PREVIEW ON ARTICLE “EcxAB is a founding member of a new family of
metalloprotease AB5 toxins with a hybrid cholera-like B subunit”
A different look for AB5 toxins
F.Xavier Gomis-Rüth
Proteolysis Lab; Molecular Biology Institute of Barcelona, CSIC; Barcelona
Science Park; Helix Building; c/ Baldiri Reixac, 15-21; 08028 Barcelona
(Catalonia, Spain).
Phone +34-934 020 186. Fax +34-934 034 979. E-mail fxgr@ibmb.csic.es.
Summary
Metzincins are a distinct clan of metallopeptidases encompassing several
families. In this issue, Ng et al. describe results of structural analysis of the
toxilysins, a novel family of metzincins employed by gastroinfective bacteria as
intracellular virulence factors following host cell invasion.
Pathogenic bacteria employ a plethora of virulence factors to disrupt the
function of infected host cells (Dubin et al, 2013). Among such factors are the AB5
toxins, which are complexes between five copies of a B subunit employed for
attachment to the host plasma membrane and a single catalytic A subunit, which
subverts cellular functions once the toxin complex has been internalized (Merritt
& Hol, 1995). Four AB5 families have been reported based on the function of the
catalytic subunit: the Shiga toxins, which interfere with protein biosynthesis; the
pertussis and cholera toxins, which affect regulation of fluid secretion; and the
subtilase cytotoxins, which have a subtilisin-type serine proteinase as A subunit
that cleaves an endoplasmic reticulum chaperone (Beddoe et al, 2010; Le Nours
et al, 2013). More recently, a fifth family was identified in diarrhea-causing
strains of Escherichia coli and Citrobacter freundii represented by toxins EcxAB
and CfxAB, respectively (Jansson et al, 2010; Karasawa et al, 2002).
In the present issue of Structure, Ng, Littler and co-workers present the crystal
structure of the E. coli EcxAB toxin complex (Ng et al, 2013), which allows them
to determine the molecular determinants of function of this heterohexameric
virulence factor: similarly to previous AB5 complex structures, the B subunits are
arranged in a ring around a central pore aligned with a fivefold axis. The A
subunit sits on top of this ring and introduces its C-terminal helical tail into the
pore of the B pentamer. The authors reveal that the catalytic EcxA subunit is a
metallopeptidase (MP) that is unrelated to any other AB5 family. It presents
structural similarity with matrix metalloproteinases, which are a family within
the metzincin clan of MPs. However, EcxA has distinct extra structural elements
that give rise to a fold different from any metzincin structurally characterized to
date (Gomis-Rüth, 2003; Gomis-Rüth, 2009). Accordingly, the authors propose
the name toxilysins to refer to a new family comprising EcxA and several CfxAtype proteins.
Metzincins were discovered back in 1993 by Bode and colleagues (Bode et al,
1993) acknowledging structural similarities among three MP prototypes that
had been structurally characterized by then, namely crayfish astacin (astacin
family), snake-venom adamalysin II (adamalysin/ADAM family), and
Pseudomonas aeruginosa alkaline proteinase (serralysin family)(Stöcker et al,
1995). Despite negligible sequence identity and distinct lengths of their
polypeptide chains, these enzymes showed topologically-equivalent structural
features: subdivison of the catalytic moieties into an upper N-terminal (NTS) and
a lower C-terminal sub-domain (CTS) by an active-site cleft harboring the
catalytic metal ion at its bottom; two helices termed the “backing helix” and the
“active-site helix” within the NTS; a five-stranded mixed parallel/antiparallel βsheet, likewise in the NTS, whose lowermost strand created the upper rim of the
active-site cleft; a “C-terminal helix” at the end of the CTS; and, most noteworthy,
a strictly conserved 1,4-turn immediately below the metal site featuring a
methionine in its third position, the “Met-turn”. In addition, the active-site helix
and the subsequent part of the polypeptide included the extended zinc-binding
consensus sequence, HEXXHXXGXXH, with the three histidines liganding the
catalytic metal ion, the glutamate acting as a general base/acid during catalysis,
and the glycine accounting for a characteristic sharp change in the direction of
the polypeptide after the active-site helix to reach the third histidine. Thereafter,
the catalytic domains of other MPs were successively reported. All featured the
aforementioned common structural traits despite minute sequence similarity, as
well as family-specific structural elements, additional ion-binding sites, inserted
domains, and upstream and downstream polypeptide extensions that caused
them to span between ~130 and ~270 residues. These MPs became the founding
members of novel families within the metzincins: matrix metalloproteinases or
matrixins (reported for their structure for the first time in 1994),
leishmanolysins
(1995),
snapalysins
(1997),
pappalysins
(2006),
archaemetzincins (2010), fragilysins (2010), igalysins (deposited with the
Protein Data Bank in 2011 though not published), and cholerilysins (2012). The
present work reveals that EcxA comprises all the characteristic elements of
metzincins (Fig. 1), and we welcome the founding member of a new metzincin
family, the toxilysins.
References
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Figure 1 – Ribbon-type plot of the A subunit of AB5 toxin. The catalytic
moiety of the founding member of the toxilysin family of metzincin MPs, EcxA, is
shown in standard orientation (Gomis-Rüth et al, 2012), i.e. with a view into the
active-site cleft. The catalytic metal ion (magenta sphere) is bound by the three
histidines (brown sticks) comprised in the currently revised extended zincbinding signature characteristic for metzincins, HEXXHXX(G/N)XX(H/D). The
general base/acid glutamate is in pink, the Met-turn with its methionine side
chain is in blue, the family specific residue is in red, and a disulfide bond, which
ligates the upstream A1 part with the downstream A2 part of the A subunit, is
depicted in yellow. Characteristic regular secondary-structure elements of
metzincins are three α-helices (in red) and four or five β-strands arranged in a
sheet in the N-terminal upper sub-domain (in yellow).