🚨Preprint! Happy to share the research from my PhD “Genome delivery of a contractile tailed phage and its superinfection exclusion mechanism”. We use cryoEM to study the genome ejection of the phage T4, revealing how the tape measure protein regulates the process.
www.biorxiv.org/content/10.6...
A jumbo cyanophage encodes the most complete ribosomal protein set in the known virosphere www.biorxiv.org/content/10.1...
"In summary, our work indicates that the Tad ATPase CpaF alternately engages its cognate platform using distinct hexameric faces, and we define the structural and evolutionary determinants of this capacity, thus positioning the Tad system as the paradigm for a new class of bifunctional motors."
Added bonus! Greg’s analysis identified opposing conformational changes in the motor during the ATP hydrolysis cycle that may influence whether major pilins are incorporated into or removed from the pilus filament depending on orientation.
We tested all the other type IV filament system motors and the only other one to adopt both orientations was the archael Epd pilus. This matches the nice work from @epcrocha.bsky.social showing that Tad pili were inherited from Archaea.
Thus, conformational changes in N-terminal helices of platform proteins dictate the orientation of the ATPase motor.
In retraction models, these N-terminal platform protein helices intersect the space occupied by the motor CpaF N1 domain in extension models, creating steric clashes that could favor inversion of the motor.
Comparing extension and retraction complex conformation reveals large changes in the platform proteins N-terminal helices.
So we have strong evidence for the Tad motor flipping which face of the toroid engages with the IM platform to drive extension vs retraction. But what drives changes in orientation?
Here is a movie of the motor-mCherry fusion.
Adding mCherry on the retraction face of the motor also gave a retraction-deficient phenotype.
Here is my favorite mutant. Yes, it’s a movie! Crazy, right?
Single residue mutation of key extension orientation to platform interaction residues gave a retraction-deficient phenotype. Yes, it's a movie. Amazing!
Here is a pili dynamics movie of wild-type.
We aligned sequences of single- and dual-orientation motors, showing significant differences in the C-terminus, corresponding to the platform protein interaction interface in the retraction conformation models. We targeted key residues for the retraction complex formation for mutagenesis.
In fact, Tad motors from multiple species are predicted to adopt either orientation with the IM platform. Yet some Tad motors were only modeled in one orientation. This provided a strategy to identify sequence features unique to dual-orientation motors.
AF3 modeling says both Tad motor orientations with the IM platform are equally confident. In fact, we get either orientation when we run predictions. It’s like flipping a coin! Crazy, right?
When you look at T4a orthologous motors, you see that they engage with the IM platform protein in opposite orientation. Here is a crazy thought: could the single Tad motor flip and engage the platform in the opposite orientation, thereby mimicking the two T4a motors? Crazy?
Do I sound desperate yet? We were still in the dark until @gregbwhitfield.bsky.social had a genius idea.
Some bidirectional motors achieve reversibility by swapping distinct sets of unidirectional motors with opposing activities; others utilize positionally-fixed, bifunctional motors that reverse activity by defined conformational transitions of regulatory subunits. No evidence Tad does this.
Our work with @guo-lab.bsky.social solving the cryoET in situ structure of the Tad motor also did not give hints about how a single motor could do both jobs. Also see cryoET work by @viollierpat.bsky.social & Grant Jensen www.biorxiv.org/content/10.1...
Structural analysis with Lynne Howell and @ianyyen.bsky.social provided strong mechanistic support for a unidirectional catalytic cycle driving pilus extension, but no clues about retraction www.nature.com/articles/s41.... Also see earlier work by @lowlab.bsky.social
@ckellison.bsky.social tried to find a non-orthologous retraction motor without success. A genetic screen showed mutations in the motor ATP binding site reduced extension & retraction in a correlated manner. So the single ATPase does both jobs, but how? www.science.org/doi/10.1126/...
The best studied type IV pili, like the T4a Pseudomonas pili, use one ATPase to extend and one to retract. But Tad pili only have a single orthologous T4P ATPase that is required for extension. So what drives the retraction?
Years ago, @ckellison.bsky.social developed a live cell pili labeling method to study pili dynamics. This work showed that Tad pili have dynamic cycles of extension and retraction. But how? www.science.org/doi/10.1126/...
🧵 Proud to present a tour de force by postdoc @gregbwhitfield.bsky.social solving the mystery of how bacterial Tad pili can extend and retract with a single motor ATPase. Great collaboration with Lynne Howell, @dr-lori-burrows.bsky.social, @ianyyen.bsky.social www.biorxiv.org/content/10.6...
This is a tour de force by @gregbwhitfield.bsky.social who I just convinced to join Bluesky.
Maybe a single image is worth a thousand thread words:
Working on a 🧵 Bacterial Tad pili extend
and retract pili using a single motor ATPase with a unidirectional catalytic mechanism. We find that the Tad motor achieves bidirectionality by physical inversion; alternating which face of the ATPase toroid engages the IM platform complex. #microsky
Weird right? It took me all those years to follow you to the dark side ;-)
