Very proud that our manuscript on the assembly and aging of actin filaments (and my first publication as a postdoc) is finally online @Nature (open access!)
nature.com/articles/s4158…
This was a great team effort from the @Intein lab @mpimoph.
A thread (1/17)
Actin is a highly abundant protein and a major component of the eukaryotic cytoskeleton. It exists in two main forms, as monomer (G-actin) and as filament (F-actin). The dynamic turnover of actin filaments is crucial for controlling cell shape, polarity and movement.
(2/17)
After polymerization, F-actin rapidly hydrolyzes ATP and undergoes transitions dependent on its bound nucleotide. 'Aged' ADP-F-actin can be depolymerized back to G-actin.
We aimed to visualize and understand F-actin assembly and aging at the highest molecular detail.
(3/17)
Through an optimized cryo-EM workflow using @SPHIRE_Package and #relion, we determined structures of Mg-F-actin in three nucleotide states (ATP-mimic, ADP-Pi, ADP) at ~2.2 Å resolution.
The high structural detail allowed for the modeling of hundreds of water molecules.
(4/17)
Mg is bound to actin in vivo. However, actin is standardly purified in buffer containing Ca. To assess the effect of the bound cation on the F-actin structure, we also determined structures of Ca-F-actin at similarly high resolutions.
(5/17)
When comparing the six nucleotide-state structures, we globally observed no major structural rearrangements.
Thus, the differences were in the details. But luckily, at these resolutions, the structures displayed a lot of detail :)
(6/17)
First, by comparing our cryo-EM structures with x-ray structures of G-actin, we could show that the G- to F-actin transition triggers the relocation of water molecules near the ATP binding site. This explains why F-actin hydrolyzes ATP within seconds but G-actin does not.
(7/17)
To recapitulate the video: In Mg-actin, water molecules are relocated during the G- to F-actin transition but the Mg-ion coordinating waters (in magenta) are not altered, retaining an octahedral coordination.
But why is the polymerization rate of Ca-actin so much slower?
(8/17)
As it turns out, in Ca-actin, water relocation necessitates the movement of a water (Wx) within the coordination sphere of Ca.
We propose that this required rearrangement of the Ca-coordination sphere could pose a kinetic barrier for the G- to F-actin transition.
(9/17)
Zooming in on ATP hydrolysis in F-actin: We observed a water hydrogen-bonded to Q137 within 3.6 Å of the Pγ-analog, which likely is the nucleophilic water (Wnuc) that hydrolyzes ATP.
The position of Wnuc is further stabilized by another water (Wbridge), D154 and H161
(10/17)
We next compared the ADP-Pi and ADP states of F-actin to investigate the mechanism of Pi release.
Pi release is predicted to involve a 'back door' of residues R177 and N111. Interestingly, the back door was previously proposed to be open in ADP-F-actin.
(11/17)
To our surprise, the back door is closed in structures of both ADP-Pi and ADP F-actin, indicating that the F-actin conformation that allows for the exit of Pi is a transient state.
We envision that the Pi release mechanism can be further explored in future experiments
(12/17)
Finally, a dogma in the field is that changes in the F-actin nucleotide are coupled to the filament periphery. Our structures now suggest a path for how the nucleotide-binding site and the F-actin C-terminus are coupled.
Please check the paper for many more details!
(13/17)
We hope that our work will be of valuable interest to all cytoskeleton enthousiasts, and hope that it will now also pave the way for high-resolution structures of F-actin bound to ABPs.
In general, these are extremely exciting time in the actin structural biology field.
(14/17)
Complementary to our work, a beautiful study by the @Alushin_Lab lab, also out in @Nature today, investigated the bending of F-actin, see: nature.com/articles/s4158…
See also the associated News and Views article discussing both our papers: nature.com/articles/d4158…
(15/17)
In addition, the Maeda and Takeda labs determined high-resolution x-ray structures of monomeric actin in an F-like conformation.
doi.org/10.1073/pnas.2…
(16/17)
I would like to thank @Intein for the invaluable support and @alexanderbelyy1, Björn & Sabrina for their great contributions.
Finally, I gratefully acknowledge the @AvHStiftung for funding my postdoctoral fellowship. Much more exciting science to come, watch this spot!
(17/17)
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