Need little suggestion regarding the effect of non-active site mutants on enzyme activity: How to approach to this issue by means of molecular dynamics simulations?
What are the appropriate analyses to be performed to address this problem?
It depends on what you mean by "enzyme activation" - that can mean a few different things. Do the distal mutations increase enzyme activity? Do they lead to changes in post-translational modification? Do they lead to some other processing mechanism (i.e. zymogens)?
Hi Justin,
Nice to have your reply. :)
Yes, some distal mutations increase enzyme activity and some of them decrease it.
I guess the binding of the ligand into the active site is affected by the mutations in that residue. However, it is far from the active site of the enzyme.
Then this is a simple matter of allostery. Collective motions within the protein are being propagated from the mutated site to the active site. PCA is your best method for analysis here; you will likely need multiple replicates of reasonably long simulations to collect nicely converged data. The differences may be subtle.
Thanks for the suggestion.
I'll be heading towards this protocol.
Should I consider analyzing protein-ligand interactions to differentiate or comment on the effect of each of the mutants on the ligand binding, at least qualitatively?
What I'm doing now is simulating each of the mutants (enzymes) with the ligand molecule sitting at the the active site and trying to figure out some conformational changes of the enzyme.
Analyzing protein-ligand interactions is a good idea. If activity is affected, then the active site dynamics have to be impacted in some way. That's the easy part. The biggest challenge is connecting those local outcomes with the dynamics of a distant site.
Yes, It is a tedious task to correlate these local changes to the dynamics of the distant site. I'm running the simulations for long time span. Let's see how we can figure it out.
Anyway, thanks again for your valuable suggestions and comments.
I'll update this discussion when I'll finish those runs and analyses.
Is there any paper which describes the correlation between the protein-ligand interaction energy and the enzyme activity?
I'm seeing a less active mutant shows higher interaction energy. Does this mean that the enzyme is less active because of tight binding of the ligand molecule in the active site?
So mutating a distant residue affects the short-range nonbonded energy between the binding site and the ligand? By how much? What interactions change?
The one thing to realize is that interaction energy is basically an artificial, force field-dependent term. Unless it was explicitly targeted as part of the force field parametrization (including the parametrization of the ligand!), it really doesn't mean anything. It might (emphasis on _might_) give some indication of enthalpy, but since you have no entropic information, you have no insight into the actual free energy of binding, which actually is a meaningful quantity. What force field are you using?
Yes.
The difference in binding energy (Coulomb Short Range) is ~150 kJ/mol.
LJ_SR energy is coming almost similar.
I'm using AMBER99-sb-ildn force field for the protein and for the ligand I use RESP charges from RED-Tools/server after some QM calculations. The bonded and non-bonded parameters are based on the standard AMBER force field parameters for similarly known molecules.
The quantity is meaningless. 150 kJ/mol is a _massive_ change in energy, and again it does not correspond to any real thermodynamic quantity.
Do I need to do some other analysis or calculations?
I see people plotting interaction energy in some protein-drug related problem.
http://pubs.rsc.org/en/content/articlehtml/2013/sm/c3sm52499j
Just because people do it doesn't make it right. For some force fields, interaction energy makes sense, for others, it absolutely doesn't. CHARMM, for instance, targets QM interaction energy with water, so the resulting values have basis in reality. One could also make an argument for Gromos force fields since they target hydration free energies.
More to the point, you have an energy difference of 150 kJ/mol - is there a clear explanation for it? Is the mutation you're making within or beyond the short-range cutoff with respect to the ligand? You are only summing short-range energies, correct? 150 kJ/mol is a huge amount of energy, and without context and a reference point (i.e. the interaction energy of the molecule in water, from which you _might_ be able to make an enthalpic argument), again I say that the value has no physical meaning.
No, the mutant residue is well far from the ligand.
Yes, herein I consider only the short range interaction energies.
Let's see how we can come up with proper explanation for the mutational effect based on some more analyses.
Thanks Justin for the fruitful discussion.
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