Extracellular Salt Bridges

GROMACS version: 2020.4
GROMACS modification: No
Dear All,

I was wondering if anyone had a similar experience and could offer some advice. I’m working with a membrane protein i.e. Nav1.5. Initially roughly about 1.5 years ago, the rat structure for Nav1.5 came out and I simulated this channel and a few mutants in NAMD. I used the CHARMM27 force field and prepared the channel in a very similar way to the potassium channel outlined in the NAMD membrane protein tutorial. One particular region I was interested in was an extracellular complex salt-bridge flanked by aromatic residues that formed cation-pi interactions. For the wild-type channel, these interactions held together quite well and they did for a positive control mutant as well. However, mutations in this salt-bridge disrupted interactions as one would expect. This was well in line with what I expected and quite good. However, one point of note was that I could only really run such a big system in NAMD for 15ns simulations or so given the number of mutations I had to run and the slowish speed of NAMD.

I was interested in running these simulations for a few structures for a longer duration (around 100ns or so). Recently the human Nav1.5 structure came out (as opposed to the rat Nav1.5 that came out earlier) so I thought about running the simulations on this structure instead of the rat structure. The human Nav1.5 sequence is almost identical to the rat Nav1.5 structure and the region I alluded to earlier was perfectly conserved between Human and rat. Looking at the cryo-EM structure of the Nav1.5 confirmed this. To simulate this system for about 100ns, I decided to try and use gromacs as opposed to NAMD. However, the major problem I’m running into is that the complex extracellular salt-bridge seems to be breaking pretty consistently and not holding together like it did in the NAMD simulations. I tried multiple simulations all to the same effect:

  • I aligned my human Nav1.5 structure to a pre-existing DPPC bilayer removing overlapping DPPC so that the protein would sit in the membrane. I then solvated and ionised as normal. I performed a production simulation for 100ns but the complex salt-bridge split apart in this case. The forcefield I used in this case was gromos53a6 with lipid parameters added as done through Prof Lemkul’s tutorial (KALP-15 in DPPC)

  • I used CHARMM-GUI to prepare my system in this case. CHARMM-GUI prepared all the files for simulation so I just ran a production run for about 5ns. However, even in this production run, the complex salt-bridge broke. The force-field used in this case was CHARMM36

There are a few papers that seem to suggest that extracellular salt-bridges are a bit finicky in MD simulations and generally hard to model given their interaction with ions and solvents and so it’s hard to reliably assess their stability. One other reason this paper suggests, https://www.biorxiv.org/content/10.1101/272799v1.full.pdf , is that CHARMM22 over-stabilises salt-bridges and that these values were corrected for in later versions of CHARMM which is why I think the complex salt-bridge may be breaking.

I’m a bit unsure about what could be causing the salt-bridge to break in the more recent simulations I’m running. It could be due to the human vs rat Nav1.5 structure but given how similar the structures are (basically identical) I don’t think that this is the case either. Does anyone have any idea what could be happening? I’d appreciate the help!

Thanks and Best Regards,

The CHARMM36 results should be a better representation of reality, as the Ahmed paper notes there are issues with C22/CMAP (aka “CHARMM27”). There are also recent NBFIXes specifically for cation-π interactions in CHARMM (https://pubs.acs.org/doi/pdf/10.1021/acs.jctc.0c00637) that should probably be investigated in your case if the balance of salt bridges and cation-π are particularly critical.