Báo cáo khóa học: The structure–function relationship in the clostripain family of peptidases potx

The structure–function relationship in the clostripain family
of peptidases
Nikolaos E. Labrou
1
and Daniel J. Rigden
2
1
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece;
2
School of Biological Sciences, University of Liverpool, UK
In this study we investigate the active-site structure and the
catalytic mechanism of clostripain by using a combination
of three separate techniques: affinity labelling, site-directed
mutagenesis and molecular modelling. A benzamidinyl-
diazo dichlorotriazine dye (BDD) was shown to act as an
efficient active site-directed affinity label for Clostridium
histolyticum clostripain. The enzyme, upon incubation with
BDD in 0.1
M
Hepes/NaOH buffer pH 7.6, exhibits a time-
dependent loss of activity. The rate of inactivation exhibits a
nonlinear dependence on the BDD concentration, which can
be described by reversible binding of dye to the enzyme prior
to the irreversible reaction. The dissociation constant of
the reversible formation of an enzyme–BDD complex is
K
D
¼ 74.6 ± 2.1 l
M
and the maximal rate constant of
inactivation is k

3
¼ 0.21Æmin
)1
. Effective protection against
inactivation by BDD is provided by the substrate N-benzoyl-
L
-arginine ethyl ester (BAEE). Cleavage of BDD-modified
enzyme with trypsin and subsequent separation of peptides
by reverse-phase HPLC gave only one modified peptide.
Amino acid sequencing of the modified tryptic peptide
revealed the target site of BDD reaction to be His176. Site-
directed mutagenesis was used to study further the func-
tional role of His176. The mutant His176Ala enzyme
exhibited zero activity against BAEE. Together with previ-
ous data, these results confirm that a catalytic dyad of
His176 and Cys231 is responsible for cysteine peptidase
activity in the C11 peptidase family. A molecular model of
the catalytic domain of clostripain was constructed using a
manually extended fold recognition-derived alignment with
caspases. A rigorous iterative modelling scheme resulted
in an objectively sound model which points to Asp229 as
responsible for defining the strong substrate specificity for
Arg at the P1 position. Two possible binding sites for the
calcium required for auto-activation could be located.
Database searches show that clostripain homologues are not
confined to bacterial lineages and reveal an intriguing variety
of domain architectures.
Keywords: active site; affinity labelling; clostripain; mole-
cular modelling; peptidase family C11.
Clostripain (EC 3.4.22.8) is a cysteine endopeptidase with

strict specificity for Arg–Xaa peptidyl bonds, isolated from
the culture filtrate of the anaerobic bacterium Clostridium
histolyticum [1]. It is heterodimeric enzyme composed of two
polypeptide chains of molecular masses 43 000 kDa and
15 400 kDa, for the heavy and light chains, respectively [2].
The two chains are held together by strong noncovalent
forces [1]. Both polypeptide chains of native clostripain are
encoded by a single gene with an ORF of 1581 nucleotides
encoding a polypeptide of 526 amino acid residues [2].
Heterologous expression experiments revealed that clostri-
pain is synthesized as an inactive prepro-enzyme. In the
presence of calcium ions, the core protein (amino acids
51–526) is able to catalyse the removal of the linker
nonapeptide (residues 182–190) [3,4]. The enzyme is
important both in sequence analysis and in enzymic peptide
synthesis, as it accepts proline in the P1¢ position [5,6].
Study of the active site of clostripain, by using protein
chemistry experiments, has shown that the Cys41 of the
heavy chain (corresponding to Cys231 of the protein, as
synthesized) is the catalytic sulfhydryl residue of the active
site [7–9]. In addition, the inactivation of clostripain by
diethylpyrocarbonate has suggested the involvement of one
or more histidine residues in clostripain activity [7]. Never-
theless, direct evidence for the involvement of a histidine
residue in the catalytic mechanism of the enzyme has not yet
been provided.
In the MEROPS classification of proteinase sequences
[10], clostripain is grouped into family C11. Although
clostripain has no significant overall sequence similarity
with other proteinase families, it has been placed in clan D,

along with cysteine peptidase families C13 (legumains), C14
(caspases) and C25 (gingipains). Several criteria supported
this grouping including shared sequence motifs, predicted
secondary structure, strong specificity for the P1 position of
the substrate peptide and immunity to inhibition by E-64
irreversible protease inhibitor [11]. Later support for the
existence of structural homology between gingipains and
caspases was provided by their common inhibition by the
Correspondence to N. E. Labrou, Enzyme Technology Laboratory,
Department of Agricultural Biotechnology, Agricultural
University of Athens, Iera Odos 75, 11855 Athens, Greece.
Fax: +30 210 5294308, Tel.: +30 210 5294308,
E-mail:
Abbreviations:BAEE,N-benzoyl-
L
-arginine ethyl ester; BDD, benz-
amidinyl-diazo dichlorotriazine dye; ChC, Clostridium histolyticum
clostripain.
Enzyme: clostripain (EC 3.4.22.8).
(Received 31 October 2003, revised 26 December 2003,
accepted 19 January 2004)
Eur. J. Biochem. 271, 983–992 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04000.x
baculovirus inhibitor p35 [12]. The separin family (peptidase
family C50) has been added to clan D [13] and the
composition, distribution and evolution of all these and
other related families analysed through sequence compar-
isons [14].
Reactive triazine dyes have been used successfully for
the purification and resolution of many proteins by affinity
chromatography and for affinity labelling of several

enzymes and proteins [15–18]. We have previously estab-
lished the use of reactive dichlorotriazine dye Vilmafix Blue
A-R as a structural probe for labelling the NAD(H) binding
site of formate dehydrogenase [16], malate dehydrogenase
[17] and the oxaloacetate binding site of oxaloacetate
decarboxylase [18].
In this study we describe the use of a reactive dichloro-
triazine dye as an affinity label for clostripain and provide
direct evidence by site-directed mutagenesis and molecular
modelling studies that His176 is part of the catalytic dyad of
clostripain. The molecular modelling, in conjunction with
sequence analysis studies, indicates the P1 specificity deter-
mining residue as Asp229 and locates possible calcium-
binding sites involved in the auto-processing.
Experimental procedures
Materials
N-benzoyl-
L
-arginine ethyl ester (BAEE), bovine pancreas
trypsin (grade III, 10 800 UÆmg
)1
)andC. histolyticum clo-
stripain were from Sigma Co. (St. Louis, MO, USA). The
plasmid pKK223-3 was from Amersham Bioscience. All
other molecular biology reagents were purchased from
Promega.
Synthesis and purification of benzamidinyl-diazo
dichlorotriazine dye
Synthesis of benzamidinyl-diazo dichlorotriazine (BDD)
was as described previously [19]. Purification of BDD was

achieved by preparative TLC on silica gel 60 plates, using
the solvent system: MeOH/H
2
O/AcCN (2.5 : 2.5 : 5; v/v/v).
Enzyme assays
Clostripain assays were performed with a Hitachi U-2000
double-beam spectrophotometer carrying a thermostated
cell holder (25 °C, 10-mm pathlength), according to a
published method [20]. One unit of enzyme activity is
defined as the amount that catalyses the conversion of
1 lmol of substrate (BAEE) to product per min. Enzyme
activity calculations were performed using molar extinction
coefficients of 1150
M
)1
Æcm
)1
at 253 nm.
Determination of protein concentration
Protein concentration was determined by the Lowry
method [21] using crystalline BSA (fraction V) as standard.
Enzyme inactivation studies
Inactivation of clostripain was performed in an incubation
mixture containing, in a total volume of 1 mL at 25 °C,
100 lmol Hepes/NaOH buffer pH 7.6, 0–148.6 nmol
BDD, 1.2 units enzyme. The rate of inactivation was
followed by periodically removing samples (10–50 lL) for
assay of enzymatic activity. Initial rates of inactivation were
deduced from plots of log (% of activity remaining) vs. time
(min) for several dye concentrations and the slopes and

intercepts of secondary double reciprocal plots were cal-
culated by unweighted linear regression analysis.
Inactivation studies of clostripain by BDD in the presence
of substrate (BAEE) was performed in a total volume of
1mL(25°C) and the reaction mixture contained 100 m
M
Hepes/NaOH buffer pH 7.6, 16.9 nmol BDD, 1 m
M
or
5m
M
BAEE and 1.2 units clostripain.
In order to calculate the pK
a
of the amino acid residue
involved in the nucleophilic modification of C. histolyticum
clostripain (ChC) by BDD, enzyme inactivation experi-
ments were performed at various pH values (6.0–8.5).
Inactivation was carried out in an incubation mixture
containing, in a total volume of 1 mL at 25 °C: 100 lmol
Mops/NaOH buffer pH 6–7, 23.1 nmol BDD, 1.2 units
enzyme, or 100 lmol Hepes/NaOH buffer pH 7–8.5,
23.1 nmol BDD, 1.2 units enzyme. Data were analysed by
the
GRAFIT
program (Erithacus Software Ltd).
Stoichiometry of BDD binding to
Ch
C
ChC(100lg) in 100 m

M
Hepes/NaOH buffer pH 7.6
was inactivated with 40.5 nmol BDD at 25 °C. The dye-
inactivated enzyme was separated from the free dye by
ultrafiltration (in an Amicon stirred cell 8050 carrying a
Diaflo YM10 ultrafiltration membrane; cut-off 10 kDa)
after extensive washing with distilled water. Further separ-
ation was achieved by gel-filtration chromatography by
applying the inactive dye–enzyme complex to a Sephadex
G-25 column (9 cm · 1.6 cm) equilibrated with water, and
collecting fractions (0.5 mL) at a flow rate of 10 mLÆh
)1
.
The solution with dye-inactivated ChC was then lyophilized
and stored at )20 °C. The lyophilized ChC–BDD covalent
complex was dissolved in 8
M
urea, and the absorbance was
determined spectrophotometrically at 387 nm using a molar
extinction coefficient of 11.4 LÆcm
)1
Æmmol
)1
determined in
8
M
urea. The protein concentration was determined by the
method of Lowry [21]; no dye interference is observed in
protein determinations.
Tryptic digestion of the BDD-clostripain covalent

complex and peptide purification using HPLC
In order to covalently block the free -SH groups, before
peptide purification, lyophilized BDD–clostripain covalent
complex (100 lg) was dissolved in Hepes/NaOH buffer
(0.1
M
, pH 7.0, 1 mL) and was denatured by the addition of
solid urea to yield 8
M
solution. To the denatured enzyme
N-ethyl-maleimide was added to a final concentration of
10 m
M
, and the solution incubated for 30 min at room
temperature. The enzyme was then dialysed against 0.1
M
ammonium bicarbonate buffer pH 8.3. The enzyme was
digested by the addition of 10 lg trypsin. The digestion was
allowed to continue overnight at 30 °C before the mixture
was lyophilized and stored dry at )20 °C. Separation of the
resulting peptides was achieved on a C18 reverse phase S5
ODS2 Spherisorb silica column (250 mm · 4.6 mm i.d.).
984 N. E. Labrou and D. J. Rigden (Eur. J. Biochem. 271) Ó FEBS 2004
Analysis was achieved by a H
2
O/acetonitrile linear gradient
containing 0.1% trifluoroacetic acid (0–80% acetonitrile
during 80 min) at a flow rate of 0.5 mLÆmin
)1
. Fractions of

0.5 mL were collected. The eluents were monitored at both
220 nm and 387 nm.
Cloning, expression, purification and site-directed
mutagenesis of
Ch
C
The gene encoding ChC was amplified by PCR from
genomic DNA using oligonucleotide primers designed
from the published gene sequence of ChC as follows [2]:
the PCR reaction was carried out in a total volume of
100 lL containing 8 pmol of each primer (5¢-ATGAACA
AAAATCAAAAAGTAACTATT-3¢ and 5¢-TTACCAT
TGGTAATGATTAACTCCTCC-3¢), 100 ng template
DNA, 0.2 m
M
of each dNTP, 10 mL 10· Pfu buffer
and 1 U Pfu DNA polymerase. The PCR procedure
comprised 30 cycles of 45 s at 95 °C, 1 min at 55 °Cand
2 min at 72 °C. A final extension time at 72 °Cfor
10 min was performed after the 30 cycles. The PCR
products were run on a 1.2% (w/v) agarose gel and the
product was excised, purified by adsorption to silica beads
and ligated to the pKK223-3 expression vector, which was
previously restricted with EcoRI and treated with T4
DNA polymerase. The resulting expression construct
pChC was used to transform competent Escherichia coli
JM105 cells. E. coli harbouring plasmid pChC were
grown at 37 °C in 1 L Luria–Bertani medium containing
100 lgÆmL
)1

ampicillin. The synthesis of clostripain was
induced by the addition of 1 m
M
isoprophyl thio-b-
D
-galactoside when the absorbance at 600 nm was 0.6.
Four hours after induction, cells (% 3g) were harvested
by centrifugation at 4000 g for 15 min, resuspended in
potassium phosphate buffer (50 m
M
,pH7.5,9mL),
sonicated, and centrifuged at 10 000 g for 20 min. The
supernatant was collected and dialysed overnight against
2 L of activation buffer (50 m
M
Tris/HCl pH 6.0, 5 m
M
DTT). The dialysate was loaded onto a column of BDD–
Sepharose, 1 mL [19] previously equilibrated with Mes/
NaOH buffer (20 m
M
, pH 6.0). Non-adsorbed protein
was washed off with 10 mL equilibration buffer, followed
by 10 mL Mes/NaOH buffer (20 m
M
, pH 6.0) containing
10 m
M
KCl. Bound ChC was eluted with equilibration
buffer containing 1 mgÆmL

)1
L-Arg. Collected fractions
(1 mL) were assayed for ChC activity and protein.
Site-directed mutagenesis was performed according to
the unique site elimination method described by Deng
and Nickoloff [22]. The oligonucleotide primer sequence
for the His176Ala mutation was as follows: 5¢-ATGGCT
AAT
GCAGGTGGTGCA-3¢ and the selection primer’s
sequence was as follows: 5¢-GAATTC
TCGTGGATCC
GTCGACCT-3¢. This primer contains a mutation in a
unique SmaI restriction site of the pChC vector. Altered
nucleotides are shown underlined. The primers were
phosphorylated before use with polynucleotide kinase.
The expression construct pChC was used as template
DNA in all mutagenesis reactions. All mutations were
verified by DNA sequencing using the DyeDeoxy
Terminator method. The mutant was expressed in
E. coli and purified as described above for the wild-type
enzyme.
Bioinformatics
Sequences homologous to clostripain were sought in the
Genpept and Unfinished Microbial Genome databases at
the NCBI using
BLAST
[23] and
PSI
-
BLAST

[24]. The resulting
sequence set was aligned with
T
-
COFFEE
[25]. Jalview (http://
www.ebi.ac.uk/$michele/jalview) was used for alignment
visualization, manipulation and the calculation of five
maximally diverse representatives of the clostripain family.
The limits of the common conserved region present in all
clostripain homologues were determined by inspection of the
alignment. This region, in diverse homologous sequences,
was submitted for fold recognition experiments at the
META
-
server [26]. The
META
-server unites most of the leading fold
recognition methods and provides consensus predictions
offering improved reliability. The most informative results in
our case were provided by the
FFAS
03 method [27], a sensitive
sequence only based method which works by alignment
of two profiles [27]. Secondary structure predictions were
carried out using
PSI
-
PRED
[28]. The domain content of the

portions, of varying lengths, flanking the common conserved
region was analysed through searches at the PFAM [29] and
SMART [30] databases, and through further
PSI
-
BLAST
and fold recognition experiments.
Modelling of the common conserved region of clostripain
was carried out with
MODELLER
6 [31] using the structures of
caspases 1 (PDB code 1bmq [32]), 3 (PDB code 1pau [33]);
and 8 (PDB code 1jxq [34]), sharing 27–36% pairwise
sequence identity over the region shown in Fig. 3, as
templates. Despite these relatively low levels of sequence
identity the regular secondary structure elements of the
three templates superimpose extremely well; significant
structural differences are confined to the connecting loops.
Catalytic and specificity-determining residues superimpose
very well. Use of multiple related templates is known to
produce better models than use of a single one. The
T
-
COFFEE
alignment was used to transfer the fold recogni-
tion alignment of the C. aurantiacus with caspases to
clostripain itself. Default regimes of model refinement by
energy minimization and simulated annealing were used.
In regions in which all three templates superimposed well,
information from each was incorporated into the modelling

process. Where the templates differed the choice of which to
use was based on local similarity in length and composition
to the clostripain sequence. For the region of 20 residues
neighbouring the site of caspase cleavage, the gingipain
structure (PDB code 1cvr [35]) was used as template.
Structural determination of gingipain showed that, despite a
lack of significant sequence similarity with the caspases, the
gingipain catalytic domain adopted the caspase-like fold
[35]. The cleaved form of clostripain, lacking the internal
nonapeptide was modelled. Given the low sequence simi-
larity between target and templates, a rigorous iterative
modelling scheme was used in which 20 models were
constructed and analysed for each variant alignment. These
models were analysed for stereochemical properties with
PROCHECK
[36] and for packing and solvent exposure
characteristics with
PROSA II
[37]. Model regions corres-
ponding to positive
PROSA II
profile peaks were treated as
possibly resulting from misalignments. Alterations in align-
ments were tested for these regions. When no further
improvements were possible the final model was taken as
Ó FEBS 2004 Clostripain family structure–function relationship (Eur. J. Biochem. 271) 985
that with the best
PROSA II
score. Protein structures were
superimposed using

LSQMAN
[38] and visualized using
O
[39].
Structural figures were produced with
PYMOL
[40]. Secon-
dary structure in experimental structures was defined with
STRIDE
[41].
Results and discussion
Kinetics of reaction of BDD with clostripain
Incubation of ChC with 5.65–148.6 l
M
BDD at pH 7.6 and
25 °C leads to a progressive loss of enzyme activity, as
shown in Fig. 1A, whereas the control enzyme (in the
absence of reagent) is stable under these conditions. The
time-dependent inactivation follows pseudo-first order kin-
etics over the first 10 min. The rate constant of inactivation
(k
obs
) exhibits a nonlinear dependence on the reagent
concentration (Fig. 1B). This indicated that the reaction
obeyed pseudo-first order saturation kinetics and was
consistent with reversible binding of reagent prior to
covalent modification according to [15–18]:
E þ BDD

!

K
D
E:BDDÀ!
k
3
E-BDD
where, E represents the free enzyme; E:BDD is the reversible
complex and E-BDD is the covalent product. The steady-
state rate equation for the interaction is [15–18]:
1=k
obs
¼ 1=k
3
þ K
D
=ðk
3
½BDDÞ
where K
D
is the apparent dissociation constant of the
enzyme:BDD complex and k
3
is the maximum rate of
inactivation at saturating concentration of the reagent. The
rate constant was measured as shown in Fig. 1A. From the
double reciprocal plot of 1/k
obs
vs.1/[BDD],shownin
Fig. 1B a value of K

D
¼ 74.6 ± 2.1 l
M
was estimated for
the dissociation constant of a reversible clostripain:BDD
complex. The observed maximum rate of inactivation at
saturating concentration of the reagent was estimated
0.21 min
)1
.
Affinity labelling is a useful tool for the identification and
probing of specific, catalytic and regulatory sites in purified
enzymes and proteins. In the present study we demonstrate
the usefulness of BDD as a structural probe for the
argininyl-recognizing protease clostripain. The 1,3,5-triazine
reactive scaffold is of special interest because of its synthetic
accessibility, by taking advantage of the temperature-
dependent successive displacement of the chloride atoms
by different nucleophiles [42]. Other advantages of synthesis
of triazine-based affinity labels are their high stability
against biological and chemical degradation and their
capacity to form hydrogen bonds with amino acid residues
within the binding site due to the presence of electron rich
nitrogen sites [42].
Specificity of a protein chemical modification reaction
can be indicated by the ability of substrate to protect against
inactivation. The substrate was added to the incubation
mixture at a concentration much higher than the known
enzyme–ligand dissociation constant in order to assess its
effect on the inactivation rates at pH 7.6 and 25 °C. For

example, for BAEE the K
m
value is 0.235 m
M
[43]. Fig. 1C
shows that the rate of enzyme inactivation by BDD
decelerated in the presence of 1 or 5 m
M
BAEE.
Fig. 1. Affinity labelling of ChC. (A) Time course for the inactivation
of ChC by BDD. Inactivation was performed at pH 7.6 and 25 °C. No
BDD (h); 5.66 l
M
(j); 11.32 l
M
(r); 16.97 l
M
(w); 37.0 l
M
(e);
148.6 l
M
(*). (B) Effect of BDD concentration on the observed rate of
inactivation (k
obs
)ofChC expressed as a double-reciprocal plot. BDD,
5.66–148.6 l
M
. The slope and intercept of the secondary double-
reciprocal plot were calculated by unweighted linear regression ana-

lysis. Inset shows the structure of BDD. (C) Effect of substrate (BAEE)
on the time course of inactivation of ChC by BDD (pH 7.6, 25 °C). No
BDD (h); BDD, 16.97 l
M
(w); BDD, 16.97 l
M
in the presence of
1m
M
BAEE (r)or5m
M
BAEE (j).
986 N. E. Labrou and D. J. Rigden (Eur. J. Biochem. 271) Ó FEBS 2004
To determine the stoichiometry of dye binding, ChCwas
completely inactivated by the dye and the dye–enzyme
covalent complex was resolved from free dye by gel
filtration on Sephadex G-25 and ultrafiltration. The molar
ratio of [Dye]:[ChC active site] was determined by
measuring the dye spectrophotometrically in urea solution,
and the protein by the method of Lowry et al.[21].The
molar ratio of dye to ChC active site was 1 : 1.1 ± 0.1,
using a molecular weight 56 000, indicating a specific
interaction between dye and protein.
BDD exhibits several characteristics of an affinity label in
its reaction with clostripain. It reacts stoichiometrically with
the enzyme. Time- and dye concentration-dependent inac-
tivation of clostripain by BDD is evident. The pseudo-first
order kinetics obtained for clostripain inactivation indicates
that the phenomenon occurs through the initial formation
of a reversible Michaelis binary complex followed by

subsequent formation of a covalent complex [16–18].
Protection against inactivation by BDD is provided by the
synthetic substrate BAEE, indicating that the dye interacts
with the enzyme at the substrate binding site.
Isolation and analysis of peptides from clostripain
modified by BDD
Modified clostripain was subjected to trypsin digestion
followed by fractionation by reverse-phase HPLC. Essen-
tially, a single yellow peak (BDD-labelled peptide) eluted
from the column. The yellow peak was freeze dried and
subjected to amino acid analysis and sequencing. The
overall recovery of modified peptide, based on the initial
amount of modified enzyme was 22%. Automated Edman
sequence analysis of the labelled peptide gave the sequence
YVLIMAN-X-GGGAR, where X indicates that no phe-
nylthiohydantoin derivative was detected in the cycle. By
comparison with the amino acid sequence of clostripain, the
X in the peptide was identified as His176, indicating that the
site chain of His is the reactive group responsible for
the nucleophilic attack on the diclorotriazine ring of the dye.
Site directed mutagenesis and pH dependence
of inactivation
The wild-type enzyme and the mutant His176Ala were
expressed in E. coli and characterized by steady-state kinetic
analysis. Assay for clostripain activity of the purified mutant
revealed that it was completely inactive. Thus both our
chemical modification and site-directed mutagenesis data
confirm the predictions made regarding clostripain’s cata-
lytic site [9]. Our data provide the first direct evidence that
catalysis by clostripain involves the Cys–His dyad almost

ubiquitously involved in cysteine peptidase mechanisms
[42,44].
The study of the effect of pH on enzyme inactivation
allows the calculation of the pK
a
of the His176 side chain
involved in the inactivation reaction. The rate of inactiva-
tion exhibited a sigmoid-shaped pH-dependence indicating
that the reaction depends strongly on the nucleophilicity of
a deprotonated group. The pK
a
value measured from this
curve was equal to 7.4 ± 0.2 (Fig. 2). This pK
a
value is
higher than the expected value for the free amino acid but is
in agreement with the expected value for a His interacting
with a thiolate [45]. In the papain family, Cys25 and His159
form a thiolate–imidazolium ion pair in which the pK
a
values of the two residues are perturbed by approximately 4
units (Cys to pK
a
4) and 2 units (His to pK
a
8.5), respectively
[45]. The absence of strong pK
a
perturbation, compared to
that observed in papain, may be related to the greater

separation of His and Cys in the caspase structures [46], and
in the clostripain molecular model (see below). The greater
separation would not allow for the degree of pK
a
pertur-
bation observed in the papain family [47].
Clostripain homologues
Previous searches for clostripain homologues and the current
state of the PFAM database revealed only the presence
of clostripain itself and three Thermotoga maritima homo-
logues [12]. Our database searches using
PSI
-
BLAST
[24], in
both GenBank and Unfinished Microbial Genome data-
bases at the NCBI ( />initially located, ignoring obviously partial sequences, 13
homologues in GenBank and three among unfinished
microbial genome data. The species in which clostripain
homologues were newly observed were C. perfringens,
C. thermoceullum, C. tetani, Methanosarcina acetivorans,
Chloroflexus aurantiacus, Geobacter metallireducens,and
Ruminococcus albus. The observation, for the first time, of a
clostripain homologue in the Archaea (M. acetivorans)is
particularly interesting in view of the interest in under-
standing the curious phyletic distributions of clostripains
and related peptidase families [9,12]. Over the alignment
section shown in Fig. 3, the archaebacterial homologue
shares 16–27% sequence identity with the other clostripain
family members. It contains all the possible functional

residues discussed later.
Alignment of these sequences enabled the location of a
common conserved region presumably containing the
catalytic domain. Of the three Thermotoga maritima
sequences found, one (GenBank, 15643282) lacked a
conserved N-terminal portion found in all the other
Fig. 2. The pH dependence of clostripain inactivation by BDD at 25 °C.
The reaction mixture contained 1.2 U enzyme, 22.1 l
M
BDD, and
100 m
M
(Mops/NaOH or Hepes/NaOH) buffer in pH values 6.0–8.5.
Ó FEBS 2004 Clostripain family structure–function relationship (Eur. J. Biochem. 271) 987
homologues. Translation of the corresponding DNA
revealed this portion lying upstream of the annotated start
but failed to highlight any alternative start codons. This
sequence was therefore not included in subsequent analysis
as possibly representing an inactivated copy. Similarly, one
of the four Chloroflexus aurantiacus sequences lacked both
the catalytic Cys47 and His residues (this work) and, since
our interest lay principally in understanding peptidase
activity in the clostripain family, was not studied further.
The appearance of inactivated copies of related peptidases
in various evolutionary lineages appears common [12].
The set of clostripain homologues was remarkably
diverse both in length and in composition. Considering
only the identified common conserved region (correspond-
ing to residues 56–446 in clostripain, see Fig. 3), no two
sequences shared more than 56% sequence identity. The

mean pairwise sequence identity among the 13 homologues
in the common conserved region was just 21%. Only six
positions were entirely conserved and another 10 were
conserved in 12 of the 13 sequences (Fig. 3).
In order to analyse the composition of the clostripain
homologues outside the catalytic domain, searches were
carried out in the PFAM [29] and SMART [30] domain
databases and more distant domain matches sought for the
remaining regions by fold recognition. The current PFAM
database shows the presence of bacterial immunoglobulin-
like domains (PFAM, PF02369; SMART, SM00634) in two
T. maritima proteins but our searches revealed a much more
diverse set of domain architectures in the family (Fig. 4). As
well as the bacterial immunoglobulin-like domains members
Fig. 3. Sequence alignment of five maximally diverse representatives of the clostripain homologue alignment with the three caspase templates used for
model construction. GenBank identification numbers and abbreviated species names are shown for the clostripain homologues (399264 is clostripain
itself), while PDB codes and enzyme names are provided for the templates. The predicted secondary structure for clostripain (obtained with
PSIPRED
[28] and clostripain numbering are shown above the alignment. The
STRIDE
[41] derived secondary structure of human caspase-1 and its numbering
are shown beneath the alignment. Shaded regions indicate portions cleaved upon activation of clostripains or caspases, although cleavage has only
been shown experimentally for clostripain, not for the homologues shown here. The boxed region indicates the single part of the clostripain
molecular model obtained from the gingipain structure (see text for details). Bold italic face is used for the catalytic His and Cys residues. Bold face
among the clostripains signifies conservation among at least 12 of the 13 sequences considered. Italic face is used to show portions of the clostripain
sequence for which reliable modelling was not possible. The figure was made with
ALSCRIPT
[53].
Fig. 4. Schematic diagram of domain architectures present among clo-
stripain homologues. Rectangles represent catalytic domains and other

shapes the additional identified domains. Only the association of clo-
stripain catalytic domains with bacterial immunoglobulin-like
domains is visible in the current PFAM database [29]. Domains were
identified through screening against PFAM and SMART [30], with the
exception of the fibronectin type 3 domain in 15644337 which was
identified by fold recognition.
988 N. E. Labrou and D. J. Rigden (Eur. J. Biochem. 271) Ó FEBS 2004
of the clostripain family contain forkhead domains
(SMART, SM00240), fibronectin type 3 domains (PFAM,
PF00041) and NHL domains (PFAM, PF00400). None of
these domain entries gives more than a clue as to the
physiological roles of the clostripain homologues but it is
interesting to note that both forkhead and NHL domains
are implicated in protein–protein interactions [48,49]. Simi-
larly, both bacterial immunoglobulin-like domains and
fibronectin type 3 domains are strongly associated with cell
adhesion [50]. Most unexpectedly, one clostripain homo-
logue from C. aurantiacus contains tandem peptidase
catalytic domains (Fig. 4) with a peptidase M37 catalytic
domain preceding the peptidase C11 domain and a fibro-
nectin type 3 domain lying between the two. The picture
that emerges is one in which clostripain itself, the only
member of the peptidase C11 family to have been experi-
mentally studied, is atypically simple in possessing the
catalytic domain alone. The peptidase C11 family contains a
large variety of domain architectures which probably reflect
a range of physiological roles that deserve further study.
Molecular modelling
Existing data showed a distant evolutionary relationship
between the clostripain family and other peptidase families

[9,12]. The characterization of the clostripain mutant
H176A and its specific chemical modification presented
here provides further support for the hypothesis. In order to
explore other aspects of the structure–function relationship
of clostripain and its homologues, a molecular model would
be invaluable. This would obviously require a reliable
alignment of clostripain, or a homologue, with a known
structure. Previous published alignments have covered only
part of the conserved common region of the clostripain
family [12], terminating shortly after the catalytic Cys and
therefore not allowing for molecular modelling. We there-
fore carried out fold recognition experiments in order to try
to obtain an alignment that would enable the construction
of a molecular model for clostripain. The recent availability
of diverse clostripain homologues would facilitate fold
recognition studies in two important ways: firstly by
enabling the limits of the catalytic domain to be identified
(thereby improving fold recognition accuracy); and sec-
ondly, as fold recognition may sometimes be successful for
one homologue but not for another, by providing several
different distantly homologous sequences to serve as input
for the fold recognition.
Fold recognition experiments with several sequences
corresponding to the common conserved region produced
initially confusing results. Strongly significant results were
obtained for the a/b hydrolase fold with the expected
caspase-like fold scoring worse. Comparison of the clostri-
pain sequences with the conserved characteristics of the a/b
hydrolase fold, such as the so-called nucleophile-elbow [51],
enabled it to be discarded as a possible fold for clostripains.

In contrast, the alignments of clostripain sequences with
caspases aligned both the Cys and His catalytic residues.
Further examination of the alignments revealed the reasons
behind the unexpected results. Firstly, the cleavage of
caspases shortly after the catalytic Cys has led to their
structures being deposited with the PDB with different
chain names for the cleaved N- and C-terminal portions.
The two pieces are therefore considered as separate chains
by the fold recognition algorithms and the clostripain–
caspase alignments covered only the caspase regions prior
to the cleavage point. The complete alignments would
presumably have scored much better. Secondly, among the
several insertions of clostripains relative to caspases is a very
large one towards the C terminus (Fig. 4), predicted to
contain four a-helices which, by chance, aligned with some
members of the a/b hydrolase superfamily containing a
similarly placed all-a excursion to the main fold.
The best alignment of a clostripain homologue with a
caspase structure (caspase-9; PDB code 1jxq [34]); was
produced for the C. aurantiacus homologue with GenBank
22972276 by the
FFAS
03 method [27] and given a highly
significant score of )7.6. Using this incomplete alignment of
clostripain with the caspases as a base, the alignment was
manually extended through matching of caspase secondary
structure with clostripain predicted secondary structure
(Fig. 3). At certain key points, residue conservation could
be used to improve confidence in the correctness of the
alignment. For example, the caspases have a serine

conserved at position 332, whose side chain forms hydrogen
bonds with both the carboxyl oxygen and the nitrogen
atoms of the backbone of the residue preceding the catalytic
Cys. The conservation of this interaction is suggestive of its
Fig. 5. The final model of the clostripain catalytic domain. The ribbon is
coloured according to secondary structure and key residues shown
using a stick representation and labelled. Residues at the catalytic site
are shown in larger face, residues of possible calcium binding sites (see
text for details) in smaller face. The model is of the cleaved clostripain
lacking the internal nonapeptide. The final residue of the resultant
a-chain, Arg181, and the first residue of the b-chain, Ala191, are also
labelled (italics). The magenta colouring towards the bottom of the
figure marks the position of the large unmodelled insertion in clostri-
pain compared to caspases towards the C terminus.
Ó FEBS 2004 Clostripain family structure–function relationship (Eur. J. Biochem. 271) 989
importance so it was reassuring that a serine, conserved with
one exception among clostripain homologues (numbered
257 in clostripain itself), could be aligned with this position
(Fig. 3). Similarly conserved caspase Trp340, lining the
catalytic site could be aligned with a conserved aromatic
residue in the set of clostripain homologues. The very
C-terminal portion of the caspase structure, around residue
400, forms a key part of the domain structure and adopts an
extended conformation which is not defined as b-structure
due to the absence of the necessary hydrogen bonds. It was
aligned with a predicted b-strand in the clostripain family.
This defined a very large insertion in clostripains relative
to the caspases which was not amenable to modelling.
However, the absence of significant sequence conservation
and variable length of the region were not suggestive of

functional importance. The match between predicted
clostripain secondary structure and actual caspase secon-
dary structure of the final alignment is very good (Fig. 3).
Nevertheless this region must be considered less reliable
than other portions of the model. Only one of the putative
functional residues discussed below is located in this region.
With the most complete caspase–clostripain alignment
available, a process of iterative model building was carried
out using as templates the highest resolution structures
available of caspases 1, 3 and 9 as described in Experimental
procedures. Over the modelled portion of clostripain the
templates shared 12–16% sequence identity with clostripain.
A particular problem was encountered for the clostripain
region near to the cleaved portion of the caspases. In all the
caspase structures, cleavage results in the segments pre-
ceding and following the site of cleavage adopting highly
extended conformations with no contacts to the compact
domain structure. In contrast, a predicted helix is present
in the corresponding, uncleaved portion of the clostripains.
For this region only, the corresponding part of gingipain,
whose structure also indicates distant homology to the
caspases [34], was used (Fig. 3). Structural similarity
between gingipain and caspases is particularly strong for
the catalytic site residues. The cleavage of clostripain with
loss of internal peptide was included in the model (Fig. 3).
During the iterative modelling scheme, several alignment
changes were found to result in improved models, as judged
by
PROSA II
[37] analysis resulting in the final alignment

shown in Fig. 3. Although the final model (Fig. 5) lacked
several insertions, too large to model effectively, it scored
)6.24 by
PROSA II
, corresponding to a near-optimal pG
value [52] of 0.99. This result confirms the correctness of
the fold used as template for modelling and is suggestive
of largely accurate alignment [52]. Eighty-six per cent of
residues occupied core regions of the Ramachandran plot in
Fig. 6. Determinants of P1 substrate specificity
in (A) clostripain (specific for Arg) (B) caspase
(specific for Asp) and (C) gingipain (specific for
Arg). The same colouring by secondary
structure is used in all panels. Key residues are
shown as ball-and-stick and coloured pink
(catalytic) or light grey (specificity-determin-
ing, experimentally determined for caspase
and gingipain, predicted for clostripain). The
caspase and gingipain structures shown (1bmq
[32] and 1cvr [35], respectively) both contain
inhibitors bound at the catalytic site and cov-
alently attached to the catalytic Cys residues
which are shown as cyan sticks. Portions of
the caspase and gingipain structures lying
outside the common conserved structural core
are coloured grey.
990 N. E. Labrou and D. J. Rigden (Eur. J. Biochem. 271) Ó FEBS 2004
the final model. There were no Ramachandran-disallowed
residues and just two located in generously allowed zones.
Model analysis

With the good objective quality of the final model estab-
lished, it was used to address issues of the structure–function
relationship in the clostripain family. The first question was
the mechanism by which clostripains specify a strong
preference for Arg at the P1 position of the substrate.
Examination of the alignment (Fig. 3) alone reveals several
conserved acidic residues, any one of which could be
responsible for substrate specificity. However, examination
of conserved residues (Fig. 3) in the context of the model
(Figs5and6),andcomparisonofthemodelwithcaspase
and gingipain crystal structures (where specificity-determin-
ing residues are understood; Fig. 6) led to a clear answer.
Residue Asp229 (clostripain numbering) is totally conserved
and well positioned to interact with substrate Arg residues
at position P1 (Fig. 6A). Even taking into account the
possibility of local alignment errors, no other conserved
acidic residue could be responsible. Interestingly, Asp229 is
structurally positioned differently to the specificity-deter-
mining Arg residues in caspases (Fig. 6B) and the Asp163 in
gingipain (Fig. 6C). However, the totally conserved caspase
Gln residue corresponding to Asp229 (numbered 283 in
caspase-9; Fig. 3) does interact with the P1 side chain of the
substrate (e.g. [33]). This provides strong additional support
for our assignment of Asp229 as specificity determinant.
Clostripain is known to undergo a calcium-dependent
auto-activation process [1–4]. Although the details are not
well understood, and it is not known if all members of the
family will behave similarly in this regard, this implies the
existence of a calcium-binding site on clostripain. Exam-
ination of the final model revealed two suggestively

positioned possibilities (Fig. 5), one positioned near the
site of cleavage, the other near to the catalytic site. The first
contains Glu212 and Glu237, both conserved in 12 of the
13 homologues, along with Asp215 found only in clostri-
pain itself. The second site contains three acidic residues
not conserved between clostripain sequences ) Glu110,
Asp114 and Asp269. The residues of the first site lie within
or near the central portion of the alignment which contains
the catalytic dyad. Here the alignment of clostripain and
the templates is particularly clear so that model quality
should be good. Each site could be relevant to calcium-
dependent auto-activation, the first through an effect on
the site of cleavage, the second through a direct influence
on the catalytic site, but the determination of which site is
truly occupied will require further experiments. Since it is
not known if all clostripain homologues undergo this auto-
activation the conservation of the first possibility within the
family does not conclusively indicate it as the likely
calcium-binding site.
Conclusions
In this study we investigate the structure–activity relation-
ship of clostripain, and its homologues in the peptidase C11
family, by affinity labelling, site-directed mutagenesis and
molecular modelling. A catalytic dyad of His176 and
Cys231 is definitively shown to be responsible for cysteine
peptidase activity in the C11 peptidase family. However, the
lack of strong perturbation of the pK
a
value of His176 is
consistent with the two catalytic residues lying further apart

than they do in papain, as indeed observed in the distantly
homologous caspases. Molecular modelling revealed the
likely source of clostripain substrate specificity and possible
sites of binding for the calcium required for auto-activation,
thus providing attractive targets for further study by site-
directed mutagenesis. The domain structures of peptidase
family C11 members are surprisingly diverse. Further study
of the family may be facilitated by the dye based labelling of
thekindusedinthiswork.
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