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

Rigden2 1 Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece; 2 School of Biological Sciences, University of Liverpool,

The structure–function relationship in the clostripain family

of peptidases

Nikolaos E Labrou1and Daniel J Rigden2

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

histolyticumclostripain The enzyme, upon incubation with

BDD in 0.1MHepes/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

KD¼ 74.6 ± 2.1 lM and the maximal rate constant of

inactivation is k3¼ 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: Lambrou@aua.gr

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)

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) and C 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/H2O/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 (25C, 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 1150M )1Æcm)1at 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 25C,

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

1 mL (25C) and the reaction mixture contained 100 mM Hepes/NaOH buffer pH 7.6, 16.9 nmol BDD, 1 mM or

5 mMBAEE and 1.2 units clostripain

In order to calculate the pKaof 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 25C: 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 theGRAFITprogram (Erithacus Software Ltd)

Stoichiometry of BDD binding toChC ChC (100 lg) in 100 mM Hepes/NaOH buffer pH 7.6 was inactivated with 40.5 nmol BDD at 25C 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-filtrsepar-ation 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 8Murea, and the absorbance was determined spectrophotometrically at 387 nm using a molar extinction coefficient of 11.4 LÆcm)1Æmmol)1determined in

8Murea 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 (0.1M, pH 7.0, 1 mL) and was denatured by the addition of solid urea to yield 8Msolution To the denatured enzyme N-ethyl-maleimide was added to a final concentration of

10 mM, and the solution incubated for 30 min at room temperature The enzyme was then dialysed against 0.1M 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 30C 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.)

Analysis was achieved by a H2O/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 ofChC

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

DNA, 0.2 mM of each dNTP, 10 mL 10· Pfu buffer

and 1 U Pfu DNA polymerase The PCR procedure

comprised 30 cycles of 45 s at 95C, 1 min at 55 C and

2 min at 72C A final extension time at 72 C for

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 37C in 1 L Luria–Bertani medium containing

100 lgÆmL)1 ampicillin The synthesis of clostripain was

induced by the addition of 1 mM isoprophyl

thio-b-D-galactoside when the absorbance at 600 nm was 0.6

Four hours after induction, cells ( 3 g) were harvested

by centrifugation at 4000 g for 15 min, resuspended in

potassium phosphate buffer (50 mM, pH 7.5, 9 mL),

sonicated, and centrifuged at 10 000 g for 20 min The

supernatant was collected and dialysed overnight against

2 L of activation buffer (50 mM Tris/HCl pH 6.0, 5 mM

DTT) The dialysate was loaded onto a column of BDD–

Sepharose, 1 mL [19] previously equilibrated with Mes/

NaOH buffer (20 mM, pH 6.0) Non-adsorbed protein

was washed off with 10 mL equilibration buffer, followed

by 10 mL Mes/NaOH buffer (20 mM, pH 6.0) containing

10 mM 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

AATGCAGGTGGTGCA-3¢ and the selection primer’s

sequence was as follows: 5¢-GAATTCTCGTGGATCC

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 usingBLAST[23] andPSI-BLAST[24] The resulting sequence set was aligned withT-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 theMETA -server [26] TheMETA-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 theFFAS03 method [27], a sensitive sequence only based method which works by alignment

of two profiles [27] Secondary structure predictions were carried out usingPSI-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 withMODELLER6 [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-COFFEEalignment 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 IIprofile 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

that with the bestPROSA II score Protein structures were

superimposed usingLSQMAN[38] and visualized usingO[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 lMBDD at pH 7.6 and

25C 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

(kobs) 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]:

Eþ BDD ƒƒƒƒƒƒ!KD E:BDD!k3 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=kobs¼ 1=k3þ KD=ðk3

where KD is the apparent dissociation constant of the

enzyme:BDD complex and k3 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/kobs vs 1/[BDD], shown in

Fig 1B a value of KD¼ 74.6 ± 2.1 lMwas 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 25C For

example, for BAEE the Kmvalue is 0.235 mM[43] Fig 1C

shows that the rate of enzyme inactivation by BDD

decelerated in the presence of 1 or 5 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 ) of ChC 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

1 m M BAEE (r) or 5 m M BAEE (j).

To determine the stoichiometry of dye binding, ChC was

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 pKaof 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 pKavalue measured from this

curve was equal to 7.4 ± 0.2 (Fig 2) This pKa 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 pKa values of the two residues are perturbed by approximately 4 units (Cys to pKa4) and 2 units (His to pKa8.5), respectively [45] The absence of strong pKaperturbation, 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 pKa 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 usingPSI-BLAST[24], in both GenBank and Unfinished Microbial Genome data-bases at the NCBI (http://www.ncbi.nlm.nih.gov/blast/), 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.

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 maritimaproteins 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.

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 FFAS03 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.

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

byPROSA 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.

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

(Figs 5 and 6), and comparison of the model with caspase

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 pKa 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 the kind used in this work

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