Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7. 1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1. 6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.
Interactions between proteins and small molecules are involved in many biochemical phenomena. Insight into these processes relies on detailed knowledge about the structure of protein/ligand complexes, e. g. how enzymes stabilize substrates and cofactors in close proximity. Moreover, almost all drugs are small-molecule ligands that interact with enzymes, receptors or channels. Accordingly, ligand-bound receptor complex structures are a critical prerequisite for understanding biological function and for structure based drug design. However, structure determination of protein/ligand-complexes can be difficult, time-consuming and expensive. Crystal structures of protein/ligand complexes are usually obtained either by co-crystallization or soaking and it is a common problem that even when conditions for crystallizing the apo-protein are well established these might not be transferable to the protein/ligand complex [1]–[4]. Particularly, conformational transitions of the receptor associated with ligand binding pose a severe challenge to the structure elucidation of holo complexes [5]–[8]. When structures of ligand-bound protein conformations are not available, structure-based drug design becomes highly challenging. Several studies showed that virtual screening to an apo-structure usually results in a poor enrichment factor (the ability to discriminate between binders and non-binders) compared to the holo-structure even when the structural difference between both is comparably small [9]–[11]. Therefore, the development of docking programs aims at allowing a certain degree of receptor flexibility either by using an ensemble of structures instead of a single receptor conformation [12]–[15] or by explicitely modeling flexibility such as sidechain variations (Autodock4 [16], [17], Gold [18], [19], FlexX [20], RosettaLigand [21]), predefined flexibility of certain parts of the structure (FlipDock [22]) and also small variations of the backbone (Glide/Prime [23], RosettaLigand [24], ICM [25], [26]). Incorporating receptor flexibility in molecular docking is a substantial progress and has been shown to enhance both enrichment factors and the ability to predict correct binding poses, particularly in cases when docking a compound to a receptor structure that has been crystallized with a different ligand (cross-docking) which is usually the case when searching for novel drugs. However, the degree of flexibility thus far is limited to either sidechain motions or small variations of the backbone and thus, the availability of a holo-structure or an apo-structure that is highly similar to the holo conformation is currently a prerequisite for a successful docking, severely limiting structure-based drug design. Particularly, receptors that undergo a substantial conformational transition upon ligand binding are currently precluded from structure based drug design. Although protein-ligand crystals suitable for diffraction might not be accessible, several experimental techniques exist to detect conformational changes. In many cases where proteins undergo domain reorientations upon ligand binding they adopt a different shape in the ligand bound state, corresponding to a change in the radius of gyration that can be studied either by NMR, where a more compact shape causes a descrease in the rotational correlation time [27]–[29] or by small-angle scattering of x-rays (SAXS) or neutrons (SANS) [30]–[33]. These shape descriptions provide invaluable information for modeling of structures [34]–[36] and macromolecular assemblies [37], [38] as well as insight into protein dynamics. Here, we present a method to predict the structure of protein/ligand complexes for proteins that undergo a large conformational change upon ligand binding. The protocol solely requires the apo-structure, a known ligand and experimental data on the shape of the holo-structure. Here, we apply the radius of gyration as shape information, a quantity that can frequently be readily assessed more easily than an x-ray structure. We developed a simulation protocol that combines biased conformational sampling, docking and molecular dynamics simulations and applied it to ten ligand-binding proteins (see Table 1). We chose cases where both, the unbound conformation and the bound conformation are known from x-ray crystallography in order to be able to a-posteriori validate the predicted receptor conformations and docking poses. The conformational changes involved range from 2. 1 to 7. 1 Å backbone RMSD (see table 1 and fig. 1) and the binding site geometries differ substantially between the apo and the holo conformations. In nine of the ten cases we predict holo receptor conformations close to the native ligand-bound conformation and in eight cases we predict ligand binding poses close to the native state, rendering our method suitable for blind predictions of protein/ligand complexes involving large conformational transitions.
Structure-based drug design has become a powerful tool in modern drug discovery pipelines. A critical prerequisite is a structure of the target protein close to its ligand bound conformation which is often difficult to determine experimentally. In many cases, a structure of the unbound receptor is available, but conformational changes with respect to the ligand-bound form preclude it from being used as a basis for structure-based drug design. We have developed a computational approach to predict the structure of protein/ligand complexes based solely on the unbound conformation, the ligand, and easy-to-assess experimental data. We tested our protocol on proteins that undergo substantial structural rearrangements upon binding a ligand and were able to predict structures of protein/ligand complexes which are in good agreement with experimentally determined structures. The ability to predict ligand bound receptor conformations based on structures in the unbound state enables structure-based drug design for cases where crystallization of the complex has not been successful so far.
biophysics/theory and simulation
biophysics/biomacromolecule-ligand interactions
computational biology/protein structure prediction
computational biology/molecular dynamics
biochemistry/drug discovery
Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations