Báo cáo khoa học: Crystal structure of human cystatin C stabilized against amyloid formation pdf

Crystal structure of human cystatin C stabilized against
amyloid formation
Robert Kolodziejczyk
1
, Karolina Michalska
1
, Alejandra Hernandez-Santoyo
1,2
, Maria Wahlbom
3
,
Anders Grubb
3
and Mariusz Jaskolski
1,4
1 Faculty of Chemistry, Department of Crystallography, A. Mickiewicz University, Poznan, Poland
2 Instituto de Quı
´
mica, Universidad Nacional Auto
´
noma de Me
´
xico, Ciudad Universitaria, Mexico
3 Department of Clinical Chemistry, Lund University, Sweden
4 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Introduction
The cystatin superfamily of cysteine protease inhibitors
is subdivided, according to structural features and
presence in the intracellular, extracellular or intravas-
cular space, into three families: 1 (stefins), 2 (cystatins),
and 3 (kininogens) [1–3]. Human cystatin C (HCC),

a member of the family 2 cystatins, is an inhibitor of
papain-like (C1) and legumain-like (C13) cysteine pro-
teases [4]. The inhibition of C1 proteases is especially
potent, with dissociation constants (in the femtomolar
range) [3] representing the strongest competitive inhibi-
tion known in biochemistry. HCC is found in all body
fluids, with particularly high concentrations in the
cerebrospinal fluid [5], and its function is to regulate
the activity of cysteine proteases, either released from
lysosomes of dying or damaged cells [6], or originating
from microbial invasion [7]. In common with other
family 2 cystatins [1,4], the 120 residue polypeptide
chain of HCC is expected to fold as a monomer with
Keywords
amyloid; crystal twinning; cysteine protease
inhibitor; protein engineering; 3D domain
swapping
Correspondence
M. Jaskolski, Faculty of Chemistry,
Department of Crystallography, A.
Mickiewicz University, Grunwaldzka 6,
60-780 Poznan, Poland
Fax +48 61 8291505
Tel: +48 61 8291274
E-mail:
Database
The atomic coordinates and structure
factors have been deposited in the Protein
Data Bank under the accession code 3GAX
(Received 4 January 2010, revised 25

January 2010, accepted 27 January 2010)
doi:10.1111/j.1742-4658.2010.07596.x
Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain-like
(C1) and legumain-related (C13) cysteine proteases. In pathophysiological
processes, the nature of which is not understood, HCC is codeposited in
the amyloid plaques of Alzheimer’s disease or Down’s syndrome. The amy-
loidogenic properties of HCC are greatly increased in a naturally occurring
L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult
life. In all crystal structures of cystatin C studied to date, the protein has
been found to form 3D domain-swapped dimers, created through a confor-
mational change of a b-hairpin loop, L1, from the papain-binding epitope.
We have created monomer-stabilized human cystatin C, with an engineered
disulfide bond (L47C)–(G69C) between the structural elements that become
separated upon domain swapping. The mutant has drastically reduced
dimerization and fibril formation properties, but its inhibition of papain is
unaltered. The structure confirms the success of the protein engineering
experiment to abolish 3D domain swapping and, in consequence, amyloid
fibril formation. It illustrates for the first time the fold of monomeric cysta-
tin C and allows verification of earlier predictions based on the domain-
swapped forms and on the structure of chicken cystatin. Importantly, the
structure defines the so-far unknown conformation of loop L1, which is
essential for the inhibition of papain-like cysteine proteases.
Abbreviations
AS, appending structure; HCC, human cystatin C; HCCAA, hereditary cystatin C amyloid angiopathy; HCC-stab, monomer-stabilized human
cystatin C; PDB, Protein Data Bank.
1726 FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS
two well-conserved disulfide bridges (Cys73–Cys83 and
Cys97–Cys117) in the C-terminal half of the sequence
[8,9]. By analogy with enzyme complexes of family 1
cystatins [10–12], and chicken cystatin docked in the

active site of papain [13], the inhibitory epitope of
HCC has been postulated to include the N-terminal
peptide Ser1–Val10, and two b-hairpin loops, L1, con-
taining a signature motif QxVxG(55–59), and L2, with
a characteristic Pro105-Trp106 sequence, all aligned in
a wedge-like fashion at one side of the molecule [4].
The need to define a precise enzyme-binding motif
was dictated by the idea of using it as a molecular tem-
plate in the design of efficient small molecule inhibitors,
targeting cysteine proteases involved in tissue-degenera-
tive diseases, such as osteoporosis or paradontosis [14],
or produced by highly virulent strains of bacteria and
viruses [15–18]. To this end, we have carried out
numerous crystallization experiments on papain–HCC
mixtures. However, single crystals of a papain–HCC
complex could never be obtained, as the incubated
samples invariably undergo proteolytic degradation,
probably because of impurities present in commercially
available papain samples. Also, despite many years of
effort, crystallization of monomeric HCC has not been
achieved. Instead, a number of crystallization condi-
tions have invariably led to crystals being formed from
HCC dimers, arising as a consequence of 3D domain
swapping [19–21], a phenomenon in which the mono-
meric fold is re-created in an oligomeric context, i.e.
from fragments of the polypeptide chain contributed
by different molecules [22]. In a closed 3D domain-
swapped dimer, the protein fold is essentially as in the
monomeric form, except in a hinge element, which has
a drastically changed conformation, allowing the

protein to partially unfold and form a mutual grip
with a similarly unfolded partner. In the HCC dimer,
the hinge element is loop L1, which assumes an
extended conformation engaged in a long intermolecu-
lar b-sheet, and the N-terminal fragment of the mole-
cule (b1–a–b2) is anchored in the domain-swapped
partner molecule (Fig. 1B). This unexpected difficulty
has opened a completely new aspect of HCC research,
connected with a naturally occurring L68Q variant of
HCC, whose extremely high propensity for dimeriza-
tion and aggregation leads to amyloid deposits in
cerebral vasculature in a lethal disease known as
hereditary cystatin C amyloid angiopathy [23,24].
The discovery of a domain-swapped HCC structure
[19] provided the first experimental evidence of 3D
domain swapping in an amyloidogenic protein, and
has rekindled interest in this phenomenon as a possible
mechanism of amyloid formation [25,26]. Since then,
3D domain swapping has been demonstrated in two
other amyloidogenic proteins, namely the prion protein
[27] and b-microglobulin [28]. The interest in HCC,
however, has not declined, partly because the dimeric
protein has been crystallized in several forms [29],
including a polymorph in which the domain-swapped
molecules have aggregated to build an infinite structure
with all b-chains in a perpendicular orientation relative
BA
Fig. 1. The fold of HCC. (A) Molecule A of HCC-stab1 (this work) folded as a monomer. The papain-binding epitope is formed by the N-ter-
minus, loop L1, and loop L2. The AS is an irregular appending structure at a ‘back side’ loop system harboring a potential legumain-binding
site. The dashed line represents a fragment of the HCC-stab1 backbone that is not visible in the electron density map of molecule A. L47C

and G69C denote the Cys mutation sites in HCC-stab1, introduced to provide a covalent disulfide link between strands b2 and b3. (B) For
comparison, the 3D domain-swapped dimer of wild-type HCC is shown (PDB code: 1G96). It has a folding domain similar to the monomeric
HCC-stab1, but composed of two molecules (blue and green). All of the structural elements of the monomeric fold are preserved, except for
loop L1, which is transformed to an extended conformation.
R. Kolodziejczyk et al. Structure of monomeric cystatin C
FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS 1727
to a common direction [21], as required by the cross-b
canon [30] of amyloid fibril architecture. Although
the present view of amyloid aggregation is more
complex [31], the interest in 3D domain swapping
remains high.
Until recently, the available experimental data were
insufficient to decide between two models relating 3D
domain swapping and amyloid structure. In one of
them, 3D domain swapping would operate in a propa-
gated, open-ended fashion linking all the molecules
into a runaway structure. According to the other view,
3D domain swapping would only be necessary for the
formation of dimeric building blocks, which would
aggregate to form fibrils using a different mechanism.
Two recently reported experiments have demonstrated
that, at least in the amyloid fibrils of T7 endonuclease
[32] and of HCC [33], 3D domain swapping in the
propagated mode is taking place.
A related research interest is focused on ways to pre-
vent oligomerization and aggregation of amyloidogenic
proteins. In one study, catalytic amounts of antibody
or enzymatically inactive papain prevented dimeriza-
tion of wild-type and L68Q HCC [34]. Also, it was
possible to inhibit dimerization, oligomerization and

amyloid formation of HCC by site-directed mutagene-
sis of its sequence. Specifically, under the assumption
that in a 3D domain-swapped dimer the organization
of the structural elements closely resembles the mono-
meric fold, the dimeric structures of HCC were ana-
lyzed in order to identify places where a covalent
crosslink would tether those structural elements of the
monomer that undergo separation during domain
swapping. Thus, pairs of juxtaposed Cys residues were
introduced into the HCC sequence, with the expecta-
tion that their connection through a disulfide bond
would provide the necessary crosslinkage [34]. Two
monomer-stabilizing disulfide bridges were introduced
in this manner, between strands b2 and b3 (monomer-
stabilized HCC-stab1 double mutant L47C ⁄ G69C) or
between the a-helix and strand b5 (HCC-stab2 double
mutant P29C ⁄ M110C).
In this work, we have crystallized the HCC-stab1
mutant and determined its 3D structure at 1.7 A
˚
resolution. The structure confirms the success of the
protein engineering experiment to abrogate 3D domain
swapping, and demonstrates for the first time HCC
folded as a monomeric protein. It allows verification
of the earlier predictions based on the domain-
swapped form or on the crystal structure of chicken
cystatin [13,35,36]. Importantly, the structure of
HCC-stab1 defines the so-far unknown conformation of
loop L1, an element that is essential for the inhibition of
papain-like cysteine proteases.

Results and Discussion
Structure solution and refinement
Initial analysis of the diffraction pattern of poorly dif-
fracting HCC-stab1 crystals (2.6 A
˚
) suggested that they
have hexagonal symmetry, with 12 protein molecules in
the unit cell. A successful run of molecular replacement
with chicken cystatin [Protein Data Bank (PDB) code:
1CEW] as a model confirmed the correctness of the P6
1
space group and, indeed, revealed two HCC-stab1 mole-
cules in the asymmetric unit. A complete model with
correct sequence was obtained after several rounds of
manual rebuilding and maximum likelihood refinement.
Although the atomic model agreed well with electron
density maps, the refinement was characterized by high
R-factors. Re-examination of the data revealed hemi-
hedral twinning with a twin law (h, –h–k,–l). The
same kind of twinning was detected in a new dataset,
with a resolution of 1.7 A
˚
, that was collected for a crys-
tal from a different crystallization trial. The subsequent
refinement, carried out in refmac5 [37] with the appro-
priate twin option, used the new data and a set of R
free
reflections selected in pairs according to the twin law.
The final value of the twinning fraction, a = 0.452,
indicates almost perfect twinning. The refinement con-

verged with R and R
free
of 0.138 and 0.167, respectively,
and with other parameters as listed in Table 1.
Description of monomeric HCC structure
Overall fold
The two HCC-stab1 molecules (A and B) in the asym-
metric unit display the canonical cystatin fold, (N)–b1–
a–b2–L1–b3–AS–b4–L2–b5–(C), with a five-stranded
antiparallel b-sheet gripped around a long a-helix
(Fig. 1). The AS is a broad irregular ‘appending struc-
ture’ positioned at the opposite end of the b-sheet
relative to the N-terminus ⁄ loop L1 ⁄ loop L2 edge,
which is the papain-binding epitope. b1 is the shortest
element of the five-stranded antiparallel b-sheet, com-
prising only two residues. In both molecules, the first
11 residues are disordered and not visible in electron
density maps. Two AS residues, different in each mole-
cule, are also disordered: Pro78-Leu79 in A and
Leu80-Asp81 in B. Some of the disulfide bridges are
evidently partially broken, an effect that can be
attributed to radiation damage.
b-Bulges
The fold of monomeric cystatin C contains four
b-bulges, which endow the molecular b-sheet with very
Structure of monomeric cystatin C R. Kolodziejczyk et al.
1728 FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS
strong curvature. Three of the b-bulges are classified
as antiparallel classic type [38], and the residues at
which the pattern of hydrogen bonds is broken are

Asp65 (at b2–b3), Glu67 (b2–b3), and Gln100 (b4–b5).
The fourth b-bulge, representing a special antiparallel
type, is created by His43 (b2–b3). An identical system
of deviations from regular b-sheet geometry exists in
the HCC dimers with 3D swapped domains, as well as
in chicken cystatin.
b-Bulges, which disrupt the continuity of b-interac-
tions, are considered to be a strategy to avoid protein
aggregation through intermolecular b-sheet extension
[39,40]. For this reason, b-bulges are desired at edge-
forming (terminal) b-strands, but are not very common
in the b-sheet interior. However, the situation in HCC
is quite the opposite. Three of the b-bulges are located
exactly within the inner b-sheet elements, namely at
the b2–b3 junction. It is very intriguing to note that it
is this very junction that gets separated during the 3D
domain-swapping event in HCC. It is therefore very
likely that the b-bulges destabilize the HCC b-sheet in
this region, thus promoting partial protein unfolding
and, eventually, oligomerization and higher-order
aggregation.
The disulfide bridges
The electron density map provides clear evidence of the
existence of the engineered Cys47-Cys69 disulfide bond
(Fig. 2), introduced to covalently link strands b2 and
b3 of one monomer. A comparison with the structures
of 3D domain-swapped HCC (e.g. PDB code: 1G69)
(Fig. 1B) or monomeric chicken cystatin (PDB code:
1CEW) shows that the presence of this extra bridge
does not perturb the conformation of the main chain in

the region of these point mutations. This is possible
because the side chain of Cys47 fills the space of the
native Leu47, and Cys69, replacing a Gly, occupies the
empty space close to the Ca atom. Considering that the
rigid fragments (i.e. the b-sheet and a-helix) of 1G69
and 1CEW agree very well with the backbone trace of
the analogous elements of HCC-stab1, we may assume
that HCC-stab1 correctly represents the structure of the
native HCC monomer.
However, it became clear in the course of the refine-
ment that the new disulfide bond had to be partially
broken to fit the 2F
o
– F
c
electron density map con-
toured at the 1.0r level. Disruption of disulfide bonds
is a common effect observed in protein crystals exposed
to intense X-ray radiation, and is caused by the reduc-
ing effect of free electrons generated in the ionization
events. Consequently, the Cys47–Cys69 bond has been
modeled with partial occupancy (40% in A, and 60%
in B), and the remaining occupancy has been allocated
to free -SH groups according to indications from an
F
o
–F
c
map contoured at the 3.0r level. As in the preli-
minary biochemical experiments (including gel filtra-

tion) there was no indication of dimerization, it must
be concluded that the disruption of the Cys47–Cys69
Table 1. Data collection and refinement statistics.
Data collection
Space group P 6
1
Cell dimensions (A
˚
) a = 76.26, c = 97.57
Temperature (K) 100
Radiation source BESSY BL14.1
Wavelength (A
˚
) 0.9180
Resolution (A
˚
) 50.0–1.70 (1.76–1.70)
a
Reflections collected 372 127
Unique reflections 35 372
R
merge
0.090 (0.896)
<I ⁄ rI> 25.9 (2.0)
Completeness (%) 99.8 (98.3)
Redundancy 10.5 (8.1)
Refinement
Resolution (A
˚
) 39.26–1.70 (1.79–1.70)

Reflections work ⁄ test set 34 199 ⁄ 1037
R
work
⁄ R
free
0.138 ⁄ 0.167
No. of protein ⁄ water atoms 2020 ⁄ 180
<B-factors> protein ⁄ water (A
˚
2
) 13.5 ⁄ 20.4
Rmsd from ideal
Bond lengths (A
˚
) 0.019
Bond angles (°) 1.75
Ramachandran statistics of u ⁄ w angles (%)
Most favored 93.7
Additionally allowed 5.8
Generously allowed 0.5
PDB code 3GAX
a
Values in parentheses are for the highest-resolution shell.
Fig. 2. 2F
o
–F
c
electron density map contoured at the 1.0r level, in
the region of the L47C ⁄ G69C mutation in molecule A, illustrating
the partial disruption of the disulfide bond.

R. Kolodziejczyk et al. Structure of monomeric cystatin C
FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS 1729
bond has occurred during the X-ray exposure of the
crystal. Another ionizing radiation-driven disruption of
a disulfide bond was found at the Cys73–Cys83 bridge
of molecule A, which was modeled with 50% occu-
pancy. In molecule B, this bond is intact. The second
native disulfide bond, Cys97–Cys117, is intact in both
molecules. The above observations illustrate the fact
that ionization-induced disruption of a disulfide bond
depends on its accessibility in both intramolecular
terms and in the crystal packing context.
The new disulfide bond is right-handed, with C–S–
S–C torsion angles (v3) of 74.2° and 75.2° in mole-
cules A and B, respectively. The remaining two, native,
disulfide bonds in molecules A and B have the
corresponding torsion angles of )89.0° and )97.9° for
Cys73–Cys83 and 114.7° and 114.5° for Cys97–
Cys117, thus defining them as left-handed and right-
handed, respectively. A survey of these bridges in the
HCC dimers shows that the C-terminal Cys97–Cys117
bond has a rigid right-handed form, whereas the
Cys73–Cys83 bond, found in the AS region, has a
variable configuration.
Conformation of loop L1
The present study reveals, for the first time, the struc-
ture of intact loop L1 of HCC, which structurally
comprises VAG(57–59). However, when referring to
loop L1 as part of the inhibitory epitope, one has to
include two additional residues at the C-terminal end

of strand b2. In this article, we follow the standard L1
nomenclature [4], referring to the QIVAG(55–59) pen-
tapeptide. The electron density in the L1 area of mole-
cules A and B is of very high quality, allowing for
modeling of the backbone and side chain conforma-
tions without ambiguity (Fig. 3). The VAG(57–59)
triplet can be classified as an inverse c-turn [41,42],
with a hydrogen bond between residues i and i +2,
and i + 1 backbone dihedral angles of )70.4° and
)89.0° (u) and 69.1° and 74.4° (w) in molecules A and
B, respectively. Of all the L1 amino acids, Val57 shows
the highest deformation, with backbone torsion angles
(u ⁄ w) that locate it in the lower left quadrant of the
Ramachandran plot, in the additionally allowed
()112.9°⁄)133.4°, molecule B) or generously allowed
()130.9°⁄)147.4°, molecule A) regions.
In chicken cystatin (PDB code: 1CEW), which is the
closest structural analog available for comparison,
most of the L1 residues have torsion angles in the pre-
ferred regions, except Ser56, which is located at the
apex of the loop in a position occupied in HCC-stab1
by Ala58, and which is an evident outlier
(u = )176.7°, w = )56.1°). This anomaly is very diffi-
cult to explain, as the two inhibitors have similar affin-
ities for the cognate enzymes. It is interesting to note
that this conformational dissimilarity is coupled with a
quite different chemical character of the residue in the
apex position of loop L1 (Ala in HCC, and Ser in
chicken cystatin). In the structure of stefin B in com-
plex with papain (PDB code: 1STF), the L1 fragment

[QVVAG(53–57)] has no outliers in the Ramachandran
plot. The residue in question, namely Val55, which is
equivalent to Val57 in the HCC sequence, adopts a
b-type conformation with u ⁄ w values ()118.7°⁄
)148.6°) very close to those found in HCC-stab1.
Structural environment of Leu68
Leu68 is of particular importance because its naturally
occurring mutation to Gln, endemic to the Icelandic
population, results in the creation of a highly amyloi-
dogenic form of HCC. Leu68 is located at the end of
strand b3 of the b-sheet, just before the polypeptide
chain enters the poorly structured and conformational-
ly variable AS. The Leu68 side chain protrudes from
the concave part of the b-sheet towards the interface
with the a-helix. The side chains of Val31 and Tyr34
(from the a-helix), Val66 (b3), Phe99 (b4), and Cys97–
Cys117 (b4–b5), and the backbone of the SRA(44–46)
segment, form a hydrophobic pocket in which the side
chain of Leu68 is nearly ideally nested.
Until now, the structural consequences of the L68Q
mutation for the stability of monomeric HCC could
only be inferred from the structures of 3D domain-
swapped dimers of HCC. The present structure pro-
vides the first possibility of visualizing the Leu68 side
chain in its true monomeric context. Comparison with
the Leu68 environment reported in the dimeric struc-
Fig. 3. 2F
o
–F
c

electron density map contoured at 1.0r in the region
of loop L1 of molecule B.
Structure of monomeric cystatin C R. Kolodziejczyk et al.
1730 FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS
tures shows an almost identical arrangement, confirm-
ing that the earlier analyses, using the dimeric struc-
ture as a template, were essentially correct [19]. The
increased size and incompatible chemical character of
a Gln side chain at position 68 would result in repul-
sive interactions destabilizing the a–b interface and
leading to an increased likelihood of a partial unfold-
ing process, in which these two main structural ele-
ments (i.e. the a-helix and the b-sheet) would separate,
thus promoting oligomerization, aggregation and, pos-
sibly, fibril formation.
The AS
The edge of the HCC molecule containing loop a⁄ b2
and the AS has been reported to harbor a site that is
important for the inhibition of legumain [4]. The AS
itself spans 26 residues, from Cys69 to Lys94, and has
an irregular conformation. The AS is located at the
opposite end of the b-sheet to the N-terminus ⁄ L1⁄ L2
epitope (Fig. 1A), so there should be no interference
between papain and legumain binding by HCC. Con-
sistent with this conclusion is the observation that the
3D domain-swapped dimer of HCC shows unaltered
inhibition of legumain, whereas it is incapable of
papain binding, because of the absence of the L1 ele-
ment of the recognition site [4]. The backbones of mol-
ecules A and B follow the same AS trace,

complementing each other in the regions with gaps
(Fig. 4A). Moreover, the conformation found in the
present structure is very similar to the backbone traces
reported in all of the dimeric structures of HCC and in
monomeric cystatin D [43] (Fig. 4B). This conforma-
tion is, however, different from the models proposed
for chicken cystatin or for human cystatin F [44], in
which, respectively, a prominent a-helix or a helical
coil is present (Fig. 4C).
Crystal packing and molecular interactions
The HCC-stab1 molecules A and B in the asymmetric
unit form crystal packing interactions leading to their
association into two types of easily recognizable assem-
blies. In the most conspicuous contact, molecules A
and B associate via their AS loops (Fig. 5), burying
about 920 A
˚
2
of the molecular surface of each partner
in a network of interactions that includes several van
der Waals contacts and four hydrogen bonds
(Table 2). The two molecules are related by an almost
perfect noncrystallographic two-fold axis along [1

1 0].
The second mode of association buries a much smaller
surface area (about 460 A
˚
2
per monomer), but it is

important because it illustrates the propensity of HCC
to undergo intermolecular b-interactions at the edge
of the molecular b-sheet. In this pairing scheme,
molecule A interacts with a crystallographic copy of
molecule B¢ (– y, x – y, z +1⁄ 3) via a pseudo-two-
fold rotation coupled with a 3 A
˚
translation (Fig. 5).
These two molecules are linked via four main chain–
main chain hydrogen bonds (Table 2) to form an inter-
molecular parallel b-sheet, utilizing their b5 elements.
Such a mode of interaction has already been observed
in the tetragonal crystal structure of dimeric HCC
(PDB code: 1TIJ) [21]. Two additional hydrogen bonds
involving side chains support these A–B¢ interactions.
The noncrystallographic symmetry axis relating the
A
C
B
Fig. 4. Superposition of HCC-stab1 and
related proteins. (A) The HCC-stab1 mole-
cules A (red) and B (blue) superpose almost
perfectly. Their AS regions (orange and
marine, respectively) complement each
other in the regions with gaps. (B) Structural
alignment of HCC-stab1 (molecule A, red)
with the folding units of 3D domain-
swapped HCC molecules (1G96, orange;
1TIJ, violet) and with cystatin D monomer
(1RN7, blue). (C) Superposition of

HCC-stab1 (molecule A, red) with chicken
cystatin (1CEW, orange) and with human
cystatin F (2CH9, blue).
R. Kolodziejczyk et al. Structure of monomeric cystatin C
FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS 1731
two molecules is aligned with the [210] direction. The
translational component of this noncrystallographic
symmetry is necessary to bring the parallel b-sheet
interactions into register (Fig. 5). It should be noted
that crystallographic symmetry requires both types of
dimer to sit on the same axis, so none of the diagonal
directions (related by the 3
1
axis) is close to a perfect
dyad. Combination of the crystallographic 6
1
axis with
the diagonal noncrystallographic dyads leads to
additional noncrystallographic two-fold axes along the
x and y directions. The molecules related by this
(nearly perfect) operation present an additional scheme
of intermolecular interactions. Specifically, there are
four hydrogen bonds between molecules A and B¢¢
(y –1, –x + y, z +5⁄ 6) at an interface formed by
loop b1 ⁄ a and the N-terminus of the a-helix. The con-
tact area is about 220 A
˚
2
per molecule. The crystallo-
graphic symmetry brings into contact molecules B and

B¢¢ (with the participation of loop L2), with only two
hydrogen bonds but many van der Waals contacts and
a buried surface area of about 450 A
˚
2
per monomer.
Similar interactions are found between molecules A
and A¢¢.
The A–B ‘dimer’ propagates along the 3
1
axis, utiliz-
ing alternating AS–AS and b5–b5 interactions, so there
is no indication of oligomeric assembly involving end-
less b-sheet formation along the crystal z-axis, as
observed in some of the dimeric HCC crystal struc-
tures [21]. The exposed b-chains at the opposite edge
of the b-sheet (b1 and b2) do not participate in any
type of intermolecular b–b interaction, so no combina-
tion of symmetry operations, real or pseudo, could
lead to such an assembly.
It is of note that the pseudosymmetric dyad along
[100] coincides with the two-fold axis relating the twin
domains, which probably promotes the prevalence and
character (a about 0.5) of the twinning phenomenon
observed for these HCC-stab1 crystals [45].
Comparison with other cystatin models
The overall fold of HCC-stab1 shows no significant
differences when compared to the available models of
type 2 cystatins, such as chicken cystatin (PDB code:
1CEW), cystatin D (PDB code: 1RN7), or cystatin F

(PDB code: 2CH9). In addition, it is possible to
compare the monomeric fold of HCC-stab1 with the
complete folding units ‘extracted’ from the 3D
domain-swapped dimers of HCC, namely 1G96 (one
copy in the asymmetric unit), 1TIJ (two copies), and
1R4C (eight copies). In all cases, the main differences
are within the AS, and concern, for example, the pres-
ence of a-helical segments in 1CEW and 2CH9
(Fig. 4C). When the AS fragment is excluded from the
alignment, the Ca superposition becomes much closer,
with, for instance, an rmsd value of 0.70 A
˚
for 1G96.
On the other hand, type 1 cystatin models, such as
the domain-swapped stefin B (PDB code: 2OCT), and
monomeric stefin A in complex with cathepsin H
(PDB code: 1NB5) or stefin B in complex with papain
(PDB code: 1STF), show very significant deviations
from HCC-stab1, mainly in the course of the a-helix
(which, in type 1 cystatins, is significantly bent), in the
positioning and curvature of the b-sheet, and in the
AS, which is essentially absent in the stefin structure.
The results of least-squares superpositions of the
HCC-stab1 molecule A and other cystatin and stefin
models are summarized in Table 3.
Manual docking of monomeric cystatin C in the active
site of papain
The structural elements of cystatins and stefins that
function as epitopes in the inhibitory interactions with
Fig. 5. Crystal packing of HCC-stab1. Molecules A (red) and B

(blue) in the asymmetric unit are related by a noncrystallographic
two-fold axis parallel to [1

10], here shown (lens symbol) in perpen-
dicular orientation to the plane of the drawing. The yellow (B¢) and
green (A¢) molecules are crystallographic copies (6
1
rotation along
c) of molecules B and A, respectively. The components of the
(equivalent) A–B¢ and B–A¢ pairs are related by noncrystallographic
symmetry involving a two-fold axis (along [210] and [120], respec-
tively), shown as an arrow between strands b5 of the intermolecu-
lar b-sheets, and an additional 3 A
˚
translation.
Structure of monomeric cystatin C R. Kolodziejczyk et al.
1732 FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS
their target enzymes are loops L1 and L2 and the
N-terminus [4]. In cystatin C, the inhibitory part of
loop L1 has the sequence QIVAG(55–59), whereas the
most important residues from loop L2 are Pro105 and
Trp106. In an unrelated family of cysteine protease
inhibitors, represented by chagasin [46–48], the
enzyme-blocking epitope is formed by loops L4, L2
and L6, which correspond to the N-terminus ⁄ L1 ⁄ L2
elements of cystatins, in that order. Loop L6 contains
conserved Pro and Trp ⁄ Phe residues at positions that
are equivalent to Pro105 and Trp106 in the HCC
sequence.
The exact modes of cysteine protease–inhibitor inter-

actions have been described, for example, for stefin B
[10] (PDB code: 1STF) and chagasin [48] (PDB code:
3E1Z) in their complexes with papain. These two struc-
tures can be used as templates for tentative modeling of
HCC-stab1–papain interactions. As a first attempt, the
HCC-stab1 molecule was superposed onto stefin B
from the 1STF complex, using a secondary structure
matching approach; this showed that the two molecules
do not align very well, with an overall Ca rmsd as high
as 2.51 A
˚
. Moreover, such an overall superposition
leads to significant discrepancies in the epitope regions,
Table 2. Interfaces formed by pairs of HCC-stab1 molecules through crystal contacts.
Interface
Hydrogen bonds
Secondary struture chID:res (atom) –
(atom) res:chID secondary structure
Distance (A
˚
)
A ⁄ B, B ⁄ B’’ or A ⁄ A’’
Surface area (A
˚
2
) Rotation (°)
Direction
cosines of
rotation axis
A-B AS A:D87 (O

d1
) – (N) V18:B b1 ⁄ a; 2.96 916 ⁄ 928 179.9 0.846
AS A:N82 (O
d1
)–(N
g2
) R24:B a; 2.78 )0.534
b1 ⁄ a A:V18 (N) – (O
d1
) D87:B AS; 2.91 )0.004
AS A:N82 (N
d1
)–(O
d2
) D28:B a; 3.09
a
A-B’ ()y,x)y,z+1 ⁄ 3) AS A:R93 (N
g2
) – (O) D119:B b5; 3.00 480 ⁄ 434 179.6 0.885
b5 A:S115 (N) – (O) S113:B b5; 3.10 0.466
b5 A:C117 (N) – (O) S115:B b5; 2.95 )0.004
b5 A:S115 (O) – (N) S115:B b5; 2.87
b5 A:C117 (O) – (N) C117:B b5; 2.98
b5 A:D119 (O
d1
) – (N) D119:B b5; 2.73
A-B’’(y)1,)x+y,z+5 ⁄ 6) b1 ⁄ a A:E19 (O) – (N) E21:B a; 3.19 220 ⁄ 228 179.9 0.466
b1 ⁄ a A:E19 (O
e1
) – (N) G22:B a; 3.03 )0.085

a A:E21(N) – (O) E19:B b1 ⁄ a; 3.09 )0.004
a A:G22 (N) – (O
e1
) E19:B b1 ⁄ a; 2.95
B-B’’(y)1,)x+y,z+5 ⁄ 6) a ⁄ b2 B:M41 (N) – (O) P105:B L2; 3.11 438 ⁄ 458 60.0 0.000
b2 B:Y42 (O
g
)–(O
e2
) E21:B a; 2.80 0.000
)1.000
A-A’’(y)1,)x+y,z+5 ⁄ 6) a ⁄ b2 A:M41 (N) – (O) P105:A L2; 3.07 462 ⁄ 453 60.0 0.000
b2 A:Y42 (O
g
)–(O
e2
) E21:A a; 2.43 0.000
)1.000
a
The side-chain amide of N82 has been flipped, as it evidently has the wrong rotation in the PDB entry 3GAX.
Table 3. Rmsd values (A
˚
) between Ca atoms of structurally aligned cystatin models, defined by their PDB codes. For the 3D domain-
swapped dimers of HCC, the superposition is calculated for one half of the dimer, corresponding to a cystatin folding unit and containing res-
idues 1–56 from one monomer (A) and 60–120 from the complementary chain (B). Explanation of PDB codes: 1G96, two-fold symmetric 3D
domain-swapped HCC dimer [19]; 1R4C, 3D domain-swapped dimer of N-truncated HCC [20]; 1TIJ, 3D domain-swapped HCC dimer [21];
1RN7, monomeric human cystatin D [43]; 1CEW, monomeric chicken cystatin [13]; 2CH9, monomeric human cystatin F [44]; 1STF, stefin B
from papain complex [10]; 1NB5, stefin A from cathepsin H complex [11].
HCC-stab1 (B) 1G96 1G96 No AS
a

1R4C (A, B) 1TIJ (A, B) 1RN7 1CEW 2CH9 1STF 1NB5
HCC-stab1 (A) 0.58 1.02 0.70 1.10 1.40 1.18 1.41 1.73 2.51 2.64
a
Residues 69–94, forming the AS loop, have been omitted from the calculations.
R. Kolodziejczyk et al. Structure of monomeric cystatin C
FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS 1733
e.g. a large deviation in the Ca traces of loop L2. Thus,
in the next modeling experiment, it was assumed that
optimal fit in loops L1 and L2 should be a priority,
and Gln55–Gly59 and Val104–Gln107 of HCC-stab1
were superposed onto their equivalents in stefin B. This
comparison, however, indicated that the coordinates of
loops L2 do not agree with each other, as illustrated by
the relative positions of the Ca atom of Pro105, which
differ by 1.77 A
˚
in the two structures (Fig. 6). Also,
forcing loops L2 to superpose worsens the alignment of
loops L1, which in a simple L1 ⁄ L1 superposition over-
lap almost perfectly (rmsd of 0.13 A
˚
). These results
clearly show that loop L2 is different in HCC and ste-
fin B. One reason for this variability may be the lack of
sequence conservation, i.e. the fact that the VPWQ
motif of HCC is replaced by LPHE in stefin B. An
additional factor may be the involvement of loop L2 of
both molecules in the HCC-stab1 structure in similar
packing interactions, which may constrain it in a non-
native conformation. The third possibility is, of course,

that the conformation of the enzyme-binding epitope,
in particular the relative disposition of loops L1 and
L2, undergoes an induced-fit adaptation on enzyme
complex formation. The first hypothesis could be partly
tested using an HCC–chagasin alignment, because
chagasin contains a Trp in a position equivalent to the
Trp106 site of HCC. Such a comparison would not be
possible by direct HCC–chagasin superposition, owing
to the completely different folds of the proteins. It
could be achieved, however, via an intermediate align-
ment of the papain components of the chagasin and
stefin B complexes. Then, when loop L2 from the
above L1 ⁄ L2 superposition of HCC-stab1 and stefin B
is analyzed, it is noted that the positions of Pro105 and
Trp106 match those of their chagasin counterparts
much better than their equivalents in stefin B (Fig. 6).
Despite this improvement, Trp106 of HCC-stab1 still
clashes with the Gln142 residue of papain, but this
obstacle could be avoided by slight conformational
adaptations of loop L2. A minor rearrangement may
also be required to bring Pro105 into an optimal posi-
tion with respect to Leu143 of papain, and to optimize
the p-type interactions between Trp106 and the cluster
of aromatic residues of papain that cap Asn175, the
last residue of the catalytic triad [46]. In the current
model of the HCC-stab1–papain complex, the interac-
tion interface buries 564 A
˚
2
and 530 A

˚
2
of solvent-
accessible surfaces of the interacting partners.
The interface between papain and loop L1 of HCC-
stab1 seems to be dominated by hydrophobic interac-
tions, and thus resembles the situation in the stefin B
complex rather than in the chagasin complex. No direct
interference with the Cys25-His159-Asn175 catalytic
triad of papain, as observed for the chagasin–papain
complex [48], is predicted from the current model.
The hypothetical enzyme-binding mode of the
N-terminus of HCC could not be analyzed, owing to
the absence of this element in the crystallographic
model of HCC-stab1. However, the backbone trace
near the visible beginning of the HCC-stab1 molecules
follows closely the equivalent fragment of stefin B, sug-
gesting that its conformation in these two proteins
could be similar. By analogy with other enzyme com-
plex structures, the N-terminal peptide of HCC-stab1
would be expected to form b-sheet interactions with
the enzyme.
The above comparisons lead to the conclusion that
the predicted interactions of HCC with its target
enzyme, based on the structure of the current HCC-
stab1 model, are compatible with various observations
reported for complexes with other inhibitors. However,
it is not possible to build, by simple target-based dock-
ing, a model that would explain all aspects of the
inhibitory interactions. Therefore, a reliable description

of HCC–papain interactions will require an indepen-
dent experimental study. Work in this direction is in
progress.
Fig. 6. The L1 and L2 epitope of HCC-stab1 docked into the active
site of papain. The gray color represents papain from the complex
with chagasin (PDB code: 3E1Z). The papain active site residues
are shown as ball-and-stick models connected by hydrogen bonds
(red dashed lines). The key residues from the L1 and L2 epitope of
HCC-stab1 (red), as well as their counterparts in stefin B (yellow)
and chagasin (cyan), are shown in stick representation. Papain resi-
dues important for interactions with loop L2 are also shown as
sticks. Labels referring to the inhibitor component correspond to
the HCC sequence. This overlay was constructed by first superpos-
ing loops L1 and L2 of HCC-stab1 (molecule A) on the correspond-
ing epitope of stefin B in its complex with papain (PDB code:
1STF). Next, the papain components of the 1STF and 3E1Z com-
plexes were superposed to bring the coordinates of chagasin into
the same system of reference.
Structure of monomeric cystatin C R. Kolodziejczyk et al.
1734 FEBS Journal 277 (2010) 1726–1737 ª 2010 The Authors Journal compilation ª 2010 FEBS
Experimental procedures
Protein expression and purification
A variant of HCC with two Cys mutations introduced at
Leu47 and Gly69 was expressed in Escherichia coli MC1061
and purified using a modified procedure of Nilsson et al.
[34]. Briefly, expression was induced at 42 °C for 3 h when
D
600 nm
was approximately 5. The protein harvested from
the periplasmic space was purified by anion exchange chro-

matography using Q-Sepharose and a buffer containing
20 mm ethanolamine (pH 9.0) and 1 mm benzamidinium
chloride. After concentration by ultrafiltration, the sample
was subjected to size exclusion chromatography using an
Amersham Biosciences FPLC Superdex HR 75 column,
equilibrated with 10 mm sodium phosphate buffer (pH 7.4),
140 mm NaCl, 3 m m KCl, and 1 mm benzamidinium chlo-
ride. In the last purification step, the protein solution was
dialyzed against 20 mm sodium citrate buffer (pH 6.5), with
1mm benzamidinium chloride. The dialyzed protein was
divided into 0.5 mg aliquots, lyophilized, and stored at
)20 °C.
Crystallization
Before crystallization, protein samples were dissolved in
150 mm sodium phosphate buffer (pH 7.5), passed through
a 0.22 l m Millipore filter, and subjected to size exclusion
chromatography using a GE Healthcare HiLoad 16 ⁄ 60
Superdex 200 prep. grade column. Fractions containing
monomeric HCC-stab1 were combined and concentrated
on Microcon filters (10 kDa cut-off) to 10 mgÆmL
)1
. The
final buffer concentration was < 20 mm.
Crystallization experiments were performed at 19 °C,
using the hanging-drop vapor-diffusion method and a grid
screen with different concentrations of ammonium phos-
phate versus pH. Drops were mixed from 1 : 1 (v ⁄ v) ratios
of the protein and precipitant solutions. The best crystals
(0.35 · 0.26 · 0.26 mm) grew above a reservoir containing
1.25 m ammonium phosphate (pH 7.0).

Data collection and processing
A preliminary X-ray diffraction dataset extending to 2.6 A
˚
resolution (data not shown) was collected at beamline I911-3
in MAX-lab (Lund, Sweden). Later, a new dataset extending
to 1.7 A
˚
resolution was collected at beamline BL14.1 of
the BESSY synchrotron (Berlin, Germany), using a
Rayonics MX-225 3 · 3 CCD detector. Crystals were cryo-
protected in mother liquor supplemented with 25% (v ⁄ v)
glycerol, and flash-vitrified at 100 K in a nitrogen gas
stream. All diffraction data were processed and scaled in the
Laue class 6 ⁄ m with hkl2000 [49]. A summary of data
collection and processing is presented in Table 1.
Structure solution and refinement
The structure was solved by molecular replacement with
chicken cystatin (PDB code: 1CEW) as a search probe,
using phaser [50]. Manual model building was performed
in coot [51], and crystallographic refinement was per-
formed with refmac5 [37]. Despite successful phasing with
molecular replacement in space group P6
1
, the refinement
had high R and R
free
values of 0.314 and 0.394, respec-
tively. Re-examination of the space group assignment and
of the diffraction data for the possibility of crystal twin-
ning, carried out in the ccp4 programs truncate and

detwin [52], as well as in cns [53], gave a strong indication
of hemihedral twinning, with a twin operation (h, –h–k,
–l) corresponding to the higher Laue class symmetry
6 ⁄ mmm. The same conclusions were reached for the second
dataset, which was used for all subsequent calculations.
The final refinement was carried in refmac
5, with the tls
[54] and twin options included. The refinement con-
verged with a final R-factor of 0.138 (R
free
= 0.167)
for all data, and a model characterized by rmsd from
ideal bond lengths of 0.019 A
˚
, with 93.7% of all resi-
dues in the most favored areas of the Ramachandran
plot (no residues in disallowed regions). The refine-
ment statistics are shown in Table 1.
Acknowledgements
This work was supported, in part, by grants from the
Polish State Committee for Scientific Research (Project
no. 4 T09A 039 25) and from the Swedish Science
Research Council (Project no. 5196), by a subsidy from
the Foundation for Polish Science to M. Jaskolski,
and by a postdoctoral fellowship (Project no. PBZ ⁄
MEiN ⁄ 01 ⁄ 2006⁄ 06) from the Polish Ministry of Science
and Higher Education to R. Kolodziejczyk. Some of the
calculations were performed in the Poznan Metropoli-
tan Supercomputing and Networking Center.
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