Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Abstract

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Angstrom structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated alpha-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

Interbacterial competition mediated by the type VIIb secretion system

“…Although only low-resolution volumes were determined, comparison with AlphaFold predictions suggest that the helical shaft comprises EsaD LXG -EsxBCD and has a cross-sectional diameter of 25–30 Å, consistent with the predicted width of the EssC channel () [109]. These findings are in agreement with the recently reported high resolution crystal structure of a TelA LXG -LapA3-LapA4 complex, with a similar helical rod conformation of ~180 Å × ~30 Å [72]. Both of these structures are consistent with AlphaFold predictions and bear a striking resemblance to structurally characterised T7SSa substrates, such as EspB or PE5-PPE4 () [111, 114, 116].…”

“…The term LXG was introduced on account of a conserved (L/F)xG sequence motif within these proteins [66, 67]. LXG domains are specific to the T7SSb and target the protein to the secretion apparatus [72]. No functions have yet been ascribed to the central regions of these proteins, which are not conserved, are of variable length, and are sometimes annotated as a ‘PT’ (pre-toxin) domain.…”

“…Here the genetic organisation is slightly different with a third protein encoded between the WXG100-like pair and the toxin (). Lap3 and Lap4 form a stable complex with their cognate TelA/B toxin, and so are predicted to function analogously to Lap1 and Lap2 [67, 72], leading to speculation that all LXG proteins might require a pair of WXG100-like proteins. In agreement with this hypothesis it was recently reported that three small WXG100-like proteins are required for secretion of the S.…”

An interbacterial lipase toxin with an unprecedented reverse domain arrangement defines a new class of type VII secretion system effector

“…While these were originally identified as effector proteins e.g. 18 , it is becoming increasingly clear that at least some of them also serve as stabilising and/or targeting factors for larger T7SS substrates, with which they are cosecreted 10,19,20,21 . The LXG proteins are large antibacterial toxins secreted by the T7SSb, which form complexes with two or three WXG100-like partners.…”

“…The LXG proteins are large antibacterial toxins secreted by the T7SSb, which form complexes with two or three WXG100-like partners. These small partners, which have been designated Lap (LXG-associated -helical protein) interact with the N-terminal helical LXG domain and are predicted to form a helical stack 19,20,21 . Larger substrates of the T7SSa include the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) proteins.…”