#11thICCS Ruth Brenk on selectivity determining features in proteins with conserved binding sites
How to rationally design selective inhibitors: steric clash, electrostatic interactions, allostery, flexibility, hydration (Huggins et al 2012). The more conserved it is, the harder it'll likely be.
N-myristoyltransferase (NMT). Co-and post-translational mods of proteins for membrane targeting. Target for cancer and African sleeping sickness.
Describes work from Frearson et al Nature 363, 7289, 728, 2010. Not selective at first, and very difficult to work in selectivity. Didn't understand why.
Selectivity of HsNMT1 (human) versus LmNMT (Leishmania - sleeping sickness)
Highly conserved binding sites though only 34% sequence identity. Shows 3d alignment of binding sites. Very similar if not identical. Why are some cmpds selective and others not?
In NMT the C terminus folds back into the binding site, slightly unusual.
First step. Get the xtal structures of cmpds in binding sites. Only 3 residues different between the two binding sites, but didn't seem to be relevant for binding. We did crossover experiments...swapping the binding sites (? I missed how).
After swapping the binding sites, the LmNMT-3x (triple mutant) was inactive, but HsNMT1-3x was active and could measure. It had the same level of inhibition as the original Lm. So the three residues were important, and specifically it was a single residue that was the origin..
Measured binding energetics. Binding energy, enthalpy and entropy. For the non-selective, the two binding sites have the same energetics on binding. For selective, there are more favourable entropic contributions for the modified binding site. Why?
We couldn't figure out why from looking at the binding site. To investigate we went to MD simulations. Determine the order parameters (S2) for bond vectors - param of 0 is fully flexible and 1 is rigid.
The flexibility does not change much for LmNMT no matter what's bound. Quite different for HsNMT1. Big change for the selective compound. Can reationalise the change in entropy due to the selective compound binding.
Looking again at the binding site, we can hypothesise why that particular residue changes its flexibility. Could be because the selective compound is bulky and thus prevents the Gln moving so much.
Now looking atl another compound with another binding mode that is also selective.
Binding affinity did not change when swapping the binding site residues. But we can see a water molecule in the Human structures but not the Leish structures, that must be expelled when binding.
Decided to change 8 residues around the water molecule in the human NMT and replace with those from Leish, to investigate this. What happens? This abolishes the selectivity. The Ki for the non-selective cmpd is unchanged but reduces Ki for the selective.
This indicates that this environment is important for the selectivity. To investigate further, we co-crystallised. 4 molecules in the asymmetric unit. We saw the water molecule in 3 of the units, but not the 4th. So it looks like the binding of the water is reduced.
Now could design selective inhibitors, based on this information. Activity not as good, but can be optimised - they are selective though..
