Helicase Review

[TomkUCL]

This section includes a living Google doc form of the review on helicases as therapeutic targets, which can be viewed and edited through the following link. https://docs.google.com/document/d/14GnyDPPl17mi56FxNwPg876HOr_SrafZYY_3j36Srg8/edit?usp=sharing

Our current tasks are:

1. Agree on the scope, outline, and length of the review (found on the first page of the google doc), adding any comments if you feel that something is missing or should be omitted.

2. The division of labour; once 1) is agreed upon, we can decide who will be responsible for each of the sections and whether there will be author overlap in any sections.

Please add any further points here for discussion.

[H-agha]

Check this recent book named RNA Remodeling Proteins, Methods and Protocols https://link.springer.com/book/10.1007/978-1-0716-0935-4

[TomkUCL]

Here is the paper discussed in today's meeting, I recommend checking the videos in the SI, which give a good indication of the movement of nsp13 within the RTC based on cryo-EM structures and MD simulations. In particular, the movement of the RecA2 domain, where our pocket is located: Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication–transcription complex. Nature Structural & Molecular Biology | VOL 29 | March 2022 | 250–260. https://www.nature.com/articles/s41594-022-00734-6.pdf

nsp13T-RTC consensus model: 41594_2022_734_MOESM5_ESM.zip

RTC RNA backtracking mechanism: 41594_2022_734_MOESM4_ESM (1).zip

This is built on earlier papers from 2020 and 2021, found here:

Architecture of a SARS-CoV-2 mini replication and transcription complex https://doi.org/10.1038/s41467-020-19770-1

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis https://www.sciencedirect.com/science/article/pii/S0092867420315336

Joe Newman’s fragments (and our pocket) are based on the nsp13 homodimer (PDB ID 6ZSL), However, the recent RTC complex (feat 2 copies of nsp13) does not seem to have our pocket – I want to overlay these structures on PyMOL – question: will our fragments bind to the nsp13 BEFORE it forms part of the RTC complex AND/OR prevent it binding to the other nsps? https://doi.org/10.1038/s41594-022-00734-6

The nsp13 RecA2 domain seems to be critical in the transition from the ‘RNA-engaged’ to ‘Apo’ state – this is the same domain where our allosteric pocket lies, the key movements in the RTC translocation from the cryoEM paper are in the videos above.

The RTC seems to show ‘backtracking’ abilities, suggesting a proofreading mechanism whereby mis-matched p-RNA 3’ nucleotides are excreted out of the NTP-entry tunnel NTP, Question: Is this an argument for targeting the ATP-binding site over the RNA binding site, particularly that the ATP binding site lies between the Rec1A and Rec2A domains? This site has been proposed as a site for allosteric inhibition in MD simulations (https://doi.org/10.1016/j.jmgm.2022.108122 ) and 10 fragments were found in this site in Joe Newman’s fragment paper.

Another useful book on the background of SARS-CoVs and potential therapeutic targets, particularly Ch18; https://onlinelibrary.wiley.com/doi/book/10.1002/9783527810697

[H-agha]

Newman paper (https://doi.org/10.1038/s41467-021-25166-6) also has a similar movie attached here. Supp Movie- Newman paper.mpg.zip

[tmw20653]

Review outline:

1. Introduction - Helicases are motor enzymes – unwind DNA and RNA - (Tim)

2. Helicases in diseases (cancer, viral, etc) - (Heba)

3. Helicases as a protein family (3 subfamilies, human, viral) - (??)

   • Who can perform the sequence analysis and generate a list of the helicases in each family (SF1-3)?
   • Need to define the key domains within each family
   • SF1 includes alphavirus nsp2 and SARS nsp13
   • SF2 includes the DEAD-box helicases
   • SF3 includes anything that does not fit SF1 or SF2 ?
   • SF1 & SF2 contain two RecA domains

4. Structures of helicases (Tom)

   • A search of PDB for 'helicase' gave 7400 entries!
   • 5892 X-ray, 1131 cryoEM, 377 NMR

5. Structural model of how helicases work - (Tom)

   • should include a cartoon and a movie

6. Known inhibitors of helicases - (Anwar)

7. New opportunities for chemical inhibition (??)

[kipUNC]

I can take a look at the structural stuff.

[tmw20653]

Updated Review outline:

1. Introduction - Helicases are motor enzymes – unwind DNA and RNA - (Tim)
2. Helicases in diseases (cancer, viral, etc) - (Heba) - Tim to check if Heba is still available to write this section
3. Helicases as a protein family (5 subfamilies SF1-5, human and viral) - (Matthieu and Lakshi)

   see Janokowski Ch1 of RSC book, Focus on the two RecA domains sequence analysis and generate a list of the helicases in SF1 and SF2 families - use Helihub output

4. Structural model of how helicases work - (Tom)

   focus on nsp13 and include a cartoon and a movie

5. Known inhibitors of helicases - (Anwar)
6. Structures of helicase/inhibitor complexes

   Expand Helihub output to map the pockets where small molecules are bound (Matthieu and Lakshi) Conservation of these pockets in nsp13 variants (Kostya)

7. New opportunities for chemical inhibition (??)