The structure of Ca2+-loaded S100A2 at 1.3-A resolutionMichael Koch1,* and Gunter Fritz2

1 Bayer Schering Pharma AG, Wuppertal, Germany

2 Department of Neuropathology, University of Freiburg, Neurozentrum, Germany

Keywords

calcium; EF-hand; p53; S100 protein; X-ray

Correspondence

G. Fritz, Department of Neuropathology,

University of Freiburg, Neurozentrum,

Breisacher Strasse 64, 79106 Freiburg,

Germany

Fax: +49 761 270 50500

Tel: +49 761 270 50780

E-mail: guenter.fritz@uniklinik-freiburg.de

*Present address

Bayer Pharma AG, Global Drug Discovery –

Lead Discovery Wuppertal, Aprather Weg

18a, Geb. 456, 42096 Wuppertal, Germany

(Received 13 December 2011, revised 24

February 2012, accepted 1 March 2012)

doi:10.1111/j.1742-4658.2012.08556.x

S100A2 is an EF-hand calcium ion (Ca2+)-binding protein that activates

the tumour suppressor p53. In order to understand the molecular mecha-

nisms underlying the Ca2+-induced activation of S100A2, the structure of

Ca2+-bound S100A2 was determined at 1.3 A resolution by X-ray crystal-

lography. The structure was compared with Ca2+-free S100A2 and with

other S100 proteins. Binding of Ca2+ to S100A2 induces small structural

changes in the N-terminal EF-hand, but a large conformational change in

the C-terminal EF-hand, reorienting helix III by approximately 90�. Thismovement is accompanied by the exposure of a hydrophobic cavity

between helix III and helix IV that represents the target protein interaction

site. This molecular reorganization is associated with the breaking and new

formation of intramolecular hydrophobic contacts. The target binding site

exhibits unique features; in particular, the hydrophobic cavity is larger than

in other Ca2+-loaded S100 proteins. The structural data underline that the

shape and size of the hydrophobic cavity are major determinants for target

specificity of S100 proteins and suggest that the binding mode for S100A2

is different from that of other p53-interacting S100 proteins.

Database

Structural data are available in the Protein Data Bank database under the accession number

4DUQ

Structured digital abstractl S100A2 and S100A2 bind by x-ray crystallography (View interaction)

Introduction

The family of S100 proteins constitutes the largest sub-

group within the EF-hand calcium ion (Ca2+)-binding

protein superfamily. Currently, more than 21 different

members of this family are known in humans [1,2].

The S100 proteins are expressed in a cell- and tissue-

specific manner, and are involved in different processes

such as cell-cycle regulation, cell growth, differentia-

tion and motility. Several S100 proteins occur in the

extracellular space where they act as pro-inflammatory

cytokines by activating the receptor for advanced gly-

cation end-products (RAGE) [3–5] and toll-like recep-

tor 4 (TLR-4) [6,7] or exert bacteriostatic function as

part of the innate immune system [8,9]. Accordingly,

dysregulation of S100 protein expression is very often

associated with a variety of severe diseases [10], among

them chronic inflammatory [11], neurodegenerative [12]

and cardiovascular [13,14] disorders, as well as cancer

[15–17]. The members of the S100 family are small

(10–12 kDa molecular mass), acidic proteins and, with

the exception of S100G (calbindinD9j), most S100 pro-

teins form homodimers and heterodimers under physi-

ological conditions. Several higher oligomers, ranging

from tetramers to octamers, have been reported for

S100B [4], S100A4 [18], S100A8 ⁄A9 [19] and S100A12

[20]. These larger assemblies appear to be important

for their extracellular cytokine-like function. Recent

Abbreviations

Ca2+, calcium ion; Na+, sodium ion; NDR kinase, nuclear Dbf2-related kinase; PEG, polyethylene glycol.

FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS 1799

findings suggest that such oligomers might represent

precursors for amyloids formed by S100 proteins [21].

Each S100 protomer is composed of two different

EF-hands: an S100-specific EF-hand is located at the

N terminus, followed by the classical EF-hand at the C

terminus [17,22]. The classical EF-hand motif comprises

a typical sequence signature of 12 residues and is present

in other Ca2+-binding EF-hand proteins such as parval-

bumin, calmodulin or troponin C [23,24]. The S100-spe-

cific motif comprises 14 residues and is unique to the

S100 proteins and to a few domains that resemble S100

proteins [17]. In both EF-hands Ca2+ is coordinated in

a pentagonal bipyramidal configuration. The six posi-

tions in the coordination sphere are denoted by X, Y, Z,

-X, -Y and -Z. Five coordinating residues are provided

by the protein and are arranged in the following motifs:

X**Y*Z**-Y****-Z in the N-terminal EF-hand and X*

Y*Z*-Y****-Z in the C-terminal EF-hand (the asterisks

represent intervening residues; see also Fig. S1). The

sixth -X position is occupied by a water molecule. An

invariant glutamate at position -Z provides a carboxy-

late group for bidentate coordination of Ca2+.

S100 proteins do not exert their biological function

alone, but interact with various target proteins to regu-

late cellular processes [25,26]. While some S100 pro-

tein–protein interactions occur in the absence of Ca2+

[27,28], a calcium-dependent conformational change is

typically required to bind effector molecules and elicit

a biological response [26].

S100A2 is expressed at high levels in different organs

such as lung, kidney, liver, heart and skeletal muscle

[29]. In the cell, S100A2 is localized almost exclusively

in the nucleus, a feature that is unique in the S100 pro-

tein family [30]. Lower levels of S100A2 are detected

in the cytoplasm where S100A2 is reported to interact

with tropomyosin and modulate the organization of

the actin cytoskeleton [31]. Its function in the nucleus

remained unknown for several years although it has

been recognized that S100A2 is involved in cell cycle

regulation [32]. S100A2 was identified first as a major

tumour suppressor in human mammary epithelial cells

[33]. Drastic down-regulation of S100A2 was also

observed in prostate adenocarcinoma [34], lung cancer

[35] and breast carcinoma [36]. A direct molecular link

between the tumour-suppressor activity and the

nuclear localization of S100A2 was established when it

was shown that S100A2 binds and activates p53 in a

Ca2+-dependent manner [37]. The results suggest a

positive regulation of p53 through S100A2. Intrigu-

ingly, other studies report an oncogenic involvement of

S100A2 in the tumorigenesis of different squamous cell

carcinomas [38,39]. This might be a result of interac-

tions of S100A2 with DNp63, which has oncogenic

and growth-stimulating activities in the development of

tumours [40]. Altogether, the studies show that

S100A2 is a potent regulator of the cell cycle, and dys-

regulation of S100A2 can lead to cancer.

Besides Ca2+, S100A2 binds zinc ion (Zn2+) with

high affinity (Kd = 25 nM) [41]. S100A2 contains two

different Zn2+-binding sites, involving cysteine residues

for coordination. We have shown that Zn2+ is a nega-

tive modulator of S100A2; binding of Zn2+ decreases

dramatically the Ca2+ affinity and thereby inhibits the

response to intracellular Ca2+ signals [41]. These find-

ings suggest that S100A2 is regulated by both Ca2+

and Zn2+ metal ions. Our objective was to understand

how the activity of S100A2 is controlled by the differ-

ent metal ions at an atomic level. High-resolution

X-ray structures provide a snapshot of the different

states and are an excellent starting point for further

site-specific mutation and computational studies. We

have already determined the X-ray structure of S100A2

in its Ca2+-free state, showing the protein in its ‘closed’

inactive state [42]. Here, we present the high-resolution

structure of the active, Ca2+-bound form of S100A2 at

1.3 A resolution, and describe the conformational

changes involved when progressing from the Ca2+-free

state to the Ca2+-bound active state.

S100A2 undergoes a large conformational change

upon Ca2+ binding. Major structural changes occur at

the C-terminal EF-hand where helix III moves out-

wards from the molecule, resulting in a reorientation

of 92� compared with Ca2+-free S100A2 [42]. This

drastic molecular reorganization is associated with the

breakage and formation of new intramolecular hydro-

phobic interactions. The movement of helix III opens

a hydrophobic cavity between helices III and IV,

which constitutes the binding site for target proteins

such as p53. This binding site exhibits unique features

compared with other Ca2+-bound S100 proteins.

Results and Discussion

Overall structure of Ca2+-bound S100A2

The final model of Ca2+-bound S100A2 consists of

two protein chains (subunit A and subunit B), each

containing residues A2-A89 and B2-B91, four Ca2+

ions, 173 water molecules and two poly(ethylene gly-

col) 400 (PEG 400) molecules. Using fully anisotropic

displacement parameters in refinement resulted in a

final Rcryst to 13.6% and Rfree to 16.8%. The stereo-

chemistry was validated by PROCHECK [43], showing

that 98% of the residues are located in the most

favoured regions of the Ramachandran plot and 2%

of the residues reside in additional allowed regions.

X-ray structure of Ca2+-loaded S100A2 M. Koch and G. Fritz

1800 FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS

Each subunit comprises 97 residues. However, the

first N-terminal methionine residue in each chain,

PheA90 to ProA97 and GlyB92 to ProB97 were not

resolved in the electron density. Similarly, the positions

of these C-terminal residues could not be resolved in

the Ca2+-free structure of S100A2 [42]. This might be

a result of structural heterogeneity caused by cis-trans

isomerization of the proline residues Pro94 and Pro97,

as indicated by an NMR study on Ca2+-free S100A2

[44].

The overall structure of Ca2+-bound S100A2 is

depicted in Fig. 1. Comparison of the two subunits

shows that the backbones adopt an almost identical

conformation, as illustrated by an rmsd value of

0.65 A for 88 Ca atoms. Strikingly, the majority of the

side chains also show almost identical orientations in

both subunits. Slight differences occur in the confor-

mation of the C-terminal EF-hand, where helix III and

helix IV adopt an interhelical angle of 119� in subunit

A and 114� in subunit B, respectively (Table 2). As a

result of this high similarity, the following description

of the structure applies for both subunits.

As in other S100 proteins, one S100A2 protom-

er consists of a pair of EF-hands linked by the

so-called hinge region [22] (Fig. 1). The N-terminal,

S100-specific EF-hand comprises helix I (4–20) fol-

lowed by the Ca2+-binding loop (Ser21-Ser30) and

helix II (31–41). Within the hinge region (42–51), resi-

dues Pro43 to Val46 form a short a-helix. Such a short

helix is also observed in other Ca2+-loaded S100 pro-

teins, for example S100A6 [protein data bank (PDB)

entry 1K96], S100A8 (PDB entry 1MR8) or S100A9

(PDB entry 1IRJ) [45–47]. The C-terminal, classical

EF-hand consists of helix III (52–62), the Ca2+-bind-

ing loop (63–71) and helix IV (72–89).

During the refinement process the electron-density

difference maps showed the presence of two PEG 400

molecules per asymmetric unit, both located at the

interface of two S100A2 dimers in the crystal. One

PEG molecule resides between the Ca2+-binding loop

of the N-terminal EF-hand from subunit B and the

Ca2+-binding loop of the C-terminal EF-hand and

helix III of subunit A from the neighbouring molecule.

The other PEG molecule is located between helix IV

from each of the subunits A of these two neighbouring

molecules. By applying crystallographic symmetry

operations, the second PEG 400 molecule was found

to consist of four carbon and three oxygen atoms,

whereby the central oxygen atom is located within the

001 face of the unit cell.

Calcium coordination

S100A2 binds four Ca2+ per dimer. The Ca2+-binding

site in the N-terminal EF-hand is formed by the back-

bone oxygen atoms of Ser20, Glu23, Asp25 and Lys28

and the carboxylate group of Glu33. An additional

water molecule completes the pentagonal bipyramidal

coordination preferred by Ca2+ ions (Fig. 2A,B). All

of these residues are conserved in S100A1 and S100B

(Fig. S1). The C-terminal classical EF-hand represents

the major target interaction site of the Ca2+-sensor

S100 proteins. Ca2+ is coordinated by Od1 of Asp63,

Od1 of Asn65, Od1 of Asp67, the backbone carbonyl

oxygen of Gln69, the carboxylate group of Glu74 and

a water molecule (Fig. 2B). The Ca2+–oxygen dis-

tances are summarized in Table 1. A similar Ca2+

coordination is also observed in the structures of other

S100 proteins [17,22].

Conformational changes upon Ca2+ binding

Recently, we have determined the structure of Ca2+-

free S100A2 [42], representing the inactive form of

S100A2. The comparison of the Ca2+-free and Ca2+-

loaded structures of S100A2 highlight the conforma-

tional changes that are required for the activation of

S100A2. In the Ca2+-free state the interhelical angle

Fig. 1. Stereo view of the Ca2+-bound S100A2 dimer. The N-terminal

S100-specific EF-hands (EF-1) from both subunits are shown in blue

and red, the C-terminal classical EF-hands (EF-2) are shown in cyan

and orange, respectively. The Ca2+ ions are depicted as green

spheres. HI, helix I; HII, helix II; HIII, helix III; HIV, helix IV.

Table 1. Distance (d ) between the Ca2+ ion and the coordinating

ligand in the two EF-hands in S100A2.

N-terminal C-terminal

EF-hand ligand d (A) EF-hand ligand d (A)

Ser20O 2.35 Asp63Od1 2.33

Glu23O 2.39 Asn65Od1 2.27

Asp25O 2.32 Asp67Od1 2.39

Lys28O 2.39 Gln69O 2.35

Glu33Oe1 2.46 Glu74Oe1 2.45

Glu33Oe2 2.53 Glu74Oe2 2.58

Water5 2.38 Water8 2.39

M. Koch and G. Fritz X-ray structure of Ca2+-loaded S100A2

FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS 1801

between helix I and helix II is 126�. The orientation

of helices I and II changed upon Ca2+ binding, result-

ing in an interhelical angle of 137� (Table 2). Interest-

ingly, the N-terminal EF-hand of Ca2+-free S100A2

harbours an Na+ ion (Fig. 2C) [42]. Na+ prefers an

octahedral coordination sphere, in contrast to the pen-

tagonal bipyramidal coordination preferred by Ca2+

ions (Fig. 2B,C) [48,49]. Nevertheless, the coordination

scheme in the EF-hand is very similar for Na+ and

Ca2+. Both ions are bound by the backbone oxygen

atoms of Ser20, Glu23, Asp25, Lys28 and a water

molecule (Fig. 2B,C). A major difference is the coordi-

nation of Ca2+ by the carboxylate group of Glu33,

whereas Na+ is coordinated by a further water mole-

cule. The binding of Glu33 to Ca2+ pulls helix II

closer to the Ca2+-binding site, shifting the entire

helix slightly (Fig. 2A). This movement reorients sev-

eral residues of helix II, which subsequently form new

hydrophobic contacts with residues on helix III.

In contrast to the small conformational changes in

the N-terminal EF-hand (Fig. 2A) the classical C-ter-

minal EF-hand undergoes a large molecular rearrange-

ment upon Ca2+ binding (Fig. 2D,E). Unlike the

almost preformed N-terminal EF-hand, several resi-

dues in the C-terminal EF-hand undergo major reposi-

tioning upon Ca2+ coordination (Fig. 2D). The side

chain of Glu74 and the backbone carbonyl group of

Gln69 display some considerable movements to bind

Fig. 2. Conformational changes in the EF-hands upon Ca2+ binding. (A) Stereo view of the N-terminal EF-hand of Ca2+-free S100A2 (blue)

and Ca2+-bound S100A2 (yellow). Only a small conformational change is observed; this involves mainly a movement of helix II. (B) Details of

Ca2+ coordination in the N-terminal EF-hand. The Ca2+ ion (green) is coordinated by backbone carbonyls, the side chain of Glu33 and a water

molecule. (C) In Ca2+-free S100A2 an Na+ ion (magenta) is bound in the N-terminal EF-hand. Like Ca2+, the bound Na+ is coordinated by

backbone carbonyls; however, the side chain of Glu33 is not directly involved and is replaced by a water molecule. (D) Structural alignment

of the C-terminal EF-hand of Ca2+-bound S100A2 (yellow) and Ca2+-free S100A2 (blue). Upon Ca2+ binding, helix III undergoes a movement

of 92�. (E) In the C-terminal EF-hand, Ca2+ is coordinated mainly by side-chain oxygens (red).

X-ray structure of Ca2+-loaded S100A2 M. Koch and G. Fritz

1802 FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS

Ca2+, and large reorientations of backbone and of

side chains are observed for residues 63–68 to accom-

modate the Ca2+ (Fig. 2D,E). Moreover, helix III has

to unwind by two residues (Leu62 and Asp63) to rear-

range Asp63-Od1 by 14 A for Ca2+ coordination.

Ca2+ binding finally causes a 92� movement of helix -

III, a feature also observed in other Ca2+-bound S100

proteins. In other EF-hand Ca2+-sensor proteins, such

as calmodulin [50] or troponin C [51], the exiting

helix IV of the EF-hand undergoes a large movement

upon Ca2+ binding instead of entering helix III. This

is readily explained by the fact that S100 proteins

form stable dimers where helix IV is involved in the

dimer interface and cannot undergo large conforma-

tional changes.

In order to describe the structural consequences of

helix reorientation and helix repacking we performed

an analysis of the changes in hydrophobic interactions

during Ca2+ binding, similarly to an analysis for

Ca2+-bound S100A1 [52]. The conformational changes

break several hydrophobic contacts of helix III with

helix IV, whereas new hydrophobic contacts between

helix III and helix II are formed (Fig. 3). In Ca2+-free

S100A2, residues Leu55, Leu58 and Leu62 of helix III

and residues Phe78, Ile82 and Met85 of helix IV form

a hydrophobic core that stabilizes the antiparallel

packing of these helices (Fig. 3A), whereas hydropho-

bic contacts between helix III and helix II are blocked

in Ca2+-free S100A2 by the side chains of Lys31 and

Lys35 from helix III (Fig. 3E). Upon Ca2+ binding,

Leu55, Ile82 and Met85 do not contribute further to

helix III–helix IV packing (Fig. 3B). Interestingly,

Ca2+ binding to the N-terminal EF-hand causes a 10�rotation of helix II, moving Lys31 and Lys35 out of

the helix II–helix III interface (Fig. 3E,F). This rota-

tion enables Leu55 of helix III to form new contacts

with Cb and Cc of Lys31 and Lys35, as well as with

Met34 of helix II (Fig. 3D). Similarly, Leu58 is

involved in Ca2+-free S100A2 in helix III–helix IV

packing. Upon Ca2+ binding the contacts to

Phe78 and Ile82 break as a result of the movement of

helix III; however, new hydrophobic contacts are

formed with Met34 ⁄Leu38 from helix II (Fig. 3D,F).

Residues Ile82 and Met85, which had contributed to

helix III–helix IV packing in Ca2+-free S100A2, are

exposed to the solvent in Ca2+-bound S100A2

(Fig. 3B) forming part of the target interaction site.

The change in the solvent-accessible surface of these

two residues illustrates readily the loss of this hydro-

phobic core upon Ca2+ binding. In Ca2+-free S100A2,

Ile82 and Met85 have solvent-accessible surfaces of

2 A2 and 17.4 A2, respectively. In stark contrast, in the

Ca2+-loaded state these residues display solvent-

exposed surfaces of 22.9 A2 and 62.8 A2, respectively.

In summary, the subtle reorientation of helix II and

the movement of helix III are crucial for the tight

helix II–helix III packing in the Ca2+-bound state. It

is likely that these interactions are essential to stabilize

the open conformation of Ca2+-bound S100A2 and

might very well explain the positive cooperativity

observed for Ca2+ binding to the two EF-hands [41].

As several of these residues are conserved among the

S100 proteins, we wondered whether the conforma-

tional changes described here for S100A2 and the

resulting hydrophobic contacts also occur in other

S100 proteins. There are structures available for

S100A1, S100A4, S100A6 and S100B in the Ca2+-free

and Ca2+-loaded states [4,18,47,52–60]. Interestingly,

the Ca2+-free structures exhibit a highly similar

helix III–helix IV packing, with Leu62 (S100A2 num-

bering) strictly conserved among these four proteins.

As in S100A2, the charged side chains of Lys31 and

Lys35 (S100A2 numbering) sterically hinder the

Table 2. Interhelical angles in EF-hands in the Ca2+-free and Ca2+-

bound states. The values represent the average between subunit A

and subunit B. In the case of S100A6, there was only one subunit

in the asymmetrical unit, and the values for S100A1 represent the

average overall models of the NMR ensemble.

Interhelical angle (�)

I to II II to III III to IV

Apo-S100A2 126 ± 1 )139 ± 3 )152 ± 1

Ca-S100A2 137 ± 1 110 ± 1 116 ± 3

Apo-S100A6 127 )142 )153

Ca-S100A6 134 114 117

Apo-S100B 133 ± 1 )149 ± 1 )166 ± 1

Ca-S100B 135.2 ± 0.2 100 ± 1 103 ± 2

Apo-S100A1 120 ± 3 )148 ± 2 )150 ± 1

Ca-S100A1 132 ± 1 125 ± 2 121 ± 2

Table 3. Spatial properties of the hydrophobic cavities of S100A2,

S100A6 and S100B.

Protein

Atoms defining

the plane

Atoms residing

in the base of

the cavity

Depth of

cavity (A)

Volume of

the central

cavity (A3)

S100A2 Phe45O Leu42Cc 7.1 99.7

Leu58Cd1

Met85Ce

S100A6 Ile44Cd1 Ile83Cd1 4.6 52.5

Leu56Cd1

Ile83Cd1

S100B Leu44Cd2 Ile36Cd1 3.9 49.5

Val56Cc2

Met79Sd

M. Koch and G. Fritz X-ray structure of Ca2+-loaded S100A2

FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS 1803

interaction of helices II and III in the Ca2+-free state

(Fig. 3E). An exception is S100B; here, the side chain

of Lys31 already points towards the solvent in the

Ca2+-free form (i.e. no rotation of helix II is required

for the formation of hydrophobic interactions). Indeed,

the binding of Ca2+ to S100B causes an axial rotation

of only 2� of helix II, in contrast to S100A2 where

helix II rotates by about 10�.In summary, the analysis of the structures of

S100A2, S100A1, S100A4, S100A6 and S100B revealed

that the Ca2+-induced movement of Lys31 and Lys35

out of the helix II–helix III interface and subsequent

hydrophobic contacts between these helices are con-

served features in the activation of S100 proteins

(Fig. 3E,F). Sequence comparison between all S100

proteins showed that the hydrophobic residues at posi-

tions 34 and 38 (S100A2 numbering) are strictly con-

served, underlining their role in S100 protein

activation (Fig. S1).

Target binding to S100A2

Sequence alignments of S100A2 with other S100 pro-

teins show that S100A2 shares high sequence homo-

logy with S100A4 (61% identity), followed by S100A1

(52%), S100A6 (47%) and S100B (45%). The five

S100 proteins have a very similar structural organiza-

tion. However, the sequence homology is not fully

reflected in structural resemblance. Although S100B

displays the lowest sequence identity, the structural

alignment of the Ca2+-bound protomers reveals that it

is most similar to S100A2 with an rmsd of 0.86 A for

75 Ca atoms excluding the Ca of the hinge region (resi-

dues 41–52). Further comparison with the subunits of

S100A6, S100A4 and S100A1 yielded rmsd values of

1.07 A (75 Ca atoms), 1.35 A (75 Ca atoms) and

1.59 A (64 Ca atoms), respectively. Major structural

differences occur in the hinge region and at the C-ter-

minal helix IV (Fig. 4). These areas represent the tar-

get protein-binding sites and the observed differences

may reflect the divergence in target molecules and

binding modes among these S100 proteins. For S100B,

several structures with peptides derived from different

target proteins have been determined [61–64]. The pep-

tide comprising the negative regulatory domain of p53

(367–392) bound to S100B adopts a helical conforma-

tion and interacts with residues from helix III, from

the hinge region and from helix IV [61]. A peptide

Fig. 3. Helix packing for Ca2+-free S100A2

(A, C, E) versus Ca2+-bound S100A2 (B, D,

F) and comparison with other S100 proteins

(E, F). In (A) and (B) the interactions

between helix III and helix IV of S100A2 are

shown, and in (C) and (D) the interactions

between helix II and helix III of S100A2 are

shown. The residues forming hydrophobic

contacts are shown in stick-type and trans-

parent van-der-Waals spheres. (E, F) Overlay

of S100A2 (cyan), S100A4 (3C1V, red)

S100A6 (1K9P, green) and S100B (2H61,

magenta). (E) Helix II and helix III in

Ca2+-free proteins (F) Helix II and helix III in

Ca2+-bound proteins. Upon Ca2+ binding,

helix II rotates slightly, by about 10�. This

rotation (counterclockwise in the figure)

removes Lys35 from the helix interface and

enables a hydrophobic contact to be made

between Leu38 and Met59 (S100A2

numbering) in the Ca2+-bound state (F).

X-ray structure of Ca2+-loaded S100A2 M. Koch and G. Fritz

1804 FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS

representing the N-terminal regulatory domain of

nuclear Dbf2-related (NDR) kinase also adopts a heli-

cal conformation in complex with S100B, and several

residues of S100B important for p53 binding are

involved in complex formation. However, the orienta-

tion of both helical peptides is noticeably different

[62]. Further variations of the binding modes are

reported for the peptide TRTK12 derived from CapZ,

where a short helix [63] and a random coil conforma-

tion were observed [64]. Similarly to S100B, S100A1

binds peptides in the same region [65,66]. A different

interaction mode was described for the complex of

S100A6 with a peptide representing the binding site of

Siah-1-interacting protein [67], where the peptide binds

to the hydrophobic region between helix III and helix

IV as well as to a region on top of the dimer interface.

S100A2, S100A6 and S100B bind to p53 [37,68–70]

in a Ca2+-dependent manner. We therefore compared

the molecular surfaces of these three proteins with

regard to electrostatic potential, shape and distribution

of hydrophobic areas (Fig. 5). As a common feature all

three proteins exhibit a net-negative surface charge.

Nevertheless, there are clear differences between these

proteins with regard to the distribution of electrostatic

surface potential. Whereas S100A2 and S100A6 display

patches of negative surface potential, S100B exhibits

areas with continuous negative potential (Fig. 5A).

Further differences in charge distribution among the

three proteins are observed around the hydrophobic

target binding pocket. Whereas S100A6 displays only a

few charged patches, continuous areas of negative

charge are observed for S100A2 and S100B. The differ-

ences in surface properties are not unexpected and

reflect different target selectivity of the three S100 pro-

teins. S100A2, S100A6 and S100B bind to p53 but each

S100 protein recognizes further target proteins (e.g.

S100A2 binds specifically to p63 [40], S100A6 to annex-

in [71] and S100B to NDR kinase [62]).

S100B recognizes a region in the tetramerization

domain of p53 (residues 325–355) as well as the nega-

tive regulatory domain of p53 (residues 362–373)

[61,72]. Binding of S100B to p53 inhibits its transcrip-

tional activation [68] by sterically blocking tetrameriza-

tion of p53 or phosphorylation ⁄ acetylation sites

essential for activation [61]. Like S100B, S100A2 recog-

nizes the C terminus of p53 [37]. In contrast to S100B,

S100A2 activates p53 [37]. Van Dieck et al. attributed

these different activities to the stabilization of either a

monomeric or a tetrameric state by S100B and S100A2

[70]. The different effect of the two S100 proteins on

p53 should be reflected in a different binding mode of

p53 in the binding pocket. We therefore characterized

depth, size and solvent accessibility of binding pockets

in greater detail. This analysis revealed that S100A2

adopts an outstanding position among these three

proteins. The cavity of S100A2, with a depth of 7 A, is

Fig. 4. Structural alignment of one protomer of S100A2 (cyan),

S100A4 (red), S100A6 (green) and S100B (magenta). Ca traces of

all four proteins are shown. Overall, the fold of the protomers of

the different S100 proteins is very similar. Noticeable deviations

occur at the hinge region and at the C terminus, which form the

target protein-binding site. Further differences are observed in

interhelical angles for the helix III–helix IV pair (Table 2). HI, helix I;

HII, helix II; HIII, helix III; HIV, helix IV.

Fig. 5. Electrostatic surface potential (A) and distribution of hydro-

phobic residues (B) for S100A2, S100A6 and S100B. (A) Negatively

charged regions are depicted in red and positively charged regions

are depicted in blue, ranging from )10 kBT ⁄ e (red) to +10 kBT ⁄ e(blue) (e, elementary charge; kB, Boltzmann constant; T, 298 K.) (B)

Residues with a positive hydropathy index, according to Kyte and

Doolittle [88], are shown in green for S100A2, in yellow for S100A6

and in purple for S100B. Molecules in (A) and (B) have the same

orientation. Cross-sections through the hydrophobic cavity of the

three S100 proteins are shown in (C). The location of the cross-sec-

tion is indicated in (B) by a black bar. (C) Left: the contours of the

cavity cross-sections of S100A6 (yellow) and S100B (purple) are

overlaid with the cross-section through the cavity of S100A2. Obvi-

ously, the cavity of S100A2 is deeper and larger than the cavity of

S100A6. The cavity of S100B appears wider, but is much shallower

than the cavities of S100A2 and S100A6.

M. Koch and G. Fritz X-ray structure of Ca2+-loaded S100A2

FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS 1805

about 60–70% deeper than those of S100A6 or S100B,

respectively (Table 3). Moreover, the cavity of S100A2

is noticeably larger than in the two other proteins

(Fig. 5C). The central cavity of S100A2 exhibits a vol-

ume of 99.7 A3, whereas the cavities in S100A6 and

S100B display volumes of 52.2 A3 and 49.5 A3, respec-

tively. Notably, the cavity of S100B is much wider and

shallower than in S100A2 and S100A6, perhaps

enabling S100B to recognize a larger number of differ-

ent targets [26]. Clearly, all three S100 proteins differ

with regard to charge and size of the target protein-

binding sites located between helix III and helix IV.

Altogether we conclude that these differences are

responsible for the different effects of the S100 proteins

on the activity of p53. Future studies on a p53362–

393 ⁄S100A2 complex might reveal a new binding mode

and will help to elucidate how S100A2 activates p53.

Materials and methods

Cloning, expression and purification

As wild-type S100A2 is prone to oxidation in air [41,73], an

S100A2-C2S-C21S-C86S-C93S variant was used for crystal-

lization experiments. S100A2-C2S-C21S-C86S-C93S was

obtained by site-directed mutagenesis and subsequently

cloned into the bacterial expression vector pMW172, as

described elsewhere [74]. Expression in Escherichia coli BL21

(DE3) and purification were carried out as described previ-

ously [73,75]. This variant is not sensitive to air oxidation.

Crystallization and data collection

Before crystallization experiments, S100A2 was loaded with

Ca2+ by passage over a NAP5 desalting column (GE

Healthcare, Munich, Germany) equilibrated with 10 mM

Tris ⁄HCl, 20 mM CaCl2, pH 7.6. The protein concentration

was 15 mgÆmL)1. Crystals were grown using the vapour dif-

fusion hanging-drop method (protein ⁄precipitant,1 lL : 1 lL). The precipitant solution was 0.1 M sodium

acetate, pH 4.0 containing 40% PEG 400. Crystals grew

within 5–10 days with dimensions up to 50 lm · 150 lm ·180 lm. The high PEG 400 concentration was sufficient as

cryoprotectant and the crystals were flash frozen in mother

liquor in the cryonitrogen stream at 100 K. Data collection

was carried out with a Swiss Light Source (PSI, Villigen,

Switzerland) at beamline X06SA using a mar225 mosaic

CCD detector (Mar Research, Norderstedt, Germany).

Data statistics are summarized in Table S1.

Structure determination and refinement

The structure of Ca2+-loaded S100A2 was determined

based on the anomalous scattering provided by 10 sulfur

atoms from methionine residues and four calcium ions present

in the structure [75]. The refinement of the model was car-

ried out with data to 1.3 A resolution using Refmac5 (ver-

sion 5.7) including anisotropic temperature factors [76,77].

Hydrogen atoms were included in refinement in riding

positions. Manual model building was performed with the

programs O [78] and Coot [79]. Data-collection and refine-

ment statistics are summarized in Table S1. Secondary

structure analysis was calculated using DSSP [80]. Struc-

ture validation was performed using SFCHECK [81] and

PROCHECK [43]. Electrostatic surface calculations were

performed using the program Adaptive Poisson-Boltzmann

Solver (APBS) [82]. Charges for amino-acid residues were

assigned using PDB 2PQR. Amber force-field parameters

for Ca2+ and Na+ were taken from Aqvist [83] for calcu-

lations of electrostatic potential. The structures were

aligned using LSQMAN [84], solvent-accessible volumes of

the cavities were calculated with CASTp using a probe

radius of 1.4 A [85] and solvent-accessible areas were

calculated using AREAIMOL [86]. For determination of

the depth of each cavity, a plane was defined by three

atoms at the border of the central cavity and the atom

inside the cavity with the largest distance to this plane

served as a reference point to define the distance between

the plane and the base of the cavity. Interhelical angles

were calculated using the INTERHLX software (K. Yap, Uni-

versity of Toronto, Toronto, Canada). Figures were pre-

pared using PyMol [87]. Structure factors and coordinates

have been deposited at the PDB database with accession

code 4DUQ.

Acknowledgements

G.F. is supported by a Heisenberg fellowship of the

Deutsche Forschungsgemeinschaft (FR 1488 ⁄3-1). Thiswork was further supported by a grant of the Deutsche

Forschungsgemeinschaft (FR 1488 ⁄ 5-1). We thank the

staff at beamline X06SA at Swiss Light Source for

excellent support.

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Supporting information

The following supplementary material is available:

Fig. S1. Multiple sequence alignment of human S100

proteins. The secondary structure of S100A2 is indi-

cated below. The Ca2+-coordinating residues are high-

lighted in red (coordination by side chains) and

magenta (coordination by backbone carbonyl). Hydro-

phobic residues at the dimer interface are highlighted

in green. All sequences were obtained from the

SWISS-PROT protein sequence database [2].

Table S1. Data collection and refinement statistics.

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X-ray structure of Ca2+-loaded S100A2 M. Koch and G. Fritz

1810 FEBS Journal 279 (2012) 1799–1810 ª 2012 The Authors Journal compilation ª 2012 FEBS