Structure of a tripartite protein complex that targets toxins to the type VII secretion system
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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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Not yet, however there are certainly hopes to explore the structure and dynamics of the complex in solution sometime in the future. We suspect dissociation of the complex is an important part of the export-mechanism, based on the precedent of PE-PPE complexes from the T7SSa.
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Yes I agree this is a fun avenue to explore! There is certainly precedent for proteins with similar high-level structural-functional descriptions (i.e. small-alphahelical proteins required for the secretion of effector substrates) within other bacterial secretion systems. However I think we need to uncover a lot more about the dynamics of the T7SS and the role these small proteins play before we can determine if these similarities extend beyond the shape of the proteins.
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We more-or-less expected this based on our previous findings published in mBio last year (PMID: 36036513), as the Alphafold-multimer models were quite convincing. However the crystallographic evidence is invaluable in proving that LXG's and their partners are structurally similar to the T7SSa targeting domains.
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Yes, we agree! It's remarkable how divergent each Lap protein sequence is from other homologues; yet a high degree of shape complementarity is almost always predicted with their partner LXG domains.
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a.
Curious if you ever examined the structure of this complex in-solution any other way? Or have any guesses about how many different folded states it can occupy in solution?
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Alphafold2 predicted structure
What about have you looked for structural cousins of any of the Lap proteins? Would be fun to see what other organisms encode these and whether it ever includes other secretion systems (or even eukaryotes)?
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many similarities to structurally characterized T7SSa effectors
Were you surprised by this?
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Remarkably, despite the relatively small size of each of the subunits within this complex, the buried surface areas between TelALXG-LapA3 and TelALXG-LapA4 are 2261.5Å2 and 1810.4Å2, respectively
This is super cool. It's amazing how much sequence diversity allows for this intimate/elongated of an interaction
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