,28.3�

2.5,20.3�

1.2

84�

684

�9

180�

92.2�

1.4,14.3�

0.5,20.5�

1.0

30�

3279

�7

57�

6Av

erag

eS100

63�

1099

�7

128�

2529

�15

(25�

8)2142�

4681

�20

CaM

10.6�

0.6,20.7�

0.7,23.2�

0.3

46�

2123�

3104�

510.9�

0.9,5.6�

1.6,21.8�

1.0

48�

4139�

489

�4

aAngles

werecalculatedusingVectorGeometry

Mapping(V

GM)methodusingapo-calmodulin(PDBentry1DMO)as

thereference.29,67

bHelices

defined

forEF1arehelix

IandII

andforEF2as

helix

IIIandIV

for:

S100B-L10–Y17,K29–E39,V53–L60,F70–V80;S100A1-L11–H18,K30–E40V54–L61,F71–L81;S100A3-I12–Y19,Q31–E41Y55–L62,F72–L82;

S100A4-M12–Y19,K31–E41F55–L62,F72–I82;S100A6-L12–Y19,K31–L41,I53-L60(#E52–D59),

F70–L80;S100A11-L13–Y20,K32–E42,L56–L63,F73–L83;S100P-I11–Y18,K30–E40V54–L61,F71–I81;S100A13-V16-F23,

V35-Q

45,L56-L63,F73-L83.

PDBentriesare

c 2PRU

d1CFP

e 1B4C

f 1K2H

g1KSO

h1M31

i 2CNP

j 1K9P

k1NSH

l 1OZO

mYUS.

Conformational Distribution for Apo-S100 Proteins

PROTEINS 35

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 � 0.73 A). This result is similar

to that observed for helices I, II and IV (1.72 � 0.48 A)

or all four helices (1.84 � 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 4Helix 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 �908about 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

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 rangeof accessible surface areas in theS100 proteins

The VGM results indicate there is a significant rangeof conformations that helices III and IV adopt withrespect to each other within the S100 family. In order to

pinpoint a rationale for this, the accessible surface areafor 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 chainsburied (see Fig. 6). Most of these residues form the

hydrophobic dimer interface in apo-S100B derived fromthe near perpendicular arrangement of helices I and I0

Figure 5Comparison 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 � 0.56 A, (B) 1.88 � 0.73 A, (C) 1.72 � 0.48 A, and (D) 1.84 � 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

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 15N relaxation77 and am-

ide proton exchange experiments48 for apo-S100B. In par-

Figure 6Comparison 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

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, 15N 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 � 9.5 and 100.3 � 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 � 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 7Helix 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 � 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

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 � 4, / 5 98 � 8) and

EF2 (y 5 25 � 2, / 5 2146 � 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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