CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ
Posted on: 3 April 2020
Preprint posted on 4 March 2020
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-020-18866-y
Weaver et al. present the first high-resolution structure of the Type IV secretin, PilQ from V. cholerae; providing insight into how it facilitates natural transformation in bacteria, and paving the way for future studies of its mechanism.
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Categories: biochemistry, biophysics, microbiology
Context
Bacteria take up DNA from the extracellular environment in a process called natural transformation. As one of several mechanisms of horizontal gene transfer in prokaryotes, this allows the rapid spread of genetic information across bacterial species, including the acquisition of antibiotic resistance genes. In Gram-negative bacteria, this process is mediated by the Type IV secretion system, which functions to import exogenous DNA. The Type IV secretion system is made up of four main protein complexes that assemble to span the bacterial cell envelope: 1) the pilus, a tube structure that projects into extracellular environment, responsible for the binding and uptake of DNA; 2) motors in the cytoplasm that drive pilus movement; 3) an inner membrane complex responsible for assembling the pilus; and 4) an outer membrane secretin, which forms a pore through which the pilus extends outside the cell. Until now, studying Type IV secretion system function has been limited by the absence of high resolution structures of its components. Here the authors solve the first structure of a Type IV secretion system secretin, PilQ from Vibrio cholerae, which provides insights into the protein complex that allows the pilus to traverse the outer membrane.
Methods
The authors solve the structure of V. cholerae PilQ to 2.7 Å resolution using single particle Cryo-EM, and compare it to the structures of secretins from the Type II and Type III secretion systems. Using structure-guided mutagenesis, the authors functionally characterize mutants that shed light on the role of the Type IV secretion system in natural transformation. The authors dock the high-resolution PilQ structure into in situ cryo-electron tomography (cryo-ET) maps of M. xanthus Type IV secretin, identifying the conformational state of the high-resolution PilQ structure in the context of native cell membranes.
Key Results
1. High-resolution Cryo-EM structure of PilQ
With a high resolution cryo-EM structure in hand, the authors compared PilQ to related secretins, identifying both similar features and notable differences. The Type IV secretin consists of 14 monomers that assemble to form a hollow tube, creating a channel through the outer membrane. This assembly is similar to Type II and Type III secretins, though the number of subunits varies in each system. Halfway along its length, the diameter of the channel constricts, and may act as a gate to restrict passage of substrates through the secretin. PilQ has a short helix connecting the N0 and N3 domains, which is unstructured in other secretins. The orientation of the N0-N3 helix causes the diameter of the PilQ pore to vary on the periplasmic side of the gate, ranging from 90Å in the N0 domain to 60Å in the N3 domain. This is in contrast to Type II and Type III secretins, where the diameter through the pore is constant. Additionally, while secretins of Type II and III secretion systems have alternating positive and negative regions internally, the Type IV PilQ is mostly negative, suggesting a possible mechanism allowing negatively charged DNA to move through its pore.
2. Locking the PilQ gate interferes with natural transformation
DNA is thought to be taken up through the pilus during natural transformation. To gain insight into how DNA uptake is controlled, two cysteine-pair mutants were introduced in the gating regions of PilQ, with the goal of reversibly locking the pore gate in a closed state through disulfide bonding. Transformation frequency was used as a measure of Type IV secretin function under the gated conditions. The “locked” mutants had decreased transformation frequency, suggesting that DNA uptake was being blocked. Upon DTT addition to break the disulfide bonds and unlock the gate, transformation frequency increased with increasing DTT concentration, suggesting that an open conformation of PilQ is required for DNA uptake.
3. High-resolution structure corresponds to the closed-gate conformation of PilQ observed by cryo-ET in situ
To determine what region of PilQ was embedded in the bacterial outer membrane, the PilQ structure was docked into a previously constructed cryo-ET of the M. xanthus Type IV system. Their structure is in good agreement with the non-piliated state of the Type IV secretion system. However, the PilQ structure does not fit into the cryo-ET reconstructions of the piliated state, suggesting that PilQ would require a significant conformational change in order to accomodate a pilus through the pore.
Impact of Key Findings
This work determined the first high-resolution structure of the Type IV secretin, PilQ from V. cholerae. This structure provides insights into how the Type IV secretion system facilitates natural transformation in bacteria, and paves the way for deeper study on the mechanisms of Type IV system-dependent gene transfer.
Open questions raised by study
- How might the secretin structure change in the presence of the pilus and other associated proteins?
- What is the structural and functional conservation of Type IV secretion across bacterial species?
- Is there a role for the N0-N3 connecting helix in regulating channel dynamics?
doi: https://doi.org/10.1242/prelights.18075
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