Electrostatic Bridge Mutations in PI Region of SpCas9
Before I learned Samson, I used Sketchup to do a geometric model of the electrostatic bridge in the PAM-interacting domain of SpCas9, based on PDB 4UN3. The point was to showcase how the spatial arrangement, and how mutations in this region relaxes the PAM specificity for NGG. Sketchup is quite useful for a geometric simplification, but you'll have to simplify a lot of the complex structures for a quick model. Sketchup now charges a fee for its license, while Samson is free for students. Samson is infinitely more powerful, but needs a gaming laptop to run smoothly. Sketchup is mainly used in the architecture/ interior design field for conceptual modelling. Much easier to learn and more fun than AutoCAD.
There is a key triad in the PAM-interacting (PI) domain of Cas9. The triad forms an electrostatic bridge : Arg1333, Arg 1335 and Glu 1219. R1333, R1335 and E1219. Arginine is positively charged, and directly interacts with G1 and G2 of the NGG PAM site on target DNA. Arginine is a common DNA interacting amino acid. Glutamic acid is negatively charged, and stabilises the arginine pair into the precise position to interact with GG. Without Glu1219, the two arginine would repel each other and move out of place. This electrostatic network is key to the NGG specificity of the PI domain. (N denotes any base.)
Now since we want to target a wider range of eukaryotic PAM sites, engineering methods focus on mutating these 3 amino acids to loosen up the electrostatic bridge, so that it can bind other sequences (e.g. NGN). We usually keep the arginines because we need them for DNA base pair binding. But if we mutate the glutamic acid to a neutral amino acid (e.g. valine), then this loosens the grip on the second arginine (R1335).
Created in Samson. Hydrogens are not shown.
This allows the guanidium group (The Y head) on R1335 to rotate on its 'hinge'. This flexibility lets it H-bond with other bases. The crucial hinge is between Cδ–Nε, also known as χ4. This is the finger joint. In hierarchical order: χ3 (Cγ–Cδ), χ2 (Cβ–Cγ) and χ1 (Cα–Cβ) also contribute to the flexibility, but to a smaller extent. χ3 is like the elbow, and χ2 + χ1 are like the shoulder. 
Now the exact hydrogens involved are unresolved experimentally, as far as I know. There is no NMR resolution PDB file for the PI region, although NMR studies have been conducted (Paula et al., 2025) .
What I understand is that the hydrogens involved can change around, depending on the configuration. What is known:
- Both nitrogens on the guanidium group are involved in DNA H-bonding
- Both nitrogens are also involved in Glu1219 H-bonding
- R1335 forms H-bonds with O6 and N7
- R1335 also forms H-bonds with E1219 (glutamate, in carboxylate form)
- In the entire electrostatic bridge, R1335 is the H donor, using up all its 4 H
So if we do a rough alignment based on 4UN3, in the engaged configuration, this is what we get. This is a reasonable guess on my part, so don't take it too literally. The 4 hydrogens exist in 3D space, not necessarily planar with the guanidium nitrogens, so the 4 H can rotate somewhat to align themselves for optimal bonding.
The next step would be to add in hydrogens to the 3D model in Samson, considering the torsional constraints.
Previous slides as background information -
References
Ruffolo, J. A., Nayfach, S., Gallagher, J.,
Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E.,
Pacesa, M., Meeske, A. J., Cameron, P., & Madani, A. (2025). Design
of highly functional genome editors by modelling CRISPR-Cas sequences. Nature, 645(8080), 518–525. https://doi.org/10.1038/s41586-025-09298-z
Moriarty, N. W.,
Liebschner, D., Tronrud, D. E., & Adams, P. D. (2020). Arginine off-kilter:
guanidinium is not as planar as restraints denote. Acta crystallographica.
Section D, Structural biology, 76(Pt 12), 1159–1166. https://doi.org/10.1107/S2059798320013534
Egli, M., & Zhang, S.
(2022). Ned Seeman and the prediction of amino acid-basepair motifs
mediating protein-nucleic acid recognition. Biophysical journal, 121(24), 4777–4787. https://doi.org/10.1016/j.bpj.2022.06.017