Mixed-solvent
molecular dynamics (MixMD) is a hotspot-mapping technique
that relies on molecular dynamics simulations of proteins in binary
solvent mixtures. Previous work on MixMD has established the technique’s
effectiveness in capturing binding sites of small organic compounds.
In this work, we show that MixMD can identify both competitive and
allosteric sites on proteins. The MixMD approach embraces full protein
flexibility and allows competition between solvent probes and water.
Sites preferentially mapped by probe molecules are more likely to
be binding hotspots. There are two important requirements for the
identification of ligand-binding hotspots: (1) hotspots must be mapped
at very high signal-to-noise ratio and (2) the hotspots must be mapped
by multiple probe types. We have developed our mapping protocol around
acetonitrile, isopropanol, and pyrimidine as probe solvents because
they allowed us to capture hydrophilic, hydrophobic, hydrogen-bonding,
and aromatic interactions. Charged probes were needed for mapping
one target, and we introduce them in this work. In order to demonstrate
the robust nature and wide applicability of the technique, a combined
total of 5 μs of MixMD was applied across several protein targets
known to exhibit allosteric modulation. Most notably, all the protein
crystal structures used to initiate our simulations had no allosteric
ligands bound, so there was no preorganization of the sites to predispose
the simulations to find the allosteric hotspots. The protein test
cases were ABL Kinase, Androgen Receptor, CHK1 Kinase, Glucokinase,
PDK1 Kinase, Farnesyl Pyrophosphate Synthase, and Protein-Tyrosine
Phosphatase 1B. The success of the technique is demonstrated by the
fact that the top-four sites solely map the competitive and allosteric
sites. Lower-ranked sites consistently map other biologically relevant
sites, multimerization interfaces, or crystal-packing interfaces.
Lastly, we highlight the importance of including protein flexibility
by demonstrating that MixMD can map allosteric sites that are not
detected in half the systems using FTMap applied to the same crystal
structures