The structural basis of pregnane X receptor binding promiscuity.

The steroid and xenobiotic-responsive human pregnane X receptor (PXR) binds a broad range of structurally diverse compounds. The structures of the apo and ligand-bound forms of PXR are very similar, in contrast to most promiscuous proteins that generally adapt their shape to different ligands. We investigated the structural origins of PXR's recognition promiscuity using computational solvent mapping, a technique developed for the identification and characterization of hot spots, i.e., regions of the protein surface that are major contributors to the binding free energy. Results reveal that the smooth and nearly spherical binding site of PXR has a well-defined hot spot structure, with four hot spots located on four different sides of the pocket and a fifth close to its center. Three of these hot spots are already present in the ligand-free protein. The most important hot spot is defined by three structurally and sequentially conserved residues, W299, F288, and Y306. This largely hydrophobic site is not very specific and interacts with all known PXR ligands. Depending on their sizes and shapes, individual PXR ligands extend into two, three, or four more hot spot regions. The large number of potential arrangements within the binding site explains why PXR is able to accommodate a large variety of compounds. All five hot spots include at least one important residue, which is conserved in all mammalian PXRs, suggesting that the hot spot locations have remained largely invariant during mammalian evolution. The same side chains also show a high level of structural conservation across hPXR structures. However, each of the hPXR hot spots also includes residues with moveable side chains, further increasing the size variation in ligands that PXR can bind. Results also suggest a unique signal transduction mechanism between the PXR homodimerization interface and its coactivator binding site.

QM/MM Prediction of the Stark Shift in the Active Site of a Protein.

Recent developments in the biophysical characterization of proteins have provided a means of directly measuring electrostatic fields by introducing a probe molecule to the system of interest and interpreting photon absorption in the context of the Stark effect. To fully account for this effect, the development of accurate atomistic models is of paramount importance. However, suitable computational protocols for evaluating Stark shifts in proteins are yet to be established. In this work, we present a comprehensive computational method to predict the change in absorption frequency of a probe functional group as a direct result of a perturbation in its surrounding electrostatic field created by a protein environment, i.e., the Stark shift. We apply the method to human aldose reductase, a key protein enzyme that catalyzes the reduction of monosaccharides. We develop a protocol based on a combination of molecular dynamics and moving-domain QM/MM methods, which achieves quantitative agreement with experiment. We outline the difficulties in predicting localized electrostatic field changes within a protein environment, and by extension the Stark shift, due to a protein site mutation. Furthermore, the combined use of Stark effect spectroscopy and computational modeling is used to predict the protonation state of ionizable residues in the vicinity of the electrostatic probe.

Recognition and reactivity in the binding between Raf kinase inhibitor protein and its small-molecule inhibitor locostatin.

The present work is aimed to provide detail on the binding process between Raf kinase inhibitor protein (RKIP) and locostatin, the only exogenous compound known to alter the function of RKIP. Understanding the basis of RKIP inhibition for use in pharmacological applications is of considerable interest, as dysregulated RKIP expression has the potential to contribute to pathophysiological processes. Herein, we report a series of atomistic models to describe the protein-ligand recognition step and the subsequent reactivity steps. Modeling approaches include ligand docking, molecular dynamics, and quantum mechanics/molecular mechanics calculations. We expect that such a computational assay will serve to study similar complexes in which potency is associated with recognition and reactivity. Although previous data suggested a single amino acid residue (His86) to be involved in the binding of locostatin, the actual ligand conformation and the steps involved in the reactivity process remain elusive from a detailed atomistic description. We show that the first reaction step, consisting of a nucleophilic attack of the nitrogen (Nε) of His86 at the sp(2)-hybridized carbon (C2) of locostatin, presents a late transition state (almost identical to the product). The reaction is followed by a hydrogen abstraction and hydrolysis. The theoretically predicted overall rate constant (6 M(-1) s(-1)) is in a very good agreement with the experimentally determined rate constant (13 M(-1) s(-1)).