Cryo-EM structure of a type IV secretion system

Macé, Kévin; Vadakkepat, Abhinav Koyamangalath; Redzej, Adam; Lukoyanova, Natalya; Oomen, Clasien J.; Braun, Nathalie; Ukleja, Marta; Lu, Fang; Costa, Tiago; Orlova, Elena V.; Baker, David; Cong, Qian; Waksman, Gabriel

doi:10.1038/s41586-022-04859-y

Cited by 72 publications

(156 citation statements)

References 77 publications

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“…The T-pilus has the required 5fold symmetry and diameter of only 76 Å. The structure is thus in agreement with the model of pilus assembly proposed by Macé et al (2022).…”

Section: Discussionsupporting

confidence: 86%

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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“…The T-pilus has the required 5fold symmetry and diameter of only 76 Å. The structure is thus in agreement with the model of pilus assembly proposed by Macé et al (2022).…”

Section: Discussionsupporting

confidence: 86%

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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“…Several T4SS proteins have been localized by immuno-localization at the cell pole (35-38). In A. tumefaciens the polar localization of VirB5 requires the presence of VirB8 and VirB9 (36), and VirB5-like proteins are also suggested to be present as in the inner membrane complex of the T4SS (4, 13). Here, we used two complementary microscopic approaches to localize TraC in permeabilized cells demonstrating accumulation at the cell perimeter and at the pole.…”

Section: Discussionmentioning

confidence: 99%

“…The general architecture of T4SS is well documented. They comprise an outer membrane core complex and an inner membrane complex that are connected by a stalk forming the translocation channel (4,(11)(12)(13). The cell surface-exposed pilus comprises the major pilin VirB2 and the minor pilus tip protein VirB5 that is believed to be involved in the recognition and adhesion to recipient bacteria during conjugation (14)(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

TraC of the conjugative plasmid pKM101 is secreted as a monomer in outer membrane vesicles and stimulates conjugation

Amro

Baron

2022

Preprint

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Gram-negative bacteria use membrane-bound type IV secretion systems to assemble pili on the cell surface, followed by cell-cell contact with recipient cells and transfer of plasmid DNA. The process at the cell-cell contact stage of conjugative DNA transfer is not well understood. We here present a biochemical and genetic characterization of the TraC protein that is a minor component of the pili determined by the IncN plasmid pKM101 from Escherichia coli. The cellular and secreted forms of TraC are monomers, TraC preferentially localizes at the cell poles and it is also detected in extracellular membrane vesicles. Purified TraC does not impact the infection with bacteriophages, but we detect binding of TraC to recipient cells and partial complementation of a traC deletion strain by the addition of purified TraC. These results suggest that the protein contributes to conjugation at the cell-cell contact stage.

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“…DeepMind has been using AF to model proteins of biomedical importance, and in partnership with European Molecular Biology Laboratory, they released 3D structures for over 200 million proteins from model organisms, human pathogens, and representative UniProt entries in the AlphaFold protein structure DataBase (AFDB) [17]. This breakthrough is transforming structural biology, where computation is becoming a key component in solving 3D structures of the most challenging and important protein complexes [18,19] and designing small molecule drugs to target specific structures [20,21]. This breakthrough in structure prediction is expected to guide the course of protein science in the near future by speeding up the discovery and characterization of proteins with novel and important functions.…”

Section: Introductionmentioning

confidence: 99%

DPAM: A Domain Parser for AlphaFold Models

Zhang

Schaeffer

Durham

et al. 2022

Preprint

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The recent breakthroughs in structure prediction, where methods such as AlphaFold demonstrated near atomic accuracy, herald a paradigm shift in structure biology. The 200 million high-accuracy models released in the AlphaFold Database are expected to guide protein science in the coming decades. Partitioning these AlphaFold models into domains and subsequently assigning them to our evolutionary hierarchy provides an efficient way to gain functional insights of proteins. However, classifying such a large number of predicted structures challenges the infrastructure of current structure classifications, including our Evolutionary Classification of protein Domains (ECOD). Better computational tools are urgently needed to automatically parse and classify domains from AlphaFold models. Here we present a Domain Parser for AlphaFold Models (DPAM) that can automatically recognize globular domains from these models based on predicted aligned errors, inter-residue distances in 3D structures, and ECOD domains found by sequence (HHsuite) and structural (DALI) similarity searches. Based on a benchmark of 18,759 AlphaFold models, we demonstrated that DPAM could recognize 99.5% domains and assign correct boundaries for 85.2% of them, significantly outperforming structure-based domain parsers and homology-based domain assignment using ECOD domains found by HHsuite or DALI. Application of DPAM to the massive set of AlphaFold models will allow for more efficient classification of domains, providing evolutionary contexts and facilitating functional studies.

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Cryo-EM structure of a type IV secretion system

Cited by 72 publications

References 77 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

TraC of the conjugative plasmid pKM101 is secreted as a monomer in outer membrane vesicles and stimulates conjugation

DPAM: A Domain Parser for AlphaFold Models

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