Cryo-EM structure of the <i>Agrobacterium tumefaciens</i> T-pilus reveals the importance of positive charges in the lumen

Amro, Jaafar; Black, Corbin; Jemouai, Zakaria; Rooney, Nathan; Daneault, Caroline; Zeytuni, N.; Ruiz, Matthieu; Bui, Khanh Huy; Baron, Christian

doi:10.1101/2022.04.28.489814

Cited by 2 publications

(2 citation statements)

References 50 publications

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“…In a contemporary study, Amro et al also resolved the structure of the A. tumefaciens T-pilus at comparable 3.2 Å resolution (Amro et al, 2022). While their structure and ours are very similar, they identified the lipid as PC.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of the Agrobacterium tumefaciens type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

Kreida

Narita

Johnson

et al. 2022

Preprint

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Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease by the horizontal transfer of oncogenic DNA that is integrated into the host’s genome. The conjugation is mediated by the conjugative VirB/D4 type 4 secretion system (T4SS). A. tumefaciens T4SS assembles an extracellular filament, the T-pilus, that is involved in the formation of a mating pair between A. tumefaciens and the recipient plant cell by a not fully understood mechanism. Here, we present a 3 Å cryo-EM structure of the T-pilus, solved by helical reconstruction. Our structure reveals that the T-pilus comprises the major pilin protein VirB2 and phosphatidylglycerol (PG) phospholipid at a 1:1 stoichiometric ratio with 5-start helical symmetry. We further show that PG-headgroups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 destabilized the VirB2 protein and completely abolished pilus formation. While our T-pilus structure shows architectural similarity with previously published conjugative pili structures, positively charged sidechains protrude into the lumen and the lumen is narrower, raising questions whether the T-pilus is a conduit for ssDNA transfer. We also show that the VirB2 subunits in T-pilus filament are not cyclic, as previously thought.

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Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of the Agrobacterium tumefaciens type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

Kreida

Narita

Johnson

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…At this time, high-resolution structures have been presented for several T4SS subunits, including the VirD4 T4CP and the VirB4 and VirB11 ATPases that energize substrate recruitment and trafficking [5,8,9]. Additionally, structures have been solved of OMCCs from several model T4SSs [10][11][12][13][14][15] and a number of conjugative pili [16][17][18][19]. Most recently, a nearly intact T4SS encoded by conjugative plasmid R388 (T4SS R388 ) was presented at near-atomic resolution [5].…”

Section: Introductionmentioning

confidence: 99%

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines

Kishida,

Li,

Ogawa-Kishida

et al. 2023

Preprint

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Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate - TraD and TraD - T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.AUTHOR SUMMARYMobile genetic elements (MGEs) comprise a diverse group of extrachromosomal plasmids or integrated DNA fragments that are widely distributed among many bacterial species. MGEs typically encode conjugation systems dedicated to their transmission to other bacteria, and also code for resistance to antibiotics or virulence or other fitness traits. The conjugation systems, along with an equally medically important group of translocators devoted to the interkingdom delivery of protein effectors by pathogenic species, comprise the superfamily of type IV secretion systems (T4SSs). Recent studies have defined many mechanistic and structural features of the T4SSs, yet there remains little understanding of how T4SSs recruit specific DNA or protein substrates, elaborate functional channels, and in some cases build attachment organelles termed conjugative pili. We explored the mechanics of T4SS machine function by systematically exchanging individual components between two distinct conjugation systems functioning in enterobacterial species. Through construction of chimeric machines, and further mutational analyses, we identified subunits or protein domains of conjugation machines specifying recruitment of distinct DNA substrates or selectively contributing to assembly of translocation channels or conjugative pili. Such features of T4SSs are prime targets for development of inhibitory strategies aimed at blocking T4SS functions for therapeutic intervention.

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Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

Cited by 2 publications

References 50 publications

Cryo-EM structure of the Agrobacterium tumefaciens type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

Cryo-EM structure of the Agrobacterium tumefaciens type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines

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