Date Published: February 01, 2017
Publisher: International Union of Crystallography
Author(s): Ben Bax, Chun-wa Chung, Colin Edge.
H atoms are ‘hard to see’ in X-ray crystal structures of protein–ligand complexes. This paper discusses the problem of identifying the correct tautomeric form(s) of protein-bound ligands.
The most famous story about tautomers in the history of science occurred in the early 1950s in Cambridge. Watson and Crick were trying to propose a structure for DNA, but had been failing for some time. They were, however, fortunate enough to be sharing an office with the American theoretical chemist Jerry Donahue. One Wednesday afternoon they discussed the possible tautomeric forms of the bases in DNA. Jerry Donahue told Watson and Crick that the literature was likely to be wrong and what the most probable tautomers for G, C, A and T were. When Jim Watson came in to work at 9.30 am on Saturday morning he had cardboard models for the four bases in the ‘correct’ tautomeric forms and, by the time that Francis Crick arrived for work at 10.30 am, Jim had worked out the classical G–C, A–T base pairing (J. Watson seminar, LMB, Cambridge, 9th June 2016). As they subsequently wrote in their famous paper If it is assumed that the bases only occur in the most plausible tautomeric forms … it is found that only one specific pair of bases can bond together(Watson & Crick, 1953 ▸). The normal Watson–Crick base-pairing for G–C is shown in Fig. 1 ▸, which also shows an unusual G–T base pair that could be made if the guanine adopted an enol tautomer (Topal & Fresco, 1976 ▸). This story illustrates that understanding tautomers can be important in understanding molecular-recognition processes, and also how valuable it can be to know a good chemist.
Macromolecular crystal structures can be refined with or without riding H atoms. However, when you deposit your structure with the PDB, part of the structure-validation process (Gore et al., 2012 ▸) is to add H atoms to the protein (with Reduce; Word et al., 1999 ▸) and to then check them with MolProbity (Chen et al., 2010 ▸; Deis et al., 2013 ▸). Ligand-validation programs (Adams et al., 2016 ▸; Emsley, 2017 ▸) will also check for clashes between the ligand and the ligand-binding pocket once both have been protonated. However, most modern refinement programs have refinement terms (Steiner & Tucker, 2017 ▸) that try to eliminate unfavourable van der Waals contacts between H atoms. If your ligand can have multiple tautomeric states or protonation states, it can be useful to try and dock and refine all possible tautomeric states and protonation states into the binding sites. For example (see below and Chan et al., 2015 ▸) we read SMILES (Weininger, 1988 ▸) strings for eight tautomers of QPT-1 into an AFITT (Wlodek et al., 2006 ▸) script, and automatically docked each of the eight into six binding sites. Computational chemistry programs such as MarvinSketch (Marvin v.16.8.15, ChemAxon; https://www.chemaxon.com) can be used to enumerate possible tautomeric and charged states.
X-rays are scattered by electrons and, as hydrogen has only one electron, hydrogen is seldom visible in a macromolecular X-ray crystal structure. Even in very high resolution X-ray crystal structures (1.2–0.65 Å) not all H atoms are visible in an electron-density map (Fisher et al., 2012 ▸). A recent paper reviewing ‘Sub-atomic resolution X-ray crystallography and neutron crystallography’ (Blakeley et al., 2015 ▸) stated that While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays.
Since Watson & Crick (1953 ▸) ‘assumed that the bases only occur in the most plausible tautomeric forms’ structural and computational studies have shown that the four bases in DNA (G, C, A and T) do indeed each have only one stable tautomer (Saenger, 1983 ▸). Nevertheless, minor tautomeric forms of the DNA bases have been speculated to play a role in mutagenic mispairings during DNA replication (Topal & Fresco, 1976 ▸; Singh et al., 2015 ▸), and in RNA biochemistry different tautomeric forms of bases can play important roles in the catalytic activity of ribozymes (Singh et al., 2015 ▸). The transfer of protons (H+) or hydride ions (H−) clearly plays a key role in many reactions catalysed by enzymes (see, for example, the proposed mechanism for ATP hydrolysis in Supplementary Fig. S5), but definitively proving such mechanisms is challenging.