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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 [email protected]. 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 Beddoe T, Paton AW, Le Nours J, Rossjohn J, Paton JC (2010). Trends in biochemical sciences 35: 411-418 Bode W, Gomis-Rüth FX, Stöcker W (1993). FEBS Lett 331: 134-140 Dubin G, Koziel J, Pyrc K, Wladyka B, Potempa J (2013). Current pharmaceutical design 19: 10901113 Gomis-Rüth FX (2003). Mol Biotech 24: 157-202 Gomis-Rüth FX (2009). J Biol Chem 284: 15353-15357 Gomis-Rüth FX, Botelho TO, Bode W (2012). Biochim Biophys Acta 1824: 157-163 Jansson L, Angstrom J, Lebens M, Imberty A, Varrot A, Teneberg S (2010). Biochimie 92: 482-490 Karasawa T, Ito H, Tsukamoto T, Yamasaki S, Kurazono H, Faruque SM, Nair GB, Nishibuchi M, Takeda Y (2002). Infection and immunity 70: 7153-7155 Le Nours J, Paton AW, Byres E, Troy S, Herdman BP, Johnson MD, Paton JC, Rossjohn J, Beddoe T (2013). J Biol Chem 288: 27505-27516 Merritt EA, Hol WG (1995). Current opinion in structural biology 5: 165-171 Ng NM, Littler DR, Paton AW, Le Nours J, Rossjohn J, Paton JC, Beddoe T (2013). Structure THIS ISSUE. Stöcker W, Grams F, Baumann U, Reinemer P, Gomis-Rüth FX, McKay DB, Bode W (1995). Prot Sci 4: 823-840 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).