VMD-L Mailing List

From: Manas Kohli (manaskohli1_at_gmail.com)
Date: Tue Jul 13 2021 - 12:02:52 CDT

Dear All,

This question doesn't really relate to a technical issue to VMD but 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 (
https://urldefense.com/v3/__http://www.mdtutorials.com/gmx/membrane_protein/02_topology.html__;!!DZ3fjg!paLDpnhMZlCo3lSIullup3Afln-ofrAIxKjM5DCTkig9gaLKP2YcmLms7PRvztQ4Dg$ )

- 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://urldefense.com/v3/__https://www.biorxiv.org/content/10.1101/272799v1.full.pdf__;!!DZ3fjg!paLDpnhMZlCo3lSIullup3Afln-ofrAIxKjM5DCTkig9gaLKP2YcmLms7PQmvH9zYQ$ , 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,
Manas