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 Å 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 α-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.

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“…4A). Recent analysis of other T7SSb LXG toxins has revealed a common mechanistic feature for secretion, involving formation of a hetero-oligomeric pre-secretion complex between the LXG domain and helical hairpin partner proteins encoded at the same genetic locus as the toxin [24, 26, 27, 37]. Logically, the TspA LXG domain should also be expected to require similar helical binding partners to generate a secretion-competent structure.…”

“…Previous work has reported that LXG proteins form a stable pre-secretion complex with their cognate helical hairpin secretion partners [23, 24, 26, 27]. To investigate whether any of LapT1, LapT2 or SACOL2601 interact with TspA, we co-produced all four proteins in E. coli .…”

“…The third protein encoded at this locus, SACOL2601, has a DUF4176 domain. Genes encoding DUF4176 domains are frequently associated with T7SS loci [3] and the involvement of a DUF4176 domain-protein in LXG toxin secretion was recently determined in S. intermedius [27].…”

“…Prior to secretion, the co-encoded WXG100-like proteins, which have been termed Laps (for L XG- a ssociated α-helical p roteins) bind to the LXG domain of the cognate toxin partner. This interaction generates a secretion-competent rod-shaped complex, and conserved sequence motifs on both the LXG domain and one of the Laps constitute a T7SSb-targeting signal [23, 24, 26, 27]. In some instances, a globular protein is also encoded at the toxin genetic locus, which also facilitates toxin secretion [24, 27].…”

“…This interaction generates a secretion-competent rod-shaped complex, and conserved sequence motifs on both the LXG domain and one of the Laps constitute a T7SSb-targeting signal [23, 24, 26, 27]. In some instances, a globular protein is also encoded at the toxin genetic locus, which also facilitates toxin secretion [24, 27].…”