doi:10.1016/j.jmb.2007.04.065 J. Mol. Biol.

(2007) 370, 887–898

The Crystal Structure of the Human (S100A8/S100A9)2

Heterotetramer, Calprotectin, Illustrates how
Conformational Changes of Interacting α-Helices Can
Determine Specific Association of Two EF-hand
Proteins
Ingo P. Korndörfer, Florian Brueckner and Arne Skerra⁎

Lehrstuhl für Biologische
Chemie, Technische
Universität München,
Freising-Weihenstephan,
Germany
*Corresponding author
The EF-hand proteins S100A8 and S100A9 are important calcium signalling
proteins that are involved in wound healing and provide clinically relevant
markers of inflammatory processes, such as rheumatoid arthritis and
inflammatory bowel disease. Both can form homodimers via distinct modes
of association, probably of lesser stability in the case of S100A9, whereas in
the presence of calcium S100A8 and S100A9 associate to calprotectin, the
physiologically active heterooligomer. Here we describe the crystal structure
of the (S100A8/S100A9)2 heterotetramer at 1.8 Å resolution. Its quaternary
structure illustrates how specific heteroassociation is energetically driven by
a more extensive burial of solvent accessible surface areas in both proteins,
most pronounced for S100A9, thus leading to a dimer of heterodimers. A
major contribution to tetramer association is made by the canonical calcium
binding loops in the C-terminal halves of the two proteins. The mode of
heterodimerisation in calprotectin more closely resembles the subunit
association previously observed in the S100A8 homodimer and provides
trans stabilisation for S100A9, which manifests itself in a significantly
elongated C-terminal α-helix in the latter. As a consequence, two different
putative zinc binding sites emerge at the S100A8/S100A9 subunit interface.
One of these corresponds to a high affinity arrangement of three His residues
and one Asp side-chain, which is unique to the heterotetramer. This struc-
tural feature explains the well known Zn2+ binding activity of calprotectin,
whose overexpression can cause strong dysregulation of zinc homeostasis
with severe clinical symptoms.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: calcium binding; calgranulin; MRP8; MRP14; zinc binding

Present addresses: I. P. Korndörfer, CrystaX
Pharmaceuticals, S.L., Barcelona Science Park, Carrer de
Josep Samitier 1-5, 08028 Barcelona, Spain; F. Brueckner,
Genzentrum, Abteilung Chemie und Biochemie,
Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25,
81377 München, Germany.
Abbreviations used: MRP, migration inhibitory factor
related protein (or myeloid related protein); r.m.s.d.,
root-mean-square deviation; NIF,
neutrophil-immobilising factor.
E-mail address of the corresponding author:
skerra@wzw.tum.de
Introduction
Changes in cytosolic calcium concentration reg-
ulate a variety of cellular processes and calcium-
binding proteins constitute key molecules in signal
transduction, differentiation, and cell cycle control.
Proteins of the EF-hand family, including the
prototypical calmodulin,1 share a common Ca2+-
binding helix-loop-helix motif, the so-called EF-
hand. They interact with target proteins in a
calcium-dependent manner and are usually part of
the signalling pathway that leads to the cellular
responses. The S100 family of proteins2,3 comprises
a subset of the EF-hand family. Contrasting with the
ubiquitous occurrence of calmodulin, gene expres-

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
888 Crystal Structure of Calprotectin

sion of the S100 proteins is cell-specific and depends
on environmental and developmental factors.
S100A8 and S100A9 (previously described as
MRP8 and MRP14) form a heterooligomer called
calprotectin,4 which occurs not only in the cyto-
plasm of neutrophils but also on the membrane of
monocytes and in plasma as well as other body
fluids.5 Together with S100A12, they form the
calgranulin subfamily,6 a group of proteins with
proinflammatory functions that are released by
phagocytes.7
Calprotectin plays a prominent role in the regula-
tion of inflammatory processes and immune
response. Upon neutrophil activation or endothelial
adhesion of monocytes, calprotectin becomes
secreted via a microtubule-mediated, alternative
pathway and can thus serve as a marker for the
influx of mononuclear phagocytes into the site of
inflammation.5,7,8 Increased serum levels of calpro-
tectin are observed in rheumatoid arthritis, various
autoimmune diseases, cystic fibrosis, chronical bron-
chitis, acute allograft rejection, gut inflammation as
well as inflammatory dermatoses and abscesses and
thus render it a relevant target for clinical diagnosis.5
In fact, calprotectin represents an established faecal
marker of inflammatory bowel disease (IBD). 9
Furthermore, this protein was recognised as a
novel player in wound repair after skin injury.10
Calprotectin also binds and sequesters zinc, thus
inhibiting matrix metalloproteinases and other zinc-
dependent enzymes as well as interfering with
microbial growth. In cases of strongly elevated
calprotectin plasma levels this may lead to severe
dysregulation of zinc homeostasis, including hyper-
zincaemia.11 Finally, it was shown that the S100A8/
S100A9 complex can inactivate casein kinase II and
thereby prevent oncoprotein E7 activation.12 Thus,
therapeutic application of calprotectin itself or
pharmacological intervention of its pathophysiolo-
gical functions is of general interest for the treatment
of inflammatory and related diseases.
Each S100 protein is composed of two EF-hand
motifs: the N-terminal motif comprising helices I
and II, and, separated by a flexible linker, the
C-terminal motif with helices III and IV, thus pro-
viding two Ca2+-binding sites.13 Human S100A8
(Swiss-Prot entry P05109) comprises 93 amino acid
residues and has a molecular mass of 10.8 kDa while
S100A9 (Swiss-Prot entry P06702) comprises 113
amino acid residues and has a molecular mass of
13.2 kDa (Figure 1). Also, an isoform of S100A9
resulting from an alternative translational start at
codon number 5 for methionine was described.14
As most other proteins of the S100 family, S100A8
and S100A9 have been shown to homodimerise both
in vitro15 and in vivo,16,17 and the resulting isoforms
may have different functions in phagocyte physi-
ology.18 However, the formation of the S100A8/
S100A9 heterodimer seems to be strongly favoured
in vivo.17 At increased Ca2+ concentration, the
(S100A8/S100A9)2 heterotetramer is specifically
formed.19 This isoform was demonstrated to be
essential in promoting tubulin polymerisation.20
The crystal structures of the individual homo-
dimers of S100A821 and of S100A922 have been
described (PDB accession codes 1MR8 and 1IRJ,

Figure 1. Structure-based amino acid sequence alignment of S100A8 and S100A9. Mutually conserved residues are
boxed. Each chain is composed of two EF-hands. EF-hand I comprises helix I (residues 4–20 in S100A8, residues 7–23 in
S100A9), the non-canonical Ca2+-binding loop (residues 21–30 in S100A8, residues 24–33 in S100A9), and helix II (residues
31–41 in S100A8 and residues 34–44 in S100A9). EF-hand II comprises helix III (residues 51–58 in S100A8, residues 56–66
in S100A9), the canonical Ca2+-binding loop (residues 59–67 in S100A8, residues 67–75 in S100A9), and helix IV (residues
68–86 in S100A8 and residues 76–94 in S100A9). The two EF-hands are separated by linker regions of different lengths in
both proteins (residues 42–50 in S100A8 and residues 45–55 in S100A9). Secondary structure assignment from the crystal
structure described here is indicated in green and pink above and below the subunit sequences, together with the
corresponding assignments in blue and red, respectively, for the S100A8 (PDB entry 1MR8) and S100A9 (PDB entry 1IRJ)
homodimer structures. The same colour scheme applies to the following Figures. A phosphorylation site at Thr113 of
S100A9, important for regulation of its activity,46 is highlighted in orange. The Cys → Ser mutations introduced into both
polypeptide chains (residue 42 in S100A8 and residue 3 in S100A9) to prevent chemical cross-linking are shown with a
grey background. The two HXXXH motifs close to the C termini of S100A8 and S100A9, which were proposed to
form Zn2+-binding sites,26 are highlighted in cyan, whereas the His and Asp residues that were found here to complement
these coordinating residues from the opposite chain are highlighted in yellow (see the text).
Crystal Structure of Calprotectin
respectively). In both homodimers, all four Ca2+-
binding sites are occupied, which is in so far
remarkable for the S100 family as the N-terminal
EF-hand usually represents a pseudo-calcium-bind-
ing site with extremely weak affinity.13 The three-
dimensional structures of the two homodimers are
fairly similar, whereby each monomer is stabilised
by a hydrophobic core of conserved amino acids.21
The two monomer subunits in each homodimer
are related by C2 symmetry and the two N and
C-terminal helices (helices I and I' and helices IV and
IV', respectively) are arranged in an antiparallel
fashion. Homodimer formation is mainly stabilised
by interactions of hydrophobic side-chains among
these helices.
Some solvent exposed hydrophobic residues in
the S100A9 homodimer22 may be responsible for the
specific binding of target molecules, e.g. endothelial
heparan sulphate proteoglycans or the cell surface
proteins CD36 and RAGE, the receptor for advanced
glycation end-products. 5,7 A cleft between the
C-terminal helices in the S100A8 homodimer (as
indicated by the presence of xenon in the crystal
structure, which was introduced for the purpose of
phase determination21) was proposed as another
binding site for target molecules.
Although homodimerisation of the individual
S100A8 and S100A9 proteins is well understood on
the structural level, nothing is known about the
precise mechanisms of heterooligomer formation,
which is preferred under physiological conditions5,7
and seems to be crucial for many pathophysiological
functions of calprotectin. Here we present the crystal
structure of the recombinant (S100A8/S100A9)2
heterotetramer in its calcium-bound form.
Results
Tertiary structures of the S100A8 and S100A9
subunits in the heterotetramer
Crystallisation of the assembled recombinant
protein was achieved after combined refolding of
S100A8 and S100A9, which had been independently
produced as inclusion bodies in Escherichia coli (each
with its free Cys residue substituted by Ser; cf.
Figure 1). Three essentially identical heterotetramers
(average r.m.s.d. of 0.152 Å for 322 Cα positions, i.e.
residues 1–78 of S100A8 and 4–86 of S100A9) were
found to occupy the asymmetric unit of space group
P21. Each of them (chains A/B/C/D, E/F/G/H,
and I/J/K/L) comprises two S100A8/S100A9 het-
erodimers (chains A/C, B/D, E/G, F/H, I/K, and J/
L, respectively) that are paired with local C2
symmetry (Figure 2).
In spite of the poor amino acid sequence conserva-
tion between S100A8 and S100A9 (merely 27%
residue identity; cf. Figure 1), the tertiary structures
of the different monomers are surprisingly similar
within the heterotetramer quaternary structure,
especially with respect to the relative arrangement
of their two EF-hand motifs (Figure 2). This is in
889
agreement with the previous description of the indi-
vidual homodimer structures.21,22 In fact, S100A8
(residues 1–42 and 51–78) and S100A9 (residues 4–45
and 59–86) from the S100A8/S100A9 heterodimer
can be superimposed with an r.m.s.d. of 1.01 Å
(average for Cα positions from all possible mutual
superpositions; cf. Figure 2(b)).
However, important conformational differences
become apparent for the linker region (also dubbed
“hinge” region; cf. Figure 1) that connects the two
EF-hands in each subunit (Figure 2(c)). In S100A9
the linker segment and the following helix III
are longer by two and three residues, respectively,
than in S100A8. This region shows the lowest
sequence conservation among the S100 protein
family22 and may form part of a specific target-
binding site in the case of calprotectin.
In the crystal structure of the S100A8 homodimer
more residues close to the C terminus assume
α-helical secondary structure than in the one of the
S100A9 homodimer (cf. Figure 1). This is probably
an intrinsic property of S100A8 and caused by
interactions between the S100A8 C-terminal helix
and the side-chains of Tyr45 and Ile46 in the linker
region, which have no counterpart in S100A9 due to
the different length and conformation of this
segment. Further stabilisation of this hydrophobic
interaction comes from the side-chain of Trp54
(Figure 2(c)), which, unlike its homologue Ile62 in
S100A9, also forms contacts with residues in the
C-terminal helix of S100A8 (i.e. with the side-chains
of Leu74 and Lys77).
Notably, in the S100A8/S100A9 heterodimer the
C-terminal α-helix IV of S100A9 is much longer than
in its homodimer structure and assumes (with slight
variations between the individual chains) almost
the same size as the α-helix of S100A8, which is of
essentially equal length in the heterodimer com-
pared with the corresponding homodimer (cf.
Figure 1). The differing extent of crystallographi-
cally ordered conformation in the C-terminal α-helix
IV of both subunits is important for the details of
heterodimer formation between S100A8 and S100A9
as discussed further below.
The S100A8 monomer from the homodimer crystal
structure21 can be superimposed with that from the
S100A8/S100A9 heterodimer with an r.m.s.d. of as
low as 0.58 Å (average from all possible mutual
superpositions for 70 Cα positions, i.e. omitting
residues 43–50 from the linker region and residues
79–89 from the C termini, which are not defined in all
molecules of the asymmetric unit; Figure 2(d)). High
structural similarity is also evident for the S100A9
monomer in its homodimeric22 and heterodimeric
form, with a slightly larger r.m.s.d. of 0.76 Å (for 70
Cα positions, homologous to those described above;
Figure 2(e)), indicating that heterodimerisation does
not require significant conformational backbone re-
arrangement in either subunit of this complex.
The structures of the two Ca2+ -binding loop
regions in both subunits are completely conserved
in the (S100A8/S100A9)2 heterotetramer with
regard to the corresponding homodimers. As in
890 Crystal Structure of Calprotectin

Figure 2. Tertiary and quaternary structure of calprotectin. (a) The (S100A8/S100A9)2 heterotetramer (chains A/B/
C/D): S100A8, green (chains A,B); S100A9, pink (chains C/D); Ca ions are shown as light blue spheres, CA501, canonical
Ca2+ binding site; CA502, non-canonical Ca2+ binding site. (b) Superposition of S100A8 (green, chain A) and S100A9
(pink, chain C) from the heterodimer. (c) Superposition of S100A8 (blue) and S100A9 (red) from the crystal structures of
the two corresponding homodimers (PDB accession codes 1MR8 and 1IRJ, respectively). The three residues in S100A8 that
contribute to structural stabilisation of its C-terminal helix and have no homologous counterpart in S100A9 are shown as
sticks and labelled. (d) Superposition of the S100A8/S100A9 heterodimer (chains A and C, green and pink) with the
S100A8 homodimer (PDB accession code 1MR8, chains A and B, blue). Only Cα positions 1–42 and 51–78 (thus omitting
the linker region) of the corresponding pair of S100A8 subunits (chains A from the two crystal structures) were used for
the least-squares calculation (r.m.s.d. = 0.58 Å). (e) Superposition of the S100A8/S100A9 heterodimer (chains A and C,
green and pink) with the S100A9 homodimer (PDB accession code 1IRJ, chains A and B, red). Only Cα positions 4–45 and
59–86 (thus omitting the linker region) of the corresponding pair of S100A9 subunits (chains C and B, respectively) were
used for the least-squares calculation (r.m.s.d. = 0.76 Å).
Crystal Structure of Calprotectin
the homodimer structures, the canonical EF-hand II,
with its highly conserved sequence throughout the
S100 family,22 is fully occupied with Ca2+ (average
B-factors of 17.4 Å2) both in the S100A8 and S100A9
subunit. The Ca ions bound to the EF-hands I, which
show more diverse sequences, are not fully occupied
(refined with an occupancy of 0.7 and average
B-factors of 22.2 Å2; cf. Materials and Methods),
similarly as in the crystal structures of the S100A8
and S100A9 homodimers.
Structure of the S100A8/S100A9 heterodimer
The S100A8/S100A9 heterodimer has a quatern-
ary structure more similar to the S100A8 homodimer
than to the S100A9 homodimer (Figure 2(d) and (e)).
While a superposition of the entire heterodimer with
homologous residues of the S100A9 homodimer
results in an average r.m.s.d. of 2.85 Å for 140 Cα
positions (chosen as described above, i.e. omitting
the hinge and C-terminal regions), a corresponding
superposition with the S100A8 homodimer results in
a significantly smaller r.m.s.d. of 0.96 Å.
The different modes of interaction can be under-
stood by considering first the S100A8 homodimer. In
this structure close contacts are made at the subunit
interface, around the dyad centre, between residues
Leu72 and Ile76 from both monomers (Figure 3(a)
and (b)). In a model for S100A8/S100A9 based on
this mode of association the much smaller Ala84
residue of S100A9 occupies the position of S100A8
Ile76 in one of the chains, which would lead to a
small structural gap at the heterodimer interface.
However, just a minor movement of the subunits is
sufficient to bring the two α-helices closer together
(Figure 3(c) and (d)) and allow S100A8/S100A9 to
associate in a mode very similar to that of the
S100A8 homodimer.
In a putative S100A9 homodimer, modelled on the
basis of the S100A8 homodimer (Figure 3(e) and (f)),
the small Ala84 would assume the position of
S100A8 Ile76 in both chains, thus resulting in a
much deeper cleft between the two subunits.
Probably to avoid this gap, and the associated
increase in exposed protein surface, a significant
rearrangement appears in the crystal structure of the
actual S100A9 homodimer (Figure 3(g) and (h)). This
includes a sliding of the two C-terminal α-helices by
one turn, corresponding to approximately four
residues, towards each other. This movement can
be accomplished by a rotation of one S100A9
subunit by 24° around an axis roughly perpendi-
cular to the interface between the two C-terminal α-
helices and passing through the pair of residues 2 or
3 of each S100A9 monomer. Due to the fact that the
residues in the N-terminal helices of both subunits
are close to this rotation axis, their interactions
(involving residues 4–20 and 7–23 for S100A8 and
S100A9, respectively; Figure 1) are affected to a
lesser extent by the differing mode of association
than those of the C-terminal α-helices.
Furthermore, the interaction of S100A9 with
S100A8 provides external stabilisation for the
891
S100A9 subunit and induces a transition for a
stretch of 6 to 14 residues (i.e. residues 86–91 and
their sequence neighbours in S100A9, with slight
variations for the different chains in the asymmetric
unit) at the end of the C-terminal α-helix from a
disordered state (as in the S100A9 homodimer
structure) to α-helical conformation with defined
electron density (see Figures 1 and 2(e)). Concomi-
tantly, new interactions are formed between this
segment and the C-terminal α-helix of S100A8, for
example involving residues Thr87, Trp88, and His91
of the former and Ile13, Leu72, and Phe68 of the
latter (Figure 4(a)). Notably, there appears a single
intermolecular salt bridge, which is unique to the
S100A8/S100A9 heterodimer, between S100A9
Asp30 and S100A8 His83 and His87 (OD1(Asp30)–
NE2(His87): 3.26 Å, OD2(Asp30)–NE2(His83):
2.68 Å; Figure 4(b)). These residues are also part of
one of the two presumed Zn2+-binding sites (see
below), whereby zinc complexation could indeed
further stabilise the heterodimer.
The heterodimer interface and tetramer
association
The observed changes in the details of subunit
association result in an increased buried surface area
at the S100A8/S100A9 heterodimer interface com-
pared with the S100A8 and S100A9 homodimers.
For S100A8 an average surface area of 1293 Å2 per
subunit is buried in the homodimer, whereas a by
116 Å2 (9%) larger surface of 1409 Å2 becomes
buried in the heterodimer with S100A9. A total of
1257 Å2 of surface per subunit is buried in the
S100A9 homodimer, whereas 1421 Å 2 become
buried in the S100A8/S100A9 heterodimer, which
corresponds to an increase by 164 Å 2 (11%).
Approximately 90% of the increase in buried surface
area is contributed by side-chains whereby 60% are
from hydrophobic residues.
For S100A8 the increase in buried surface area can
be interpreted as result of a large number of small
changes, where some amino acids appear more
exposed but many others are more buried (Figure 5).
For S100A9 a few more prominent changes are
observed (Figure 5(c) and (d)): (i) in its N-terminal
EF-hand I most residues are less buried at the
heterodimer interface. However, three residues in
the N-terminal α-helix I and the following loop have
a strongly increased buried surface in the hetero-
dimer. Residue S100A9 Gln7 becomes buried due to
the relative rotation of the S100A8 subunit with
respect to the homologous chain in the S100A9
homodimer as explained further above. Residues
Ile16 and Asp30 of S100A9 become buried by the
now extended C-terminal end of α-helix IV from
S100A8. (ii) While some residues of S100A9 are also
less buried in the C-terminal EF-hand II in case of the
heterodimer (e.g. residue Met83), several residues
become ordered as part of the much longer α-helix
in the S100A8/S100A9 heterodimer compared with
the S100A9 homodimer (e.g. Thr87, Trp88, and
His91).
892 Crystal Structure of Calprotectin

Upon association to the heterotetramer, additional the three tetramers in the asymmetric unit). This
surface areas of 740 Å2 and 514 Å2 become buried for corresponds to about half or less of what is buried in
S100A8 and S100A9, respectively (averaged for the corresponding heterodimer contacts (approxi-

Figure 3 (legend on next page)

Crystal Structure of Calprotectin 893

Figure 4. Side-chain interactions at the interface of the S100A8/S100A9 heterodimer and putative Zn2+ binding sites
of calprotectin. (a) Residues involved in interactions specific for heterodimer formation between S100A8 (green and cyan)
and S100A9 (pink and yellow). (b) Arrangement of the two different Zn2+ binding sites in the S100A8/S100A9
heterodimer. The HXXXH motifs are coloured cyan whereas the complementing side-chains from the opposite subunit
are coloured yellow (cf. Figure 1). The resulting His3Asp tetradentate Zn2+ coordination site formed by His residues 83
and 87 from S100A8 and His20 as well as Asp30 from S100A9 is specific for the calprotectin heterodimer. (c) Close-up of
the His3Asp zinc binding motif in calprotectin. (d) The His4 zinc binding motif in calprotectin. (e) The homologous His4
zinc binding motif in the (S100A8)2 homodimer.

mately 1400 Å2; see above). Interactions between the (cf. Figure 1) of S100A8 (e.g. Asn25, His27, Ile60,
pair of heterodimers comprise hydrophobic as well Asn61, Asp63, Gln69 each have more than 50 Å2 of
as polar side-chains from both Ca2+-binding loops their surface area buried) and the regions flanking

Figure 3. Molecular mechanism for different modes of association observed in (S100A8)2, (S100A9)2, and (S100A8/
S100A9)2, i.e. calprotectin. Colouring of subunits is similar to the previous Figures while modelled structures are shown in
grey. Side-chains depicted as stick models always correspond to pair-wise homologous residues. (a) Molecular surfaces of
the monomers in the (S100A8)2 homodimer. (b) Details of interaction between the two C-terminal α-helices in the
(S100A8)2 homodimer. (c) and (d) Similar representation of the S100A8/S100A9 heterodimer from the crystal structure
described here. The calprotectin heterodimer associates in a similar manner as observed for the S100A8 homodimer and
can avoid a small gap between its different subunits that would arise from replacement of Ile76 by the much smaller
residue Ala84 in S100A9 via small shifts in the mode of helix association. (e) and (f) Putative (S100A9)2 dimer modelled on
the basis of the crystal structure of the (S100A8)2 homodimer. The gap in the hypothetical homodimer structure, which is
mostly caused by the substitution of Ile76 of S100A8 by Ala84 in S100A9, is indicated for the surface representation with a
broken ellipse and leads to a visible groove between the two subunits. Arrows in the ribbon diagram indicate the relative
movement of the α-helices at the dimer interface that is required to obtain the crystallographically observed homodimer.
(g) and (h) The crystal structure of the (S100A9)2 homodimer shows no gap at the subunit interface, which is a
consequence of a register shift of the two antiparallel C-terminal α-helices (cf. panels above) and closer packing between
them.
894 Crystal Structure of Calprotectin

Figure 5. Histogram of buried surface area per residue at the subunit interface in the S100A8/S100A9 heterodimer
and in the homodimers of S100A8 and S100A9. Key secondary structure elements of the molecules are indicated below the
residue numbers shown on the abscissa. Amino acid residues that exhibit particularly pronounced differences in
accessible surface area between homo- and heterodimers are labelled. (a) S100A8 surface area A(heterodimer) per residue
buried in the heterodimer contacts of calprotectin. (b) Differences A(heterodimer) – A(homodimer) of buried surface area
for S100A8. (c) S100A9 surface area A(heterodimer) per residue buried in the heterodimer contacts of calprotectin. (d)
Differences A(heterodimer) – A(homodimer) of buried surface area for S100A9.

loop 2 (with the canonical, tighter Ca2+ binding site
II) from S100A9 (e.g. Asp65, Thr68, Glu77, Met81
and Arg85).
It was previously described that disturbance of the
Ca2+ binding sites, e.g. by mutagenesis of S100A9
Asn69 and Asp78, both involved in coordination of
the bound Ca2+, has no influence on dimerisation
but prevents tetramerisation.20 The apparent struc-
tural involvement of the Ca2+-binding loops in the
formation of the heterotetramer as well as their
participation in a large number of crystal packing
contacts may explain why crystal growth was only
observed in the presence of 2 mM Ca2+, whereas no
crystals were obtained in the presence of similar
concentrations of lanthanides (e.g. Gd3+, Yb3+ or
Ho3+), which were alternatively applied with the
purpose of anomalous phasing.
Discussion
Two important roles have been assigned to
calprotectin: (i) this signalling protein plays a
prominent role in the regulation of inflammatory
processes, immune response, and wound repair; (ii)
the (S100A8/S1009)2 heterotetramer binds and
sequesters zinc, thus inhibiting zinc-dependent
enzymes as well as microbial growth. The crystal
structure of calprotectin reveals a dimer of S100A8/
S100A9 heterodimers and represents, to our knowl-
edge, the first experimental structure reported for a
heterodimer of S100 proteins to date.13 In conjunc-
tion with a deeper analysis of the homodimerisation
modes observed for the isolated S100A8 and S100A9
proteins, the present crystal structure explains their
specific heteroassociation to form the intact calpro-
tectin and the concomitant emergence of a physio-
logically important high affinity binding site for
zinc.
Zinc binding
The high affinity for Zn2+ is a property character-
istic of calprotectin but absent both in the individual
S100A8 and S100A9 proteins.23 Various studies
have indicated that the antimicrobial as well as
apoptosis-inducing activities of the S100A8/S100A9
heterooligomer are attributable to its Zn2+-chelating
capacity.23–25 Two HXXXH motifs (residues 83–87 in
S100A8 and residues 91–95 in S100A9; cf. Figure 1)
were proposed to be responsible for Zn2+ binding.26
Neither for the S100A8 or S100A9 homodimers nor
for their heterooligomer crystallographic data in the
presence of bound Zn2+ are available. However, in
all three crystal structures, the HXXXH motifs are
located at the interface between the respective
S100A8 and S100A9 subunits.
In the (S100A8/S100A9)2 heterotetramer de-
scribed here His83 and His87 from the HXXXH
motif of S100A8 are complemented by His20 from
Crystal Structure of Calprotectin
the opposite S100A9 molecule (Figure 4(c)). To-
gether with the side-chain of Asp30 in spatial
neighbourhood this would allow the formation of
a His3Asp Zn2+ binding motif, homologous to the
zinc and copper-binding sites observed for psoria-
sin, S100A7 (His86/His90/His17/Asp24),27 and
for the calgranulin S100A12 (His85/His98/His15/
Glu25).28 Similar zinc binding motifs are commonly
observed, for example, in matrix metalloproteases
(for a review see Harding29 and Supplementary
Material therein). Both S100A7 and S100A12 form
homodimers in a manner more similar to S100A8
than to S100A9.
The homologous residues His91 and His95 from
the HXXXH motif of S100A9 are complemented by
His17 from S100A8 (Figure 4(d)). In this case,
however, His27 from S100A8 would be the likely
fourth candidate, instead of Asp, to be involved in
tetrahedral metal coordination. A His4Zn2+ binding
site has, to our knowledge, only been observed for
one natural protein, the fungal transcription factor
HAP1 (PDB accession code 1HWT),30 where it
appears as part of an interchain packing contact in
the crystal structure. The function of such a His4
binding site was investigated in a genetically
engineered variant of carbonic anhydrase II, where
it led to reduced metal affinity.31
In native carbonic anhydrase the catalytically
active Zn2+ is coordinated by His residues 94, 96,
and 119 as well as a hydroxide anion. The variant
T199E exhibits tetrahedral coordination of the Zn2+,
with three His residues and one Glu side-chain
involved, and a 200-fold increase in affinity towards
Zn2+ compared with the wild-type enzyme. This
corresponds to the situation for the first zinc binding
site in the S100A8/S100A9 heterodimer described
above, where the carboxylate metal ligand is pro-
vided by an Asp side-chain. In contrast, the carbonic
anhydrase variant T199H, which would correspond
to the His4 binding site, has a 20-fold lower affinity
than the native enzyme. In this case, the artificial
fourth His side-chain rotates away from the bound
Zn2+, as shown by X-ray crystallographic analysis,
indicating that this kind of coordination sphere is
energetically disfavoured for zinc.
In the S100A8 homodimer, both HXXXH motifs
(residues 83–87) are complemented by two His
residues from the respective opposite chain
(Figure 4(e)) to form such a His4 zinc binding
motif with presumably low affinity. In case of the
S100A9 homodimer the C-terminal helix IV is
destabilised and shorter (see above), such that the
HXXXH motif (residues 91–95) is not resolved in
the crystal structure. Hence, the S100A9 homo-
dimer should not display significant affinity for
Zn2+ either, due to its inappropriate mode of
association and the entropic cost of ordering α-
helix IV to create a structurally defined binding
site. Consequently, only in calprotectin one of two
putative binding sites, i.e. comprising residues
His83 and His87 of S100A8 and His20 and Asp30
of S100A9, can give rise to a typical high affinity
Zn2+ binding site.
895
Specific subunit heteroassociation of S100
family members in calprotectin
Inspection of the published crystal structures of
S100A8 and S100A9 homodimers21,22 indicates that
variations in their EF-hand linker regions lead to
different stabilisation of their C-terminal α-helices
and thus to distinct modes of dimerisation. Inter-
action of the S100A8 and S100A9 subunits in the
S100A8/S100A9 heterodimer structure better re-
sembles the one observed for (S100A8)2 and reveals
more extensive contacts than in the homodimer of
S100A9.
Although S100A9 exhibits in its amino acid
sequence the longest C-terminal extension (cf.
Figure 1) among the S100 family of proteins,22
only a much shorter α-helical region appears to be
conformationally ordered in the crystal structure of
its homodimer. In contrast, the S100A8 homodimer
with its shorter polypeptide reveals a considerably
longer C-terminal α-helix.21 In the crystal structure
of the S100A9 homodimer the main-chain was
described to make a turn towards the solvent at
the end of the C-terminal region.22 It was proposed
that, due to the presence of several Gly and Pro
residues in the C-terminal polypeptide segment, the
formation of a defined secondary structure cannot
be expected. The present crystallographic analysis
demonstrates that specific heterodimer association
of S100A9 with S100A8 actually results in a
structural stabilisation of S100A9 and significant
elongation of its C-terminal α-helix to approxi-
mately the same length as observed for S100A8.
The C-terminal region of S100A9 is identical with
the N-terminal segment of neutrophil-immobilising
factor (NIF), which is involved in interactions with
neutrophils and prevents their random migration
and chemotaxis.32 For the region comprising resi-
dues Arg85 to Met94 a role in heterodimer interac-
tion was proposed before. 17,18,33 In the crystal
structure of S100A8/S100A9, up to six residues of
the amino acid sequence that is in common with
NIF, Ala89 to Gly107, assume α-helical secondary
structure and participate in the formation of the
heterodimer. Apart from that, three consecutive His
residues (His103–His105) in this region were shown
to be involved in arachidonic acid binding.34 In
many cases target recognition takes place via short
linear motifs that lack a defined conformation.35
Possibly, the association with S100A8 and concomi-
tant stabilisation of this region may be sufficient to
restrict sterical accessibility of the remainder of the C
terminus and, thus, to modulate or even prevent the
interaction with possible target molecules as sug-
gested by Itou et al.22
While heterodimerisation between S100A8 and
S100A9 seems also possible in the absence of
calcium,36 mass-spectrometric analyses and gel
filtration studies have shown that the (S100A8/
S100A9)2 heterotetramer only forms in the presence
of Ca2+.36,37 Our crystallographic analysis illustrates
the importance of the Ca2+-binding loops, especially
the canonical ones, for the association of the two
896
S100A8/S100A9 heterodimers to form the calpro-
tectin heterotetramer (cf. Figure 2(a)).
While the S100A8 protein alone was shown to
promote tubulin polymerisation,38 no such effect
could be observed for the isolated S100A9 protein.
Instead, S100A9 appeared to act as a regulatory
subunit in the S100A8/S100A9 complex with
respect to tubulin polymerisation, which is an
important calcium-dependent process during pha-
gocyte migration. Our structure of calprotectin
allows for the first time a rationalisation of this
effect: while S100A8 assumes a rather similar
tertiary structure both as homo- and as heterodimer,
the much shorter C-terminal α-helix of S100A9 in the
case of its homodimer should lead to a weaker
homoassociation. Both proteins experience a gain in
buried surface area upon heterodimerisation, an
effect that is approximately 50% more pronounced
for S100A9 than for S100A8.
Thus, the S100A9 which is probably present in
locally high concentrations in the physiological
environment is readily recruited for formation of
the energetically favoured and functionally impor-
tant heterooligomer with S100A8, i.e. calprotectin.
It might be possible to confirm this model via
measurement of the precise thermodynamic para-
meters for the association/dissociation equilibria
using suitable biophysical methods, even though
this will be difficult because of the various molecular
species or complexes that are involved. Together
with more detailed data on the differential expres-
sion patterns of S100A8 and S100A9, both spatially
and temporally, our structural model should sig-
nificantly contribute to understanding the regula-
tion of inflammatory processes and may provide a
basis for the rational development of therapies.
Materials and Methods
Recombinant protein production and purification
Expression vectors harbouring the coding sequences for
human S100A8 and S100A9 proteins (pET36-S100A8 and
pET36-S100A9, respectively) were kindly provided by
SWITCH Biotech AG (Neuried, Germany). On these
plasmids, the complete coding sequences for both proteins
(SwissProt accession numbers P05109 and P06702, respec-
tively) were inserted between the NdeI and BamHI
restriction sites of pET36b(+) (Novagen, Madison, WI).
To avoid the formation of non-physiological disulphide
cross-links, the mutants S100A8(C42S) (cf. Figure 1) and
Table 1. Data collection statistics
Space group, P21
Unit cell parameters (Å, °) 48.56, 78.68, 176.85,
90.00, 94.98, 90.00
No. of reflections 99,507 (3119)
Resolution (Å) 30.00–1.80 (1.90–1.80)
Completeness of data (%) 81.0 (69.9)
I/σ(I) 3.8 (1.4)
Mosaicity 0.60
Redundancy 6.2 (6.1)
Rmeas 0.13 (0.52)
Crystal Structure of Calprotectin
Table 2. Refinement statistics
Resolution (Å) 30.00–1.80 (1.85–1.80)
R/RFree 0.158/0.206 (0.185/0.254)
Protein residues visible 1076
No. of solvent molecules 1059
Other compounds 24 Ca2+, 4 Cl−, 1 citrate
r.m.s. deviations from 0.012
ideality of bond lengths (Å)
r.m.s. deviations from 1.479
ideality of bond angles (°)
S100A9(C3S) were constructed via polymerase chain
reaction with the forward primers 5′-GAAGAAATTGC-
TAGAGACCGAGTCTCCTCAGTATATC-3′ and 5′-GGA-
GATATACATATGACTAGCAAAATGTCGCAGCTGG-3′,
respectively, each together with the reverse primer 5′-
GCTAGTTATTGCTCAGCGGTGGCAGC-3′. The result-
ing gene fragments were digested with BsaI/XhoI and
NdeI/XhoI, respectively, and recloned on the correspond-
ing expression vectors.
Both proteins were separately produced in E. coli BL21
(DE3). Transformed bacteria were cultivated at 37 °C in
shaker flasks with LB medium containing 70 mg/l of
kanamycin, induced with 0.1 mM isopropyl-β-D-thioga-
lactopyranoside at A550 ≈ 2.5, and further incubated for
24 h. Cells were harvested by centrifugation, stored at
−20 °C, and used for disruption, whereby 10 g of each
sediment was resuspended in 50 ml 20 mM Tris–HCl (pH
8.0) and sonicated. To isolate inclusion bodies the cell
lysate was centrifuged and the sediment was resuspended
in washing buffer (20 mM Tris–HCl (pH 8.0), 0.5% (v/v)
Triton X-100, 10 mM EDTA), sonicated, and sedimented
again by centrifugation. This procedure was repeated five
times, followed by another three times with omission of
Triton X-100 and EDTA.
For protein renaturation and reconstitution of the
S100A8/S100A9 heterodimer, equal amounts of the two
inclusion body preparations were resuspended in 20 mM
Tris–HCl (pH 8.0) (5 ml/g inclusion body protein) and the
mixture was slowly dripped into 6 M guanidine–HCl
(100 ml/g). After clarification by centrifugation, the
resulting solution was dialysed three times against
20 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM EGTA
(1.5 l/g). Following centrifugation and sterile filtration
(0.45 μm), the protein solution was applied to a
RESOURCE Q column (Amersham Pharmacia, Uppsala,
Sweden), followed by elution with an NaCl gradient from
0–100 mM in the same buffer that was used for dialysis.
Fractions containing the renatured S100A8/S100A9 mix-
ture were concentrated by ultrafiltration to 10 mg/ml,
applied to a Superdex 75 HiLoad 16/60 gel filtration
column (Amersham Pharmacia) in the presence of 20 mM
Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, and eluted in essentially one homogeneous peak,
corresponding to the heterodimer. The yield was approxi-
mately 50 mg of purified protein per 1 l of E. coli culture.
Mass spectrometric analysis indicated that the start Met
residue of the recombinant S100A8(C42S) was present and
partially N-formylated whereas the N-terminal residue of
S100A9(C3S) was fully cleaved.
Crystallisation and data collection
Crystals of the recombinant calprotectin were grown
using the hanging drop vapour diffusion technique39 with
32% (v/v) 2-methylpentane-2,4-diol and 1 M sodium
citrate (pH 4.2) as precipitants. Two μl of the protein
Crystal Structure of Calprotectin
solution (15 mg/ml in 20 mM Tris–HCl (pH 8.0), 2 mM
CaCl2) were mixed with 2 μl of the precipitant solution on
a siliconised glass cover slip, followed by equilibration
against 0.5 ml of the precipitant solution at 12 °C.
Calprotectin crystallised in space group P21 with six
S100A8/S100A9 heterodimers in the asymmetric unit
(Table 1) over the course of one to two weeks.
Protein crystals were harvested directly from the drop
using nylon loops (Hampton Research, Aliso Viejo, CA)
and frozen in a 100 K nitrogen stream (Oxford Cryosys-
tems, Oxford, UK). A native dataset was collected at the
synchroton X-ray source PX I (SLS, Villigen, Switzerland)
to a resolution of 1.8 Å (Table 1). Reflection intensities were
processed with MOSFLM and SCALA.40 All crystals grew
as thin plates and displayed a strong anisotropy in their
X-ray diffraction patterns such that it was not possible to
process reflections close to the plane of the plates, which
accounts for the limited completeness of the dataset.
Structure determination and analysis
The crystal structure was solved by molecular replace-
ment with PHASER41,42 using the monomers of the
published structures of S100A8 (PDB accession code
1MR8) and S100A9 (PDB accession code 1IRJ) as search
models. Model building with QUANTA43 and translation,
liberation, screw-rotation (TLS)/maximum likelihood
refinement in REFMAC540 using non-crystallographic
symmetry restraints (“tight” restraints for core residues,
“medium” restraints for residues on the surface) led to a
final model with good stereochemistry and an R-factor of
0.158 at 1.8 Å resolution (Table 2). While the Ca ions
occupying the canonical Ca2+ binding site in the loop of
EF-hand II could be modelled with full occupancy, the
occupancies of the second Ca2+ binding site in EF-hand I
were set to 0.7 in order to yield B-factors for the metal ions
approximately corresponding to those of their liganding
protein side-chains.
Graphical representations of molecular models were
prepared with PyMOL†. Secondary structure assignments
were made with DSSP.44 Surface accessibility calculations
were carried out with AREAIMOL and SURFACE.40
Protein Data Bank accession code
The crystal structure coordinates of calprotectin were
deposited at the RCSB Protein Data Bank45 under
accession code 1XK4.
Acknowledgements
The authors thank Boris Bieger and Andreas
Goppelt, SWITCH Biotech AG, Neuried, Germany
for inspiring this project, experimental advice, and
providing cDNAs of the native S100A8 and S100A9
proteins. The authors are furthermore grateful to
Joseph Danzer for assistance with protein purifica-
tion and crystallisation, Holger Stalz (Lehrstuhl für
Chemie der Biopolymere, TU München) for mass
spectrometric analyses, and the staff at the PX I of
† http://www.pymol.org
897
the Swiss Synchrotron Light Source (Villigen,
Switzerland) for support during data collection.
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Edited by R. Huber
Available online 3 May 2007