So, people like me spend their time trying to make small molecules that will bind to some target protein. So what happens, anyway, when a small molecule binds to a target protein? Right, right, it interacts with some site on the thing, hydrogen bonds, hydrophobic interactions, all that – but what really happens?
That’s surprisingly hard to work out. The tools we have to look at such things are powerful, but they have limitations. X-ray crystal structures are great, but can lead you astray if you’re not careful. The biggest problem with them, though (in my opinion) is that you see this beautiful frozen picture of your drug candidate in the protein, and you start to think of the binding as. . .well, as this beautiful frozen picture. Which is the last thing it really is.
Proteins are dynamic, to a degree that many medicinal chemists have trouble keeping in mind. Looking at binding events in solution is more realistic than looking at them in the crystal, but it’s harder to do. There are various NMR methods (here's a recent review), some of which require specially labeled protein to work well, but they have to be interpreted in the context of NMR’s time scale limitations. “Normal” NMR experiments give you time-averaged spectra – if you want to see things happening quickly, or if you want to catch snapshots of the intermediate states along the way, you have a lot more work to do.
Here’s a recent paper that’s done some of that work. They’re looking at a well-known enzyme, dihydrofolate reductase (DHFR). It’s the target of methotrexate, a classic chemotherapy drug, and of the antibiotic trimethoprim. (As a side note, that points out the connections that sometimes exist between oncology and anti-infectives. DHFR produces tetrahydrofolate, which is necessary for a host of key biosynthetic pathways. Inhibiting it is espccially hard on cells that are spending a lot of their metabolic energy on dividing – such as tumor cells and invasive bacteria).
What they found was that both inhibitors do something similar, and it affects the whole conformational ensemble of the protein:
". . .residues lining the drugs retain their μs-ms switching, whereas distal loops stop switching altogether. Thus, as a whole, the inhibited protein is dynamically dysfunctional. Drug-bound DHFR appears to be on the brink of a global transition, but its restricted loops prevent the transition from occurring, leaving a “half-switching” enzyme. Changes in pico- to nanosecond (ps-ns) backbone amide and side-chain methyl dynamics indicate drug binding is “felt” throughout the protein.
There are implications, though, for apparently similar compounds having rather different effects out in the other loops:
. . .motion across a wide range of timescales can be regulated by the specific nature of ligands bound. Occupation of the active site by small ligands of different shapes and physical characteristics places differential stresses on the enzyme, resulting in differential thermal fluctuations that propagate through the structure. In this view, enzymes, through evolution, develop sensitivities to ligand properties from which mechanisms for organizing and building such fluctuations into useful work can arise. . .Because the affected loop structures are primarily not in contact with drug, it is reasonable to envision inhibitory small-molecule drugs that act by allosterically modulating dynamic motions."
There are plenty of references in the paper to other investigations of this kind, so if this is your sort of thing, you'll find plenty of material there. One thing to take home, though, is to remember that not only are proteins mobile beasts (with and without ligand bound to them), but that this mobility is quite different in each state. And keep in mind that the ligand-bound state can be quite odd compared to anything else the protein experiences otherwise. . .