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The Motions of a Protein

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. . .

3 comments on “The Motions of a Protein”

  1. Anonymous BMS Researcher says:

    Another connection between Oncology and Antibiotics is that in both fields multi-drug-resistance from efflux pumps is a major issue. And of course in these fields and inAntivirals the evolution of resistance is a major concern.

  2. channeljockey says:

    As a biologist who became a computational chemist, I have a strong interest in protein motion, since my view of the drug discovery process starts with the protein target and then zooms in from there. However, in speaking with chemists, especially during employment interviews, I find that generally they are not interested in protein dynamics, only in H-bond interactions at the binding site. This is true even when I have presented very compelling results from molecular dynamics simulations that help to explain why compound X or compound Y did not make it to the binding site. My conclusion is that chemists sometimes have a rather limited view of the drug discovery world, and that world is centered around the binding site without regard for anything else that might be going on in the target protein.
    Any synth/med/org chemists out there wish to comment?

  3. John says:

    Dear Derek,
    Thank you for this Blog. Through your voice and the voices of your commentators, you help bring clarity to an otherwise highly complex, verbose, seemingly alien, trivia-laden field of study. You should start a radio/TV program….
    I have some basic questions: the X-Ray crystal structures (solids) and NMR solution structures (which often occur at micro to milligram/mL concentrations, dissolved in a non-physiological solvent i.e. alcohol), how much do you use these structures in your drug design strategy? How much do companies like Merck and Pfizer invest towards solving X-Ray and NMR protein structures?
    Would you recommend anyone become a protein NMR spectroscopist today? Are the structures an essential part of the small molecular ‘rational design’ strategy or are they theoretical afterthoughts? I heard that medicinal chemists use combinatorial libraries to inhibit or activate proteins. What thought process goes into making a ‘combinatorial library’? How fundamental is the NMR or X-ray structure to that thought process?
    From what I understand, in vivo, proteins certainly don’t exist in crystalline states; they exist in solution usually at ng/mL concentrations (not ug/mL to mg/mL concentrations) and they exist in complex cytosolic, extracellular, lipid bilayer, etc solution environments (not pure alcohol, fluoroalcohol, or some singular type of micelle environment). Since we know that environment strongly dictates protein fold, HOW USEFUL/RELIABLE are these structures? The ultimate utility of all this structural information is to model protein-ligand interactions (using physical/mathematical equations) thus saving tons of time and money on dead-end ‘needle in the haystack’ laboratory benchwork … but how far away are we from sitting down at a computer and solving diseases (misfolded proteins) based on current X-ray and NMR structures. How good can the protein-ligand modeling be? How good can it be (despite whatever brilliant physics/math is applied) when WE KNOW that the structural model is NOT representative of the in vivo structure (because the in vitro environment we used is alien to the in vivo)! How do medicinal chemists working at Merck and Pfizer reconcile this basic limitation with their modeling work? I realize that “some picture is better than no picture” and that due to the limitations of our spectroscopy and labeling techniques, we’re stuck – there is no “in vivo spectroscopy” available; but why waste a penny on current X-ray and NMR methods then? Just to pretend? Or do you get invaluable information from these structures. Can you recommend a good website that will alleviate my disillusionment/cynicism towards this stuff?
    Perhaps I’m totally out to lunch on this stuff. I don’t have any scientists as soundboards in my life. Thank you for your time. Thank you for your free communication and knowledge. In fact, God bless you for your donated time, down-to-earth communication and free knowledge. I would appreciate anyone else’s feedback on these questions.
    Sincerely,
    John

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