It’s been a while since we’ve touched on some of the hazards of interpreting crystal structures (see here, here, and here). In a recent issue of J. Comput. Aided Mol. Des., Alpeshkumar Malde and Alan Mark of the University of Queensland, Australia describe some mishaps taken from the literature, and how molecular modeling could have avoided them.
The authors start by noting that although protein structure determination using crystallography has been highly optimized, small molecule ligands are a different matter. Part of the problem is that small molecules may show more disorder than protein side chains, thus making it more challenging to fit the model into the observed electron density. Moreover, the parameters for refining protein structures do not always transfer to small molecules: electrostatic interactions are frequently ignored, as are alternative conformations.
As an example, the authors revisit the structure of noradrenochrome bound to an enzyme that synthesizes adrenaline. A racemic mixture of the ligand was used during crystallization, and when the crystal was solved at modest resolution it was possible to fit the ligand within the electron density in eight different orientations – four for each enantiomer. Despite this ambiguity, only a single structure was deposited in the protein data bank (pdb). Malde and Mark ran molecular dynamics (MD) simulations and free energy calculations and found that this structure is likely incorrect: it is higher energy than other structures and binds in a different orientation than the natural ligand, whose structure had previously been solved. In fact, the conformation suggested by MD is the opposite enantiomer from that deposited in the pdb and rotated 180 degrees.
In another example, the authors examine a high-resolution structure of a pyrazole-containing compound bound to the kinase CDK2. Pyrazoles can adopt two different tautomers in which the hydrogen is on either of two adjacent nitrogens, and in this particular case the original paper suggested that both tautomers were present in equal amounts, and both were deposited in the pdb. However, computations suggested that one tautomer is 7 kJ/mol higher energy than the other, and Malde and Mark suggest that in fact probably just a single tautomer is present in the structure.
Finally, the authors describe cases where a primary amide or primary sulfonamide group is in the wrong orientation. In most cases it is difficult to distinguish between a nitrogen and oxygen atom on the basis of electron density alone, and given that there are about 1000 ligands containing a -CONH2 group and about 200 containing a -SO2NH2 there are probably many mistakes.
The authors acknowledge that the examples they present are relatively simple, and one could argue that some of them would have been caught if they were critical structures in a lead optimization program. Nonetheless, the fact that they weren’t suggests that one must always be on guard, particularly in virtual screening where dozens or hundreds of structures are used in an automated fashion to develop or validate docking algorithms. Malde and Mark also note that, in the case of fragment screening with very small low-affinity ligands, one needs be especially cautious.
There is something extremely attractive about a crystal structure: it looks so real that it is easy to lose sight of the fact that it is just a model. Checking one’s assumptions with a bit of computation can prevent costly mistakes.