Inherent versus induced protein flexibility: Comparisons within and between apo and holo structures

Understanding how ligand binding influences protein flexibility is important, especially in rational drug design. Protein flexibility upon ligand binding is analyzed herein using 305 proteins with 2369 crystal structures with ligands (holo) and 1679 without (apo). Each protein has at least two apo and two holo structures for analysis. The inherent variation in structures with and without ligands is first established as a baseline. This baseline is then compared to the change in conformation in going from the apo to holo states to probe induced flexibility. The inherent backbone flexibility across the apo structures is roughly the same as the variation across holo structures. The induced backbone flexibility across apo-holo pairs is larger than that of the apo or holo states, but the increase in RMSD is less than 0.5 Å. Analysis of χ1 angles revealed a distinctly different pattern with significant influences seen for ligand binding on side-chain conformations in the binding site. Within the apo and holo states themselves, the variation of the χ1 angles is the same. However, the data combining both apo and holo states show significant displacements. Upon ligand binding, χ1 angles are frequently pushed to new orientations outside the range seen in the apo states. Influences on binding-site variation could not be easily attributed to features such as ligand size or x-ray structure resolution. By combining these findings, we find that most binding site flexibility is compatible with the common practice in flexible docking, where backbones are kept rigid and side chains are allowed some degree of flexibility.Author summary: Here, we examine how ligand binding affects protein flexibility by analyzing over 4000 crystal structures, an order of magnitude more than previous studies based on apo-holo pairs. A debate exists in the literature over how flexible binding sites are in proteins. Studies that conclude there is little motion upon ligand binding tend to measure backbone RMSD, but studies that show larger conformational change base their analyses on side-chain orientations. None of these studies have used the same proteins, so it is unclear how much the different conclusions are due to the chosen analyses versus the different datasets used. Furthermore, many studies have used apo-holo pairs to measure conformational change in proteins, but none have examined the inherent flexibility across the apo and holo states themselves. The induced change seen in an apo-holo pair must be placed in context of how variable the apo and holo states are. Our analyses reconcile any existing debate by confirming inherently different behavior for backbones and side chains, where backbones tend to sample very little conformational space and side chains are frequently pushed into new conformations upon ligand binding.