Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

Jäger, Franziska; Lamy, Anaïs; Sun, Wei-Sheng; Guerini, Nina; Berntsson, R.P.-A.

doi:10.1016/j.str.2022.03.013

Cited by 3 publications

(4 citation statements)

References 73 publications

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“…We speculate that PrgL binds PrgK to localize it to the T4SS channel during channel assembly. This is in line with the previous hypothesis that PrgL, as other VirB8-like proteins, acts as a scaffold during the biogenesis of the T4SS 35 .…”

Section: Discussionsupporting

confidence: 93%

“…To validate the predicted interaction between PrgK and PrgL, we tested whether the purified extracellular domains of the two proteins (PrgKEC and PrgL32-208) could form a complex and co-elute from size exclusion chromatography. PrgL32-208 forms elongated dimers in solution 35 , which gives it a similar elution volume as PrgKEC (Fig. 2B).…”

Section: Prgk Interacts With Prgl In Vitromentioning

confidence: 84%

“…This analysis suggested a PrgK:PrgL heterodimer with reasonable confidence. In this AF model, the VirB8-like domain of PrgL (PrgL32-208) 35 interacts with the SLT domain of PrgK (Fig. 2A & S4A-B).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we report the crystal structure of the LytM domain and combine AlphaFold models with biochemistry to analyze the remaining part of the extracellular domains. Alphafold modelling suggested that the extracellular domain of PrgK interacts with that of PrgL, another pCF10 encoded T4SS protein that plays a role in T4SS biogenesis 35 . We confirm this interaction in vitro and show that PrgK can also dimerize in a redox dependent manner.…”

Section: Introductionmentioning

confidence: 99%

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Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Sun,

Torrens,

Beek

et al. 2024

Preprint

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Conjugative Type 4 Secretion Systems (T4SS) are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient mating bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has 3 predicted extracellular hydrolase domains, LytM, SLT and CHAP. Here, we report the structure of the LytM domain, and show that its active site is degenerate and lacks the active site metal. Further, we show that only the predicted SLT domain is functional, and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Further, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information towards understanding the function of Gram-positive T4SSs.

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Section: Discussionsupporting

confidence: 93%

Section: Prgk Interacts With Prgl In Vitromentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Sun,

Torrens,

Beek

et al. 2024

Preprint

Self Cite

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A new twin expands the VirB8-like protein family

2022

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Breaking barriers: pCF10 type 4 secretion system relies on a self-regulating muramidase to modulate the cell wall

Sun,

Torrens,

ter Beek

et al. 2024

mBio

View full text Add to dashboard Cite

Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs. IMPORTANCE Antibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria.

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Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

Cited by 3 publications

References 73 publications

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

A new twin expands the VirB8-like protein family

Breaking barriers: pCF10 type 4 secretion system relies on a self-regulating muramidase to modulate the cell wall

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