The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases.

Abstract

Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg(2+) or Ca(2+) , which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg(2+) or Ca(2+) binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs.

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The structure of the catalytic domain of Tannerella forsythia
karilysin reveals it is a bacterial xenologue of animal matrix
metalloproteinasesmmi_7434 119..132
Núria Cerdà-Costa,1†Tibisay Guevara,1†
Abdulkarim Y. Karim,2Miroslaw Ksiazek,2
Ky-Anh Nguyen,3,4Joan L. Arolas,1Jan Potempa2,5**
and F. Xavier Gomis-Rüth1*
1Proteolysis Lab; Department of Structural Biology;
Molecular Biology Institute of Barcelona, CSIC;
Barcelona Science Park; Helix Building; c/ Baldiri
Reixac, 15-21; E-08028 Barcelona, Catalunya.
2Department of Microbiology; Faculty of Biochemistry,
Biophysics and Biotechnology; Jagiellonian University;
PL-Krakow 30-387, Poland.
3Institute of Dental Research, Westmead Centre for Oral
Health, Sydney, NSW 2145, Australia.
4Faculty of Dentistry, University of Sydney, Sydney,
NSW 2006, Australia.
5University of Louisville; School of Dentistry; Oral Health
and Systemic Disease; Louisville, KY 40202, USA.
Summary
Metallopeptidases (MPs) are among virulence factors
secreted by pathogenic bacteria at the site of
infection. One such pathogen is Tannerella forsythia,
a member of the microbial consortium that causes
peridontitis, arguably the most prevalent infective
chronic inflammatory disease known to mankind. The
only reported MP secreted by T. forsythia is karilysin,
a 52 kDa multidomain protein comprising a central
18 kDa catalytic domain (CD), termed Kly18, flanked
by domains unrelated to any known protein. We
analysed the 3D structure of Kly18 in the absence and
presence of Mg2+or Ca2+, which are required for func-
tion and stability, and found that it evidences most of
the structural features characteristic of the CDs of
mammalian matrix metalloproteinases (MMPs). Unex-
pectedly, a peptide was bound to the active-site cleft
of Kly18 mimicking a left-behind cleavage product,
which revealed that the specificity pocket accommo-
dates bulky hydrophobic side-chains of substrates as
in mammalian MMPs. In addition, Kly18 displayed a
unique Mg2+or Ca2+binding site and two flexible seg-
ments that could play a role in substrate binding.
Phylogeneticand sequence
revealed that Kly18 is evolutionarily much closer to
winged-insect and mammalian MMPs than to poten-
tialbacterial counterparts
sequencing projects. Therefore, we conclude that this
first structurally characterized non-mammalian MMP
is a xenologue co-opted through horizontal gene
transfer during the intimate coexistence between T.
forsythia and humans or other animals, in a very
rare case of gene shuffling from eukaryotes to
prokaryotes. Subsequently, this protein would have
evolved in a bacterial environment to give rise to
full-length karilysin that is furnished with unique
flanking domains that do not conform to the general
multidomain architecture of animal MMPs.
similaritystudies
found bygenomic
Introduction
Metallopeptidases
protein and peptide hydrolases with major endogenous
roles in the physiology and pathology of living organisms.
In addition, MPs have exogenous functions in the venoms
used by predators, such as in poisonous snakes, scorpi-
ons and spiders, or, conversely, in poison-mediated
defence strategies against predators such as in scorpion
fish (Carrijo et al., 2005; King, 2007; Fox and Serrano,
2009). Another source of exogenous MPs is bacteria,
which secrete them as virulence factors during host
infection. In this condition, MPs damage host tissues by
cleaving cell-surface and tissue proteins, either directly or
indirectly by activating host peptidases. MPs also inacti-
vate key proteins in host defence, recruit nutrients, and
activate other bacterial virulence factors. These strategies
are required for bacterial invasion, survival, proliferation
and colonization of the host in a hostile environment
(Lantz, 1997; Miyoshi and Shinoda, 2000; Potempa and
Pike, 2009). One such human infection is periodontitis,
which causes chronic inflammation of the gums and
affects 10–15% of adults worldwide, leading to gingival
(MP)are mostlyzinc-dependent
Accepted 8 October, 2010. For correspondence. *E-mail: fxgr@ibmb.
csic.es; Tel. (+34) 934 020 186; Fax (+34) 934 034 979; **E-mail:
jspote01@louisville.edu; Tel. (+1) 502 852 5572; Fax (+1) 502 852
5572.†These authors contribute equally and share first authorship.
Molecular Microbiology (2011) 79(1), 119–132 ?
doi:10.1111/j.1365-2958.2010.07434.x
First published online 2 November 2010
© 2010 Blackwell Publishing Ltd

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tissue destruction and tooth loss (Fox, 1992; Cutler et al.,
1995). Due to their inflammatory and infective character,
severe forms of the disease further contribute to the
development of systemic pathologies such as cardiovas-
cular disease, stroke, rheumatoid arthritis and diabetes
(Jordan, 2004; Pihlstrom et al., 2005; Behle and Papa-
panou, 2006; Persson, 2006; Wegner et al., 2010).
Current treatment and curettage of severe periodontal
disease is only partially effective. Furthermore, it entails
the intensive usage of antibiotics, which contributes to the
spread of antibiotic resistance (Haffajee et al., 2003; Pihl-
strom et al., 2005; Suvan, 2005). Consequently, there is a
need for innovative and specific therapeutic approaches
to this pathology, and detailed structural information of
participating virulence factors may contribute to this
development (Mittl and Grütter, 2006).
Periodontitis is caused by bacteria that grow on tooth
surfaces and in the gingival crevice, and three species,
Porphyromonas gingivalis, Treponema denticola and
Tannnerella forsythia, are the major periodontopathogens,
collectively referred to as the red complex (Socransky
et al., 1998). These pathogens secrete a vast armamen-
tarium of proteolytic enzymes, which has been thoroughly
characterized for the first two organisms (Potempa et al.,
2000;EleyandCox,2003).Incontrast,theonlypeptidases
identified to date in T. forsythia are a cysteine proteinase,
PrtH (Saito et al., 1997), and the MPkarilysin (Karim et al.,
2010). The latter is a 472-residue secretory protein com-
prising a 20-residue signal sequence, a 14-residue pro-
peptide, an 18 kDa catalytic peptidase domain (CD) and a
30 kDa C-terminal domain of unknown function. The latter
has been found only in other putative secretory proteins of
T. forsythia (Karim et al., 2010). The recombinant full-
length enzyme gives rise to karilysin CD – hereafter Kly18
–throughsequentialautolysis.ThefirstcutatAsn34–Tyr35
(numberingaccordingtothecompletegenesequence,see
UniProt D0EM77) removes the pro-peptide and generates
a fully active 48 kDa variant. Subsequent autolytic cleav-
age events happen downstream of the CD and do not
affect activity (Karim et al., 2010).
Whereas the flanking sequences do not resemble any
other structurally or functionally analysed protein, Kly18 is
similar to mammalian matrix metalloproteinases (MMPs)
(see fig. 2 in Karim et al., 2010). These constitute a sepa-
rate family within the ‘metzincin’ clan of MPs (Bode et al.,
1993; Stöcker et al., 1995; Gomis-Rüth, 2003; 2009; Visse
and Nagase, 2003; Murphy and Nagase, 2008; Tallant
et al., 2010b) and participate in both broad-spectrum turn-
over of extracellular-matrix components and selective
limited proteolysis to activate or inactivate other proteins
and enzymes. When dysregulated, their degrading poten-
tial leads to uncontrolled proteolysis and tissue destruc-
tion,apoptosis andinflammation,
periodontitis among other conditions (Overall and López-
asobservedin
Otín, 2002). MMPs have mainly been studied in mammals,
and 23 paralogues are present in humans [see http://
degradome.uniovi.es/met.html#M10 (Puente et al., 2003;
Quesada et al., 2009)]. They are secreted as soluble or
membrane-bound mosaic proteins comprising several
inserts and domains, which include a signal peptide for
secretion,an~80-residuezymogenicpro-domainincluding
a conserved cysteine engaged in latency maintenance
(Rosenblum et al., 2007), a ~165-residue zinc- and
calcium-dependentCD,alinkerregionanda~200-residue
haemopexin-like C-terminal domain for collagen binding,
pro-MMP activation and dimerization (Gomis-Rüth, 2004;
Maskos, 2005). In addition to mammals, MMPs have been
studied (at least at the mRNA level) in several classes of
animals and plants (Table 1). As to lower eukaryotes and
prokaryotes, sequences similar to MMP CDs have been
found in the genomes of fungi, viruses, archaea and bac-
teria, but to our knowledge they have not been studied
(Gomis-Rüth, 2003). At the structural level, only human,
mouse and pig MMP CDs have been analysed so far (see
Tallant et al., 2010b and http://pfam.sanger.ac.uk/family/
PF00413#tabview=tab8).
In order to explore the mechanisms of action of karilysin
and to provide a high-resolution scaffold for the design of
specific inhibitors against this bacterial virulence factor,
we studied structure and function of Kly18, in the absence
and presence of alkaline-earth metal ions. In addition,
phylogenetic studies and evolutionary considerations ren-
dered a plausible explanation for the origin of this MP.
Results and discussion
Endopeptidolytic activity of Kly18
Thedistinct(autolytic)variantsofT.forsythiakarilysinhave
beenpreviouslycharacterizedfortheirproteolyticpotential
(Karim et al., 2010; Koziel et al., 2010). These studies
revealed that the enzyme requires calcium for activity and
thermal stability and that it cleaves bonds preferentially
N-terminally of bulky hydrophobic residues. It efficiently
degraded elastin, fibrinogen and fibronectin, thus pointing
to possible roles in periodontitis progression. In addition,
karilysin inactivated the antimicrobial peptide, LL-37,
which is a component of the immune system that targets
bacteriaandothermicroorganisms,thusfurthersupporting
a role of this MP in T. forsythia virulence. Here, we further
studied the endopeptidolytic potential of Kly18 in front of
two fluorogenic peptide substrates of seven and 10 resi-
dues respectively.These were developed to be cleaved by
distinct mammalian MMPs, with kcat/Km values ranging
between 1E4 and 6E5 M-1s-1(Knight et al., 1992; Nagase
et al., 1994; Fields, 2001). These substrates were effi-
ciently cleaved by Kly18, with kcat/Km values of 4895 and
14 709 M-1s-1for the two substrates, respectively, thus
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indicating that the bacterial enzyme and its mammalian
counterparts evince comparable substrate specificity and
efficiency.
Structure of Kly18
The structure of Kly18 was solved both in the absence
and in the presence of magnesium, hereafter ‘unbound’
and ‘magnesium-bound’ variants respectively. These vari-
ants crystallized in distinct space groups and contained
two and one protein molecules, respectively, in the crys-
tallographic asymmetric unit. Calcium had been reported
to be important for karilysin activity and thermal stability
(Karim et al., 2010), and so this cation was added to the
protein prior to crystallization to produce a calcium-bound
form. However, in the crystal structure, which was
obtained to significantly higher resolution than the
unbound form (Table 2), only one partially occupied mag-
nesium site was found as inferred from electron density
maps and distances to ligands, probably owing to the
excess of the latter over calcium in the crystallization
conditions (see Experimental procedures).
The Kly18 molecule is roughly spherical, with a diam-
eter of ~40 Å. Carved into its frontal surface, a shallow
active-site cleft traverses the molecule left (non-primed
side) to right (primed side) and divides it into a large
N-terminal (NTS, Tyr35–Gly162) and a small C-terminal
subdomain (CTS; Ile163–Ser201; see Fig. 1A and B).
Both the N- and the C-terminus are on the lower left of the
molecule surface. The N-terminal a-amino group, in the
front, is salt-bridged to the side-chain ofAsp187 within the
‘C-terminal helix’ aC of CTS and thus anchored to the
peptidase moiety. The NTS consists of a twisted five-
stranded b-sheet (strands bI–bV), whose strands are all
parallel to a substrate bound to the cleft except for outer-
most strand bIV, which forms the upper rim of the crevice
and runs antiparallel. Two helices, the ‘backing helix’ (aA)
and the ‘active-site helix’ (aB), nestle into the concave
side of the sheet (Fig. 1A and C). On the convex side of
the latter, three loops protrude from the molecular surface:
the loop segment connecting bII and bIII (LbIIbIII), LbIVbV
and LbIIIbIV. The latter, termed ‘S-loop’ due to its sinuous
chain trace, encompasses a structural zinc binding site, at
which the cation is tetrahedrally co-ordinated by three
histidines and an aspartate (His102, Asp104, His117 and
His133; Fig. 1D). The final stretch of the S-loop is termed
‘bulge-edge segment’ and it shapes the upper rim of the
active-site cleft on its primed side (Fig. 1A). This loop
Table 1. MMPs reported from non-mammalian organisms.
Kingdom Animalia
Amphibia
African clawed frog Xenopus laevis (Yang et al., 1997); bullfrog Rana catesbeiana (Gross and Lapière, 1962; Oofusa et al., 1994);
Japanese newt Cynops pyrrhogaster (Suzuki et al., 2001); Eastern newt Notophthalmus viridescens (Vinarsky et al., 2005); Mexican axolotl
Ambystoma mexicanum (Yang and Bryant, 1994); green-striped burrowing frog Cyclorana albogutta (Hudson et al., 2007); Pacific tree frog
Pseudacris regilla (Veldhoen et al., 2006)
Reptilia
Spectacled caiman Caiman crocodilus apaporiensis (Shintani et al., 2007); tiger keelback snake Rhabdophis tigrinus tigrinus (Komori
et al., 2006)
Aves
Chicken Gallus gallus (Yang and Kurkinen, 1998); common canary Serinus canaria (Kim et al., 2008)
Actinopterygii (fish)
Rainbow trout Oncorhynchus mykiss (Saito et al., 2000); zebrafish Danio rerio (Wyatt et al., 2009); Atlantic salmon Salmo salar (Skugor
et al., 2008); Japanese pufferfish Takifugu rubripes (Tsukamoto et al., 2007); Amur catfish Silurus asotus (Cho et al., 2002); medaka fish
Oryzias latipes (Matsui et al., 2000); channel catfish Ictalurus punctatus (Yeh and Klesius, 2008)
Hydrozoa
Hydra Hydra vulgaris (Leontovich et al., 2000)
Echinoidea
Sea urchins Paracentrotus lividus and Hemicentrotus pulcherrimus (Gache et al., 2004)
Secernentea (nematodes)
Microbivorous roundworm Caenorhabditis elegans (Wada et al., 1998); parasitic nematode Gnathostoma spinigerum (Uparanukraw et al.,
2001); soybean cyst nematode Heterodera glycines (Kovaleva et al., 2004); potato cyst nematode Globodera rostochiensis (Kovaleva et al.,
2004)
Insecta
Fruitfly Drosophila melanogaster (Llano et al., 2000; 2002); wax moth Galleria mellonella (Altincicek and Vilcinskas, 2008); red flour beetle
Tribolium castaneum (Knorr et al., 2009)
Bivalvia (mollusks)
Eastern oyster Crassostrea virginica (M. Gomez-Chiarri, pers. comm.); abalone (sea snail) Haliotis diversicolor (Wang et al., 2008)
Kingdom Plantae
Soybean Glycine max (Ragster and Chrispeels, 1979); thale cress Arabidopsis thaliana (Maidment et al., 1999); cucumber Cucumis
sativus (Delorme et al., 2000); sugarcane Saccharum spp. (Ramos and Selistre-de-Araujo, 2001); barrel medic Medicago truncatula
(Combier et al., 2007); loblolly pine Pinus taeda (Ratnaparkhe et al., 2009); tobacco plant Nicotiana tabacum cv. BY-2 (Schiermeyer et al.,
2009)
Structure of an odontopathogenic metalloproteinase
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opens out into the active-site helix, which includes the first
half of a zinc-binding consensus sequence, HEXXHXXG/
NXXH/D (amino acid one-letter code; X for any residue),
which is characteristic of MMPs in particular and metz-
incins in general (Gomis-Rüth, 2003; 2009; Tallant et al.,
2010b). The first two histidines of the motif, His155 and
His159, bind the catalytic zinc ion through their Ne2
atoms. Imbedded between them is the general-base/acid
required for catalysis, Glu156. After this helix, the NTS
ends at the glycine of the consensus sequence (Gly162),
and the polypeptide enters the CTS. The latter residue
allows for a sharp turn in the trajectory of the polypeptide
to reach the third zinc ligand of the motif, His165.After this
residue, Ser166 forms a hydrogen bond with Asp187,
Table 2. Crystallographic data.
Data setKly18 with Mg2+
Kly18 without Mg2+
Space group/cell constants (a, b, c, in Å; b in ° if ? 90)
Wavelength (Å)
No. of measurements/unique reflections
Resolution range (Å) (outermost shell)a
Completeness (%)
Rmergeb
Rr.i.m. (= Rmeas)b/Rp.i.m.b
Average intensity (<[<I>/s(<I>)]>)
B-factor (Wilson) (Å2)/average multiplicity
Resolution range used for refinement (Å)
No. of reflections used (test set)
Crystallographic Rfactor (free Rfactor)b
No. of protein atoms/solvent molecules/ligands/ions
I4/82.79, 85.79, 53.48
0.9790
153 136/21 225
30.3–1.70 (1.79–1.70)
99.0 (95.9)
0.034 (0.121)
0.036 (0.139)/0.013 (0.065)
37.1 (10.8)
18.9/7.2 (4.4)
•–1.70
20 403 (820)
0.158 (0.186)
1328/196/
1 tripeptide (AFT)/2 Zn2+, 1 Mg2+, 1 Cl-
C2/121.4, 53.1, 86.3, 134.6
0.8726
62 645/15 411
45.3–2.40 (2.53–2.40)
99.3 (95.9)
0.088 (0.511)
0.101 (0.608)/0.050 (0.322)
14.8 (2.5)
38.4/4.1 (3.4)
•–2.40
14 703 (708)
0.189 (0.259)
2644/115/
1 tris, 1 tetrapeptide (AFTS)/2 Zn2+
Rmsd from target values
Bonds (Å)/angles (°)
Bonded B-factors (main-chain/side-chain) (Å2)
Average B-factors for protein/peptide ligand atoms (Å2)
Main-chain conformational angle analysisc
Residues in favoured regions/outliers/all residues
0.006/1.03
0.46/1.23
16.6/19.6
0.013/1.32
0.60/1.46
33.3/50.2
160/0/166315/0/332
a. Values in parentheses refer to the outermost resolution shell.
b. For definitions, see table 1 in Mallorquí-Fernández et al. (2008).
c. According to MOLPROBITY (Davis et al., 2007).
Fig. 1. Structure of Kly18.
A. Richardson-type plot of Kly18 in the commonly accepted standard orientation for MPs, i.e. with the view into the active-site cleft, which runs
horizontally from left [non-primed side; it accommodates substrate residues upstream of the cleavage bond at subsites S1, S2, S3, etc.
according to Schechter and Berger (1967)] to right (primed side; it binds downstream residues at S1′, S2′, S3′, etc.). The regular secondary
structure elements are shown as green arrows (b-strands bI to bV) and orange ribbons (helices aA to aC) and labelled. Residues participating
in catalytic-zinc binding, as well as the general base/acid glutamate, the Met-turn methionine, and the residues engaged in anchoring of the
N-terminus to the protein moiety are displayed as sticks and labelled. The cleavage-product peptide bound to the primed side of the active-site
cleft is shown as deep purple sticks. The zinc and magnesium ions are shown as magenta and blue spheres respectively. Chain segments
characteristic of MMPs and discussed in the text are further pinpointed. Helix aA is inserted after strand bI and runs across the back surface
of the molecule from top right to bottom left. Loop LbIaA is flexible at Ser54–His57 and was traced on the basis of weak electron density
maps. Helix aB follows strand bV and runs horizontally across the centre of the molecule, contributing to the back wall of the active-site cleft.
The two aforementioned helices contact each other through the side-chains of Ala70 (aA) and Ala154 (aB), which participate in a central
hydrophobic core together with residues from the sheet. In addition, a significant part of the ‘bulge-edge segment’ was likewise flexible in
Kly18 and was traced on the basis of weak electron density maps at Asp109–Thr112. After the S-loop and strand bIV, LbIVbV includes three
prolines in a row (Pro121–Pro123), which adopt a 1,4-turn structure and are followed by an alanine and two glycine residues. Thereafter, the
polypeptide enters strand bV and a large loop, LbVaB, which contributes to cleft delimitation and substrate binding.
B. Topology scheme of Kly18 illustrating the regular secondary structure elements and cations with the same colour code as in (A). Hydrogen
bonds and salt bridges are displayed as blue dashed lines.
C. Close-up view of (A) showing the active site and residues framing the specificity pocket or S1′ subsite.
D. Close-up view of (A) centred on the structural zinc ion.
E. Superposition in stereo of the unbound (the two molecules in the asymmetric unit are shown as cyan and purple sticks respectively) and
magnesium-bound (yellow sticks) Kly18 structures with the corresponding cleavage-product peptides as blue/deep purple and orange sticks
respectively. Variation between the magnesium-bound and -unbound structures was found at LbIaA (slight variation) and within the bulge-edge
segment of LbIIIbIV (large variation), which appears as essentially two conformers. The latter segment is folded inward in the
magnesium-bound structure and outward in the unbound structure molecules, leading to a maximal displacement of ~13 Å (measured at
Asn111 Ca). As the bulge-edge segment contributes to substrate binding on the primed side of the cleft, an outswung conformation should
enable binding of longer substrates. This resulted in that the peptide found in the cleft could be traced for one more residue, a tentative
serine, in the unbound structure due to additional electron density.
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which also binds the N-terminus of the molecule (see
above). Thereafter, four residues lead to a tight 1,4-turn of
type I (Ala171–Tyr174), termed the ‘Met-turn’ due to an
invariable methionine found at the third turn position in all
MMPs and metzincins (Gomis-Rüth, 2003; 2009; Tallant
et al., 2010a,b). This turn is stabilized by a double main-
chain interaction with the side-chain ofAsp188 within helix
aC (Fig. 1C). Between the Met-turn and this helix,
segmentPro175–Ty177,
segment’, shapes the lower rim of the cleft on its primed
side. Thereafter the chain enters the ‘specificity loop’
termed‘S1′-wallforming
(Gly179–Gln183), which delimitates the back of the S1′
subsite or specificity pocket of Kly18 (Fig. 1A and C). At
Asn186, the chain enters the C-terminal helix, aC, which
leads into the C-terminus on the molecular surface.
Kly18 was obtained as a serendipitous complex with a
peptide, which mimics a slightly shifted C-terminal cleav-
age product bound to the primed side of the cleft (Fig. 1A,
C and E). The high resolution of the electron density maps
enabledustoassignatripeptideofsequenceAla–Phe–Thr
in the magnesium-bound structure. The N-terminus of this
peptide is bound to both the catalytic zinc ion and the
Structure of an odontopathogenic metalloproteinase
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