Báo cáo khoa học: Crystal structure of the parasite inhibitor chagasin in complex with papain allows identification of structural requirements for broad reactivity and specificity determinants for target proteases pptx

Crystal structure of the parasite inhibitor chagasin in
complex with papain allows identification of structural
requirements for broad reactivity and specificity
determinants for target proteases
Izabela Redzynia
1,
*, Anna Ljunggren
2,
*, Anna Bujacz
1
, Magnus Abrahamson
2
, Mariusz Jaskolski
3,4
and Grzegorz Bujacz
1,4
1 Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland
2 Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Sweden
3 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland
4 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Papain (EC 3.4.22.2) from the latex of the papaya fruit
(Carica papaya) was one of the first known proteolytic
enzymes, and its digestive properties were already
being utilized in the 19th century. Detailed biochemical
studies in the 20th century peaked with efforts in the
1960s, defining the chemistry of the enzymatic mecha-
nism, delineating the concept of specificity for protein
substrate recognition [1–3], and with elucidation of the
Keywords
Chagas disease; cruzipain; cysteine
proteases; papain; protein inhibitors

Correspondence
G. Bujacz, Institute of Technical Biochemis-
try, Faculty of Biotechnology and Food
Sciences, Technical University of Lodz, ul.
Stefanowskiego 4/10, 90-924 Lodz, Poland
Fax: +48 42 636 66 18
Tel: +48 42 631 34 31
E-mail:
M. Abrahamson, Department of Laboratory
Medicine, Division of Clinical Chemistry and
Pharmacology, Lund University, University
Hospital, SE-221 85 Lund, Sweden
Fax: +46 46 130064
Tel: +46 46 173445
E-mail:
*These authors contributed equally to this
paper
Database
Atomic coordinates, together with structure
factors, have been deposited in the Protein
Data Bank under the accession code 3E1Z
(Received 13 October 2008, revised 15
November 2008, accepted 1 December
2008)
doi:10.1111/j.1742-4658.2008.06824.x
A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and
papain, a classic family C1 cysteine protease, has been crystallized. Kinetic
studies revealed that inactivation of papain by chagasin is very fast
(k
on

= 1.5 · 10
6
m
)1
Æs
)1
), and results in the formation of a very tight,
reversible complex (K
i
=36pm), with similar or better rate and equilib-
rium constants than those for cathepsins L and B. The high-resolution
crystal structure shows an inhibitory wedge comprising three loops, which
forms a number of contacts responsible for the high-affinity binding. Com-
parison with the structure of papain in complex with human cystatin B
reveals that, despite entirely different folding, the two inhibitors utilize very
similar atomic interactions, leading to essentially identical affinities for the
enzyme. Comparisons of the chagasin–papain complex with high-resolution
structures of chagasin in complexes with cathepsin L, cathepsin B and falci-
pain allowed the creation of a consensus map of the structural features that
are important for efficient inhibition of papain-like enzymes. The compari-
sons also revealed a number of unique interactions that can be used to
design enzyme-specific inhibitors. As papain exhibits high structural simi-
larity to the catalytic domain of the T. cruzi enzyme cruzipain, the present
chagasin–papain complex provides a reliable model of chagasin–cruzipain
interactions. Such information, coupled with our identification of specifi-
city-conferring interactions, should be important for the development of
drugs for treatment of the devastating Chagas disease caused by this
parasite.
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 793
crystal structure of the enzyme, one of the first protein

structures to be determined [4]. Since then, papain has
been used as a model protein in many studies, and is
the founding member of the large C1 family of
papain-like cysteine proteases [5]. Approximately 12
mammalian cysteine proteases are evolutionarily clo-
sely related to papain and hence belong to this family
(e.g. cathepsins B, H, L, S and K). Enzymes from the
C1 family generally function in every cell as compo-
nents of the lysosomal degradation system, participat-
ing in the turnover of proteins, but, in addition, have
been shown to participate in a number of specialized
functions, such as proteolytic cleavages activating pro-
hormones, regulation of antigen presentation, etc. C1
family proteases are evolutionarily old, are found in
both prokaryotic and eukaryotic organisms, and in
many cases show activity that is indispensable for the
organism. The unicellular parasite Trypanosoma cruzi
is an example of such an organism, in which the
papain-like enzyme, cruzipain, is essential for the life-
cycle of the parasite and also acts as a virulence factor
when the parasite infects its human host, causing the
devastating Chagas disease [6,7].
In a variety of species, from mammals, plants and
insects to simpler eukaryotes such as the filarial
parasites Onchocerca volvulus and Acanthocheilonema
viteae, C1 family cysteine proteases are in equilibrium
with protein inhibitors belonging to the cystatin fam-
ily, I25 [5,8–10]. Most cystatins, such as human cysta-
tin B, are single-domain proteins of 100–120 residues
with a characteristic wedge-like epitope consisting of

the N-terminus and two b-hairpin loops, which blocks
the active site cleft of the target enzyme, thereby inhib-
iting the activity in a reversible manner [11,12]. Cysta-
tins show high affinity for their target enzymes due to
a large binding area, with dissociation constants (K
i
)in
the range 10
)9
–10
)11
m. In extreme cases, such as the
human cystatin C–papain complex, K
i
values as low as
10
)14
m have been reported [13].
Trypanosomatids, such as various Trypanosoma and
Leishmania species, produce inhibitors of their own
family C1 proteases [14]. Chagasin, a tight-binding
inhibitor of cruzipain found in T. cruzi [15], exhibits
no sequence similarity with cystatins (GenBank ⁄ EMBL
[16] accession number AJ299433), despite its similar
size (110 residues). Molecular modeling studies pre-
dicted an immunoglobulin-like fold for chagasin [17],
which was essentially confirmed by subsequent NMR
[18] and crystallographic studies [19,20]. Recently, crys-
tal structures of chagasin in complex with human cath-
epsins L and B [20,21], and additionally with falcipain

from the malaria parasite [22], have been determined.
The complex structures demonstrate that the enzyme-
binding epitope of chagasin consists of three loops
(L4, L2, L6) that together form a wedge-like enzyme-
binding epitope.
In this study, we present a high-quality crystal
structure of chagasin in complex with papain, the
model C1 family cysteine protease and one of only
two enzymes in the family for which structural infor-
mation for a cystatin complex is available [23,24].
Based on the amino acid sequence and structure-based
alignment, papain has been shown to be a close
homolog of cruzipain [25]. Our results confirm map-
ping of the enzyme-binding epitope to the three loops,
as in chagasin complexes with mammalian enzymes,
and illustrate the degree of structural adjustments as
well as precise atomic contacts formed during enzyme
binding. Moreover, comparative analysis of several
chagasin complexes has revealed a strikingly similar
core structure involved in enzyme binding, which
results in sub-nanomolar K
i
values and rate constants
for inactivation in the 10
5
–10
6
m
)1
Æs

)1
range in all
cases. Additionally, several contacts unique to the
individual enzyme complexes could be identified, rais-
ing the prospect of accurate structure-aided design of
specific inhibitors of cruzipain and cathepsins.
Detailed knowledge of the structure and inhibition
mode of chagasin should be valuable in guiding the
development of drugs for the prevention and treat-
ment of Chagas disease.
Results
Function of chagasin as an inhibitor of papain
Chagasin used in this study was expressed in Escheri-
chia coli and purified to homogeneity as reported
previously [20]. The recombinant protein contains five
extra N-terminal amino acid residues from the expres-
sion construct, and has a mass of 12 440 Da as
expected [20]. The protein shows almost 100% activity
as a protease inhibitor based on titration of a papain
solution with known activity, forms stoichiometric
1 : 1 complexes with cathepsin L or B, and is not
cleaved by these proteases [20,21].
Kinetic parameters for the interaction of chagasin
with papain at pH 6.0 were determined in a continuous-
rate assay using the sensitive fluorogenic substrate car-
boxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide, with
a sufficiently high inhibitor concentration for the
binding reaction to be of pseudo-first order. The k
on
value was determined to be 1.5 · 10

6
m
)1
Æs
)1
, very simi-
lar to that determined for cathepsin L and higher than
that for cathepsin B under the same conditions
(Table 1). The equilibrium constant for dissociation
Chagasin–papain complex structure I. Redzynia et al.
794 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS
(K
i
) of the chagasin–papain complex was calculated
from the results of similar assays, under conditions
when steady-state enzyme rates could be determined
before and after addition of chagasin to a specific con-
centration. The K
i
value for the papain–chagasin com-
plex, corrected for substrate competition in the assays,
was estimated as 36 pm, again similar to that of cathep-
sin L [20] and significantly lower than the values for
wild-type cathepsin B or for a cathepsin B variant with
an H110A substitution in the occluding loop, for which
the structure of its chagasin complex is known [21]
(Table 1).
Crystallization and structure determination
A complex between chagasin and papain was formed
by incubating the proteins in a 1.3 : 1 molar ratio for

approximately 4 h before setting up crystallization
drops. Single crystals of the chagasin–papain complex
were obtained using Crystal Screen II in Hepes buffer
at pH 7.5 without further optimization. The crystal
structure of the complex was solved to 1.86 A
˚
resolu-
tion by molecular replacement using the chagasin–
cathepsin L model (PDB code 2NQD) [20] as a probe.
The initial atomic coordinates of the chagasin–papain
complex were obtained by rigid-body substitution of
cathepsin L by a papain model (PDB code 1KHQ)
[26]. After least-squares refinement, the main-chain
traces of the chagasin and papain molecules were visi-
ble in 2F
o
–F
c
electron density maps without breaks at
the 1.7 r level, except for the N- and C-termini of the
chagasin molecule. All side chains, as well as both ter-
minal segments, are clearly visible when the electron
density maps are contoured at the 1.0 r level. The
GPLGS peptide introduced as an N-terminal extension
of the recombinant chagasin is totally disordered and
not visible in the electron density maps. In addition to
298 water molecules, the model includes 10 formate
ions from the crystallization buffer. The refinement
statistics are presented in Table 2. The residues of the
inhibitor are labeled without a chain designator. The

residues of the enzyme are marked ‘e’. When cystatin
sequences are discussed in this paper, amino acid num-
bering according to the chicken cystatin sequence is
used, as in the original papain–cystatin B structure
[23]. To convert to human cystatin C numbering,
Table 1. Function of chagasin as an inhibitor of papain and other
C1 family enzymes. Equilibrium constants for dissociation (K
i
)of
chagasin–papain complexes were determined under steady-state
conditions at pH 6.0 as described in Experimental procedures. Cor-
responding values for the papain-like cysteine proteases cathep-
sin L, cathepsin B and falcipain, with known inhibitor complex
structures [20–22], as well as for the papain complex with human
cystatin B [12], are included for comparison. The K
i
values pre-
sented were corrected for substrate competition in the assays, as
described in Experimental procedures. ND, not determined.
Enzyme
K
i
(nM) k
on
(M
)1
Æs
)1
)
Chagasin Cystatin B Chagasin

Cathepsin L 0.039 ND 2.5 · 10
6
Cathepsin B 0.93 16 8 · 10
4
H110A cathepsin B 0.35 ND 5 · 10
5
Papain 0.036 0.034 1.5 · 10
6
Falcipain 1.7
a
ND ND
Cruzipain 0.018
b
ND ND
a
Determined under slightly different assay conditions than in the
present study [22].
b
Determined for a recombinant variant of
chagasin with a 16 residue N-terminal extension [15].
Table 2. Data collection and structure refinement statistics.
Data collection
Radiation source X13, EMBL Hamburg
Wavelength (A
˚
) 0.8086
Temperature of measurements (K) 100
Space group I422
Cell parameters (A
˚

) a = 99.1, c = 159.5
Resolution range (A
˚
) 60.0–1.86 (1.93–1.86)
a
Reflections collected 350 034
Unique reflections 33 263
Completeness (%) 98.4 (88.8)
Redundancy 10.5 (6.8)
<I> ⁄ <rI> 22.2 (2.1)
R
int
b
0.090 (0.557)
R
pim
c
0.028 (0.191)
Refinement
Number of reflections in the
working ⁄ test sets
31 568 ⁄ 1694
R
d
⁄ R
free
(%) 16.4 ⁄ 20.8
Number of atoms
(protein ⁄ solvent ⁄ Zn ⁄ other)
rms deviations from ideal

2561 ⁄ 298 ⁄ 1 ⁄ 30
Bond lengths (A
˚
) 0.017
Bond angles (°) 1.61
<B>(A
˚
2
) 27.6
Residues in Ramachandran plot (%)
Most favored regions 89.7 (98.1)
e
Allowed regions 10.3
PDB code 3E1Z
a
Values in parentheses correspond to the last resolution shell.
b
R
int
=
P
h
P
j
| I
hj
)<I
h
>| ⁄
P

h
P
j
I
hj
, where I
hj
is the intensity of
observation j of reflection h.
c
R
pim
=
P
h
(1 ⁄ n
h
)1)
P
j
|I
hj
)<I
h
>| ⁄
P
h
P
j
<I

hj
> [42], calculated using SCALA [43] (from data
processed using Denzo).
d
R =
P
h
||F
o
|)|F
c
|| ⁄
P
h
|F
o
| for
all reflections, where F
o
and F
c
are observed and calculated
structure factors, respectively. R
free
is calculated analogously for
the test reflections, randomly selected and excluded from the
refinement.
e
Ramachandran ‘favored’ region, as defined by
MolProbity [50].

I. Redzynia et al. Chagasin–papain complex structure
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 795
which is widely used, ‘2’ should be added to all residue
numbers, so that G9 in cystatin B corresponds to G11
in cystatin C [27].
A strong residual peak in the F
o
–F
c
electron density
map, in close proximity to H72, H74, E23 and one of
the formate ions, was interpreted as a zinc cation. This
interpretation is supported by the bond valence test
[28,29] and by the tetrahedral coordination of this
cation. Although chagasin inhibition is not dependent
on any cofactors, this site at the surface of the mole-
cule may be of structural significance, as the same his-
tidine residues in the cathepsin B complex were found
to bind a phosphate ion [21].
The chagasin–papain interface
The papain chain in the present complex starts with
residue I1e, which is well defined in the electron den-
sity map. The last residue, N212e, is also clearly visible
because the side chain is stabilized by hydrogen bonds
with D108e and I148e, and the C-terminal carboxylate
forms a salt bridge with R188e, the latter two inter-
actions involving a symmetry-related molecule. The
enzyme used for crystallization was in an inactive
form, with the catalytic C25e residue protected by
carboxymethylation. The blocking group is clearly

visible in the electron density maps.
The overall conformation of the chagasin molecule
in the present complex is similar to that found for free
chagasin (PDB code 2NNR) [20]. The C- and N-termi-
nal residues of chagasin are somewhat flexible, but the
contour level of 1 r for the 2F
o
–F
c
electron density
maps was sufficient for unambiguous modeling. The
first visible residue at the N-terminus is S2, which is
anchored by a side-chain hydrogen bond to N64e from
a symmetry-related molecule. The C-terminal N110
residue points to a water channel.
In overall shape, the present complex is similar to
the previously described complex structures of chaga-
sin with cathepsins L and B [20,21], resembling an
inverted mushroom, with the stalk formed by the
cylindrical chagasin molecule and the cap by the glob-
ular papain (Fig. 1). The C25e-H159e-N175e catalytic
triad of papain is located at the bottom of a long cleft
running across the width of the molecule, dividing it
into the L and R domains [30].
The binding region of chagasin formed by the loops
L2 (N29–F34), L4 (P59–G68) and L6 (R91–S100) is
docked very tightly to the papain molecule (Fig. 2A).
The main hydrogen bonds between chagasin and
papain observed in the complex are listed in Table S1.
All three loops are located in the catalytic groove, with

the 3
10
tip of loop L2 inserted directly into the cata-
lytic center. Loops L4 and L6 embrace the enzyme
molecule from both sides.
The interactions of each loop have different charac-
teristics. Loop L6 forms three types of interactions
with the enzyme: hydrogen bonds (R91), hydrophobic
contacts (P92) and p interactions (W93), which ‘probe’
different elements of the catalytic apparatus. First,
W93 interacts with a cluster of aromatic residues
(F141e, W177e, W181e) that serve to position the
N175e element of the catalytic triad (C25e-H159e-
N175e) through N-H p hydrogen bonds. R91 assumes
a fully extended conformation reaching to the catalytic
site of the enzyme and loop L2 of chagasin. The R91
guanidinium group forms two hydrogen bonds with
the carbonyl group of T32 in loop L2, which is located
next to the active-site-blocking residue, T31 [20,21].
The other segment of the guanidinium group of R91
forms a pair of hydrogen bonds with the oxygen atom
of the side-chain amide group of N18e. It is interesting
to note that the equivalent position in cruzipain is
occupied by an aspartate, making the interaction with
Fig. 1. Stereoview of the chagasin–papain
complex. The chagasin molecule is colored
green and papain is colored pink. The
surfaces of both proteins are marked
correspondingly. The view is along the
catalytic cleft of papain and corresponds to

the standard orientation used for cysteine
proteases, with the L and R lobes on the
left and right, respectively.
Chagasin–papain complex structure I. Redzynia et al.
796 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS
R91 even stronger. Finally, the guanidinium group of
R91 is also hydrogen-bonded to the carbonyl group of
G20e. The third element of L6, P92, shapes the loop
for optimal interactions with the enzyme by forming
hydrophobic contacts with the side chain of L143e
(Fig. 2B). In addition to the direct interactions of loop
L6 described above, there are also contacts mediated
by water molecules.
The interactions of loop L4 with the enzyme are
based on formation of an antiparallel intermolecular
b-sheet. Two residues from chagasin, G66 and L65,
interact with the papain main-chain atoms N64e–G66e
(Fig. 2C). In addition, the side-chain carbonyl Od1
atom of N64e forms a water-mediated contact with
the main-chain N atom of G68, and the main-chain
nitrogen of G66 of chagasin forms a water-mediated
A
B
C
D
Fig. 2. Interactions of chagasin and cystatin B with papain. Color coding: chagasin (green)–papain (pink); cystatin B (brown)–papain (gray).
(A) Stereoview of aligned molecules created by superposition of the Ca atoms of papain from the crystal structures of its complexes with
cystatin B (PDB code 1STF) and chagasin (this work). The upper panel emphasizes the different angle of approach of the two inhibitors in
the standard orientation of papain. The lower panel, rotated by 90° (papain R domain at the front) emphasizes the similar shape of inhibitory
elements (loops and the cystatin B N-terminus). (B) Zoom-in view of the interactions of papain with loop L6 of chagasin and loop L2 of cysta-

tin B. (C) Zoom-in view of the interactions of papain with loop L4 of chagasin and the N-terminal segment of cystatin B. (D) Zoom-in view of
the interactions of papain with loop L2 of chagasin and loop L1 of cystatin B.
I. Redzynia et al. Chagasin–papain complex structure
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 797
contact with the main-chain carbonyl group of D158e
from papain.
Compared to the very strong and extended interac-
tions of loops L4 and L6, the interactions of loop L2
are very limited. A repulsive contact is seen between
the carbonyl O atom of T31 and the Nd1 atom of the
imidazole ring of the catalytic H159e residue. A much
longer, attractive contact exists between the same T31
carbonyl and the Ne1 atom of W177e (Table S1). The
hydroxyl group of T31 interacts with the main-chain
carbonyl of D158e (Fig. 2D). The four additional
atoms of the carboxymethyl modification of the cata-
lytic C25e residue are easily accommodated at the
inhibitor–enzyme interface. The oxygen atoms of
the carboxymethyl block form contacts with both the
enzyme (main-chain N of C25e and side chain of
Q19e) and the inhibitor (OH group of T31).
The inhibition mode of chagasin
The best-studied group of cysteine protease inhibitors
are the cystatins, which are small proteins with a
molecular mass of 11–14 kDa [27]. The structure of
papain in complex with cystatin B (PDB code 1STF)
[23] offers an excellent opportunity for comparison of
the mode of interaction of the two very different inhib-
itors with the same target enzyme.
Although chagasin and cystatin B show essentially

identical affinity for papain (Table 1), superposition of
the two complexes based on Ca alignment [31] of the
enzyme portions shows a completely different fold for
the two inhibitors (Fig. 2A). The characteristic b-sheet
grip around a long a-helix, characteristic of cystatins,
contrasts with the all-b structure of chagasin.
However, despite their different overall fold, the
epitope presented by both inhibitors to the enzyme is
arranged similarly. The L4–L2–L6 wedge of chagasin
overlaps with a similar wedge of cystatin B formed by
the N-terminal segment and two b-hairpin loops, L1
and L2 (Fig. 2A–D). This similarity does not extend
beyond the active site, and, in fact, the two molecules
approach the enzyme from a different angle. We have
defined the angle of approach, s (Table 3), as the
dihedral angle between two planes, one (a) dividing the
Table 3. Comparison of various enzyme complexes of chagasin. The superpositions of Ca atoms were calculated using ALIGN [31] for the
entire complex (c), for the enzyme molecule only (e), and for the chagasin molecule only (ch). Each superposition is characterized by the root
mean square (rms) deviation in A
˚
and the number of aligned Ca atoms (in parentheses). For comparison, superpositions with the crystallo-
graphic models of cruzipain and free chagasin (molecules A and B) are also included. Where appropriate, a number in square brackets shows
the level of sequence identity (%) between the compared enzymes. The last two rows characterize the chagasin complexes by giving the
contact area (in A
˚
2
) calculated using Areaimol [32] and by specifying the angle of inhibitor approach s (in degrees) relative to the enzyme
framework (see definition in the text).
Chagasin–cathepsin B
Chagasin–cathepsin L Chagasin–papain Chagasin–falcipainForm I Form II

Cruzipain 1.38 (198) 1.46 (197)
[27.8%]
0.81 (190)
[43.4%]
1.07 (191)
[35.8%]
1.10 (192)
[37.7%]
Chagasin
A 0.44 (98) 0.55 (101) 0.48 (100) 0.44 (92) 0.38 (99)
B 0.37 (98) 0.54 (105) 0.52 (103) 0.35 (91) 0.37 (102)
Chagasin–cathepsin B
Form I c 1.16 (348)
e 0.54 (233)
ch 0.46 (100)
c 1.25 (301)
e 1.28 (198)
ch 0.43 (102)
c 1.15 (287)
e 1.32 (192)
ch 0.43 (96)
c 1.15 (294)
e 1.30 (193)
ch 0.37 (101)
Form II c 1.55 (300)
e 1.31 (196)
ch 0.55 (107)
[28.2%]
c 1.10 (278)
e 1.42 (191)

ch 0.42 (101)
[29.7%]
c 1.19 (293)
e 1.35 (190)
ch 0.52 (107)
[24.1%]
Chagasin–cathepsin L c 1.18 (297)
e 0.79 (188)
ch 0.61 (101)
[40.6%]
c 1.19 (300)
e 1.00 (189)
ch 0.44 (105)
[35.9%]
Chagasin–papain c 0.87 (274)
e 1.18 (187)
ch 0.34 (93)
[37.7%]
Contact area 1221 1373 972 922 984
Angle of approach s 7.2 2.3 11.3 5.8 5.4
Chagasin–papain complex structure I. Redzynia et al.
798 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS
enzyme into the R and L lobes along the catalytic
groove, defined by the Ca atoms of three papain resi-
dues, I40e, Y67e and W177e (or their equivalents in
other enzymes), and the other (b) created by three Ca
atoms defining the inhibitor framework and passing
along the inhibitory wedge. In the case of chagasin,
plane (b) is triangulated by the tips of the peripheral
loops L4, L6 and the C-terminus, or specifically by the

Ca atoms of G66, R91 and A109. The corresponding
Ca atoms of cystatin B are located in residues G9 (in
the N-terminal binding segment, according to chicken
cystatin numbering [23]), D68 (a loop from the oppo-
site pole) and L102 (loop L2). The s angle in the
chagasin–papain complex is 5.8°, indicating that the
chagasin molecule is slanting towards the R domain.
The angle of approach of cystatin B is )12.7°, and the
inhibitor molecule is inclined towards the L domain of
the enzyme. The difference in the angles of approach
between chagasin and cystatin B is 18.5°. It is also of
note that the sequential epitope of cystatins corre-
sponds to a non-sequential binding site of chagasin.
The contact area [32] is similar for both complexes,
and is 853 and 922 A
˚
2
for the cystatin B–papain and
chagasin–papain complexes, respectively.
The three crucial residues of loop L6 of chagasin
(R91, P92 and W93) correspond to L102, P103 and
H104, respectively, in the cystatin B molecule
(Fig. 3A). It is noteworthy that the pattern Pro–aro-
matic residue is conserved in chagasin-like inhibitors
and in cystatins (where it is predominantly PW),
despite the lack of overall sequence similarity. The
role of the proline residue appears to be to maintain
the specific shape of the loop. The aromatic residue,
on the other hand, interacts with the aromatic clus-
ter of the enzyme (Fig. 2B). The residue preceding

the Pro–aromatic motif, which is invariably an argi-
nine in chagasin-type inhibitors of protozoan origin,
is replaced by an aliphatic residue in cystatins
(Fig. 3A). This difference may be an important ele-
ment regulating the enzyme specificity of these two
groups of inhibitors. The R91 residue of chagasin
provides direct communication between loops L6 and
L2, and also interacts with the crucial D18e⁄ N18e
residues of cruzipain ⁄ papain. The role of the L102
residue of cystatin B is different, and supports inter-
action with the aromatic cluster of the enzyme
(Fig. 2B). An additional interaction between loops
L2 and L6 of chagasin is provided by the carbonyl
group of the main chain of M90 and the nitrogen
atom of A35. A similar stabilizing contact between
cystatin B loops L2 and L1 is formed by the main-
chain carbonyl of Q101 and the peptide nitrogen
atom of T58.
The interaction of loop L4 of chagasin with papain
is based on formation of an intermolecular b-sheet
(Fig. 2C). There is an analogous interaction between
the N-terminus of cystatin B and papain. G9 from the
N-terminal cystatin B segment and G66 from loop L4
of chagasin provide a degree of flexibility, thus allow-
ing optimal interactions between the two main chains.
The same role is played by G65e of papain. The short
antiparallel b-sheet interaction is formed by only one
residue, G66e, of papain with L65 or S66 of chagasin
or cystatin B, respectively. This antiparallel interaction
is supported by a water molecule linking the N atom

of G66 of chagasin and the main-chain O atom of
D158e of papain. In the cystatin B complex, an equiv-
alent carbonyl is involved in a water-mediated interac-
tion with the N atom of A10.
The L2 loop of chagasin and the corresponding loop
L1 of cystatin B interact with the catalytic center of
papain (Fig. 2D). Our structural alignment (Fig. 3A)
shows that loop L2 of chagasin is one residue longer,
and thus T31 has no equivalent in loop L1 of cysta-
tin B. Loop L2 of chagasin not only interacts with
loop L6 but also with loop L4, by forming a hydrogen
bond between the side-chain amide of N29 and main-
chain carbonyl of G66. A similar interaction is
observed in cystatin B, where the side-chain amide of
Q53 forms a hydrogen bond with the main-chain car-
bonyl of G9, stabilizing the interaction between loop
L1 and the N-terminus. Although the conformation of
these two loops is somewhat different, in both cases
they have the same, substrate-like, polarity. There is a
surprisingly small number of specific interactions with
the catalytic residues of the enzyme for both chagasin
loop L2 and cystatin loop L1, which explains why
chagasin (and also cystatins) can bind with high affin-
ity to cysteine proteases with the catalytic -SH group
protected by a carboxymethyl group. The repulsive
interactions between the chagasin loop L2 and the cat-
alytic site of papain, described above, are reproduced
in the cystatin complex.
Discussion
Comparison of the existing structures of

chagasin complexes with cysteine proteases
In addition to the chagasin–papain complex presented
in this paper, four additional crystal structures of
chagasin complexes with other cysteine proteases are
available in the Protein Data Bank. The target
enzymes for chagasin in these complexes are cath-
epsin L (PDB code 2NQD) [20], cathepsin B in two
crystal forms (PDB codes 3CBJ and 3CBK) [21] and
I. Redzynia et al. Chagasin–papain complex structure
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 799
falcipain (PDB code 2OUL) [22]. These structural data
form an excellent platform for comparison of the inter-
actions between chagasin and the targeted proteolytic
enzymes. The residues from the catalytic cleft of vari-
ous cysteine proteases that interact with chagasin are
structurally aligned (Fig. 3B). The inhibitor binds
papain and cathepsin L with essentially the same, very
high, affinity (K
i
approximately 0.03 nm); the affinity
for cathepsin B is approximately one order of magni-
tude lower, and that for falcipain is yet another order
of magnitude lower, although still in the nanomolar
range (Table 1).
The contact surface area for chagasin–cysteine pro-
tease complexes varies between 922 and 1373 A
˚
2
(Table 3), and does not directly correlate with the effi-
ciency of inhibition. The extra contact area found in

both crystal forms of the chagasin–cathepsin B com-
plex is created by the additional and unique occluding
A
B
Fig. 3. Structure-based sequence alignment
of the interacting residues of cysteine prote-
ases and their inhibitors. (A) Alignment of
structurally equivalent residues forming the
enzyme-binding epitopes of chagasin-like
(L4, L2 and L6) and cystatin-like inhibitors
(N-terminus, L1 and L2). The following
protein sequences have been used:
inhibitors, Trypanosoma cruzi (GenBank
accession number AJ299433),
Trypanosoma brucei (AJ548777),
Leishmania mexicana (AJ548776),
Leishmania major (AJ548878),
Entamoeba histolytica (AJ634054),
Pseudomonas aeruginosa (AAG04167) [53],
Gallus gallus cystatin (J05077), Homo
sapiens cystatin B (BC010532), H. sapiens
cystatin C (BC110305); proteases, Carica
papaya papain (M15203), H. sapiens cathep-
sin L (X12451), H. sapiens cathepsin B
(BC010240), Plasmodium falciparum falci-
pain (AAF97809), T. cruzi cruzipain (X54414).
(B) Alignment of structurally equivalent resi-
dues from the catalytic groove of various
cysteine proteases, based on the crystal
structures of their complexes with chagasin,

except for cruzipain, for which a complex
with a small-molecule inhibitor (PDB code
1ME3) is used. The residues crucial for
interactions with chagasin are color-coded
as yellow (catalytic triad), red (aromatic
cluster), green (residues forming hydrogen
bonds) and blue (hydrophobic contacts).
Chagasin–papain complex structure I. Redzynia et al.
800 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS
loop of this enzyme. The inhibition of cathepsin B by
chagasin is relatively weak, which may be due to the
fact that some of the binding energy has to be invested
in pushing the occluding loop out of the catalytic cleft.
The angle of approach, calculated in the way described
above, has the lowest value for the tetragonal form of
the chagasin–cathepsin B complex and the highest for
the chagasin–cathepsin L complex (Table 3). The dif-
ference of 9° between these complexes may be corre-
lated with the variation of the rate of binding and
affinity for chagasin of these enzymes. On the other
hand, in the two crystal forms of the chagasin–cathep-
sin B complex, the difference is 5°, showing that there
is some degree of variability in inhibitor–enzyme dock-
ing, resulting either from inherent freedom of move-
ment or adaptability to environmental factors, such as
crystal packing interactions.
The rms deviations for the four enzyme-bound
chagasin molecules are 0.34–0.61 A
˚
, a range that is

similar to that for comparisons of the two crystal
structures of native chagasin (0.35–0.55 A
˚
). These
results show that the chagasin molecule has a rigid
conformation and does not change upon complex for-
mation. This contradicts the conclusions drawn from
an NMR study that predicted a high level of flexibil-
ity of the chagasin molecule [18]. A superposition of
all the chagasin molecules from the complex and
native structures is shown in Fig. 4A. A different
conformation is only visible for a few N-terminal resi-
dues. Additionally, a small difference between native
and complexed chagasin is observed at loop L4,
which is rich in Gly residues, where a conformational
change is responsible for adjustment of the inhibitor
to the enzyme in the b-sheet-forming motif. The
C-terminus has a relatively stable conformation,
although the last two residues protrude from the pro-
tein surface. The C-terminal end of chagasin is a
good marker of the variable angle of approach of the
inhibitor relative to the enzyme, as illustrated in
Fig. 4B.
L4
A
B
L2
L6
L4
L2

L6
Fig. 4. Stereoview of aligned chagasin com-
plexes. (A) Superposition of the Ca atoms of
the chagasin molecules from the complexes
of papain, cathepsins L and B, and falcipain
with native chagasin. (B) Alignment of all
above chagasin complexes based on
superposition of the Ca atoms of the
enzyme components. Color code: chagasin
(green)–papain (pink) (this work), chagasin
(gold)–cathepsin L (dark blue) (PDB code
2NQD), chagasin (orange)–cathepsin B (mid-
green) (monoclinic form, 3CBJ), chagasin
(yellow)–cathepsin B (lime green) (tetragonal
form, 3CBK), chagasin (light blue)–falcipain
(gray) (2OUL). The additional two molecules
of native chagasin (2NNR) are colored dark-
green (chain A) and purple (chain B).
I. Redzynia et al. Chagasin–papain complex structure
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 801
Considering the stability of the chagasin structure,
the similarity or dissimilarity of its complexes with
various enzymes may be regarded as the result of
two factors: (a) the overall similarity of the enzy-
matic part, and (b) the variability of the angle of
approach of the inhibitor relative to the catalytic
cleft of the enzyme. The latter factor may reflect not
so much the geometry of the catalytic site itself,
which is highly conserved, but rather the general
shape of the peripheral regions surrounding the

active site of the enzymes, which may guide the
inhibitor molecule during its docking. The data in
Table 3 show that the Ca traces of cathepsin L,
papain, falcipain and cruzipain have rms deviations
in the range 0.79–1.18 A
˚
. Much higher deviations are
observed for cathepsin B (1.28–1.42 A
˚
), in agreement
with the view that it is the most unique member of
this group of enzymes.
Although the complexes include a variety of enzyme
sequences and differ in the angle of inhibitor
approach, they have a relatively similar shape; the rms
deviations for the entire complexes range from 0.87 A
˚
for the chagasin–papain ⁄ chagasin–falcipain pair, to
1.55 A
˚
for the superposition of chagasin complexes
with cathepsins L and B.
Core structural elements explaining the broad
inhibition profile of chagasin
Chagasin displays a broadly-reactive inhibition profile,
and inhibits all the investigated C1 family proteases.
This efficient binding is achieved despite some differ-
ences in the architecture of the active site clefts of the
enzymes, which are especially evident for cathepsin B
[21]. What are the principal elements utilized by chaga-

sin that enable it to become such a broadly-reactive
inhibitor? Correct identification of these core elements
would be useful for guiding the rational design of
efficient cysteine protease inhibitors.
In all the presented structures, the inhibitory loops
creating the enzyme-binding epitope have the same
architecture except for loop L6 in the C-terminal frag-
ment from the chagasin–papain complex, which adopts
a slightly different conformation in comparison with
the other structures. The different shape of this loop is
caused by formation of a hydrogen bond between the
side chain of D99 and the main-chain N atom of S21e
(Fig. 2B).
Residue R91 of loop L6 forms important hydrogen
bonds in all chagasin complexes, both with the N⁄ D
residue at position 18e (papain numbering) and the
G ⁄ K residue at position 20e (Fig. 3B), and is an
important core elements explaining the broad inhibi-
tion profile of chagasin. The crucial aromatic W ⁄ F
residue at position 93 of chagasin is conserved as
W ⁄ H in cystatins. This residue interacts with the
aromatic cluster that is present in all cysteine prote-
ases as an extension of the catalytic triad. The pro-
line residue at position 92 in chagasin, which is
responsible for the shape of the L6 loop, is also con-
served in cystatins.
The shape of loop L4 is very similar in all com-
plex structures. Conserved interactions formed by
loop L4 include those of residues L64–A67, which
participate in both the antiparallel intermolecular

b-sheet and hydrophobic contacts with Y67e and
P68e (Fig. 3B).
Although papain, cathepsin L and cruzipain show
moderate sequence identity (36–43%), the residues
responsible for the interaction with chagasin in the cat-
alytic groove are conserved. Chagasin thus utilizes a
few conserved residues in the active site cleft of C1
family enzymes to become a broadly-reactive inhibitor
with quite similar affinity for all these enzymes. From
a biological perspective, it appears that these residues
in C1 family enzymes have been conserved to allow
binding by chagasin- or cystatin-type inhibitors, result-
ing in a means by which the organism can regulate
cysteine protease activity as required.
Enzyme-specific interactions of chagasin
Chagasin also utilizes some enzyme-specific interac-
tions, explaining why it binds more tightly to papain,
cathepsin L and cruzipain than to cathepsin B and
falcipain. Identification of these interactions is now
possible based on structural and functional data.
Detailed comparison of the various complexes reveals
a few contacts, all positioned close to the consensus
elements of the L4–L2–L6 loops, that are unique to
each of the papain, cathepsin L and cathepsin B
complexes (Fig. 3B). For papain, this is the contact
of S21e, for cathepsin L the contacts of Y72e and
E141e, and for cathepsin B the contacts of E194e
and D224e with chagasin residues boxed in Fig. 3B.
The role of the conserved residues indicated by our
structural data is consistent with published mutagene-

sis studies [33]. The identified characteristic interac-
tions appear promising for use when designing
specific inhibitors to a particular enzyme. A struc-
ture-based sequence alignment of the residues from
the catalytic groove of cysteine proteases that inter-
act with chagasin is shown in Fig. 3B. These residues
are also preserved in cruzipain, which justifies our
suggestion that these interactions are also maintained
in a chagasin–cruzipain complex.
Chagasin–papain complex structure I. Redzynia et al.
802 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS
Interaction of chagasin with cruzipain
The crystal structure of the possibly most physiologi-
cally relevant chagasin complex, that with the T. cruzi
enzyme cruzipain, has not yet been presented. How-
ever, the available models can be used to predict the
interactions of the parasite protease–inhibitor pair. The
rms deviations for the superimposed known structures
are presented in Table 3. Additionally, Table 3 com-
pares the inhibitor part of each complex with unbound
chagasin (PDB code 2NNR) [20] and cruzipain (PDB
code 1ME3) [34] with the enzyme portions of the com-
plexes. A graphical illustration of the aligned complexes
based on enzyme superposition is presented in Fig. 4B.
Three recently published studies [18,20,21] have
attempted to model the interactions between chagasin
and cruzipain so as to elucidate the binding
interactions utilized in order to achieve efficient
enzyme inhibition. These previous conclusions are
corroborated by superposition of the cruzipain model

on the papain portion of the complex described in the
present study.
Conclusions
The chagasin–papain complex presented here is the
third high-resolution structure for the T. cruzi inhibitor
chagasin in a cysteine protease complex, completing a
series that started with the human C1 family enzymes
cathepsins L and B [20,21]. Together with detailed
functional studies of chagasin binding to the three
target enzymes, the results provide a platform for a
thorough understanding of the basic elements required
for efficient inhibition by this parasite protease inhibi-
tor and explain its broadly-reactive properties. The
core determinants of the broad reactivity of chagasin
appear to be based on a rigid protein structure, opti-
mally presenting an epitope formed by three loops, L4,
L2 and L6, which precisely match their interaction
partners in the substrate-binding cleft of the C1 family
enzymes. Smaller adaptations, connected with adjust-
ment of the angle of chagasin approach during enzyme
docking, optimize the interactions of loops L4 and L6
at the periphery of the enzyme-binding epitope with
the distal elements in the enzyme’s active-site clefts
[21,35], to create a large contact area (922 A
˚
2
), result-
ing in sub-nanomolar K
i
values and rate constants for

inactivation in the 10
5
–10
6
m
)1
Æs
)1
range. A few unique
interatomic interactions in each complex could
additionally be identified and are interpreted as further
improving the binding efficiency to papain and cathep-
sin L versus the other enzymes, with the hydrogen
bond between D99 in loop L6 of chagasin and S21e of
papain providing an illustrative example. A compari-
son of the present chagasin–papain complex with the
structure of papain in complex with the human inhibi-
tor cystatin B reveals that, despite their entirely differ-
ent folds, the two inhibitors utilize very similar atomic
interactions for papain binding, leading to essentially
identical affinities for the enzyme. As papain shows a
high structural similarity among the C1 family prote-
ases to the catalytic domain of the T. cruzi enzyme cru-
zipain, the present chagasin–papain complex structure
provides a reliable starting point for a model of the
interactions of the two T. cruzi proteins, which,
together with our identification of the specificity-con-
ferring interactions, will be important for development
of drugs for the prevention and treatment of Chagas
disease.

Experimental procedures
Expression, purification and characterization of
recombinant chagasin
Chagasin was produced using a glutathione S-transferase
gene fusion protein system with the vector pGEX-6P-1
(Amersham Biosciences, Uppsala, Sweden), as described
previously [20]. Following purification to homogeneity, the
chagasin solution was concentrated using a Vivaspin col-
umn with a cut-off limit of 5 kDa (Vivascience, Lincoln,
UK) to a final concentration of 5 mgÆmL
)1
. The protein
was characterized as correctly expressed GLPGS-chagasin
by mass spectrometry, N-terminal protein sequencing and
electrophoretic analyses [20].
Preparation of papain
Active papain was prepared from the commercial papaya
latex enzyme (Sigma-Aldrich, St Louis, MO, USA) through
affinity purification on Sepharose 4B (GE Healthcare Bio-
Sciences AB, Uppsala, Sweden), to which Gly-Gly-Tyr-Arg
had been covalently coupled, as described previously [36,37].
Purified this way, the enzyme could be activated to at least
65% of its original activity after storage at )80 °C for up to
3 months.
Protein analyses
Protein concentration was estimated using a Coomassie
protein assay (Pierce Biotechnology Inc., Rockford, IL,
USA). N-terminal sequencing was performed after electro-
phoresis in agarose gels, blotting to a poly(vinylidene diflu-
oride) membrane (Millipore, Bedford, MA, USA) and

staining with 0.05% Coomassie blue. Edman degradation
was performed using 470A gas–liquid solid-phase seq-
uencer (Applied Biosystems, Foster City, CA, USA) at the
I. Redzynia et al. Chagasin–papain complex structure
FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 803
Department of Clinical Chemistry, Malmo
¨
University Hos-
pital, Sweden. MALDI-TOF mass spectrometry analysis
using a Reflex III mass spectrometer (Bruker Daltonics
Inc., Billerica, MA, USA) was used to verify the correct
mass of recombinant chagasin as described previously [38].
Enzyme inhibition assays
Active-site titration of papain [with E-64 (l-trans-epoxy-
succinyl-leucylamido-(4-guanidino)butane), using Bz-dl-
Arg-NHPhNO
2
(a-N-benzoyl-dl-arginine p-nitroanilide) as
substrate; Bachem Feinchemikalien, Bubendorf, Switzerland]
and titration of the molar papain-inhibitory concentration
of chagasin were accomplished as described previously for
cystatin analysis [39]. Active inhibitor concentrations deter-
mined in this way were used for calculation of the K
i
values.
The fluorogenic substrate used for determination of equilib-
rium constants for dissociation (K
i
) of complexes between
chagasin and papain or other family C1 cysteine peptidases

was carboxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide
(10 mm; Bachem) and the assay medium was 100 mm sodium
phosphate buffer, pH 6.0, containing 1 mm dithiothreitol
and 1 mm EDTA. Steady-state velocities were measured
before and after addition of varying amounts of chagasin,
and the K
i
values were calculated as described previously
[40]. Corrections for substrate competition were made using
K
m
values determined for the substrate batch used, under the
assay conditions employed (60, 55, 3.2 and 1.9 lm for
papain, cathepsin B, cathepsin L and cruzipain, respec-
tively). To determine the association rate constant for the
chagasin–papain interaction, the pseudo-first-order rate
constants (k
obs
) in continuous-rate assays with various con-
centrations of chagasin were determined by non-linear
regression. The association rate constant (k
on
) was then
calculated from the slope of a plot of k
obs
versus inhibitor
concentration.
Crystallization
All crystallization experiments were performed at 18 °C
using the hanging-drop vapor diffusion method and Crystal

Screens I and II and PEG ⁄ Ion Screen from Hampton
Research (Aliso Viejo, CA, USA). The crystals of the
chagasin–papain complex used for diffraction experiments
were grown by mixing chagasin at 4 mgÆmL
)1
concentra-
tion with papain at 0.7 mgÆ mL
)1
concentration in a 1.3 : 1
molar ratio. The complex was incubated for approximately
4 h. Drops from a 1.5 lL protein solution and 1 lL of pre-
cipitant solution containing 2.0 m ammonium formate and
0.1 m Hepes, pH 7.5, were mixed. Single crystals of the
complex in the form of thin square plates reached maxi-
mum dimensions of 0.16 · 0.16 · 0.01 mm in approxi-
mately 30 days. A mixture of the well solution with 50%
v ⁄ v poly(ethylene glycol) (PEG 400) in a 1 : 1 volume ratio
was used as cryoprotectant.
X-ray data collection and analysis
X-ray diffraction experiments were performed at 100 K
using the X13 EMBL beamline of the DESY synchrotron
(Hamburg, Germany). Diffraction data extending to 1.86 A
˚
resolution were indexed, integrated and scaled using Denzo
and Scalepack from the HKL program package [41]. Addi-
tionally, R
pim
[42] was calculated using SCALA [43].
Table 2 shows the data collection and processing statistics.
Structure solution and refinement

The structure of the complex was solved by molecular
replacement. An initial molecular-replacement solution was
obtained using the chagasin–cathepsin L complex (PDB
code 2NQD) as the search model in MolRep [44]. The
complete model of the chagasin–papain complex was
obtained by superposition of papain (PDB code 2KHQ) on
the oriented cathepsin L portion as the target. Manual
model rebuilding was subsequently performed using coot
[45]. refmac5 [46] was used for structure refinement with
TLS (translation, libration, screw motion – parameters
describing anisotropic motion of rigid body molecules) [47]
parameters defined separately for papain and chagasin.
Water molecules were introduced manually using coot [45].
R
free
[48] was monitored using a randomly chosen subset of
reflections comprising 5% of the unique data set. Side
chains of a number of residues were modeled in two con-
formations. The quality of the final structure was assessed
using procheck [49] and molprobity [50]. The final refine-
ment statistics are shown in Table 2. All crystallographic
calculations were performed using the CCP4 suite of pro-
grams [51]. Molecular illustrations were prepared using
pymol [52].
Acknowledgements
This work was supported in part by grant N-N404-
2348-33 from the Polish Ministry of Science and
Higher Education to G. B. The work in Lund was sup-
ported by grants to M. A. from the Crafoord and
A. O

¨
sterlund Foundations, the Swedish Research
Council (project number 09915), the Swedish Cancer
Society (project number 07 0030) and the Research
School in Pharmaceutical Chemistry (‘FLA
¨
K’), Lund
University, Sweden. This paper is dedicated to Profes-
sor Zofia Kosturkiewicz on the occasion of her 80th
birthday.
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Supporting information
The following supplementary material is available:
Table S1. Hydrogen bonds between chagasin and

papain in the complex, with donor – acceptor distances
in A
˚
.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
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sponding author for the article.
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806 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS