A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways.

Quinazolinones, indolo- and pyrrolopyrimidines with inhibitory effects toward ABCB1 (P-gp) and ABCC1 (MRP1) transporters were studied by pharmacophore modeling, docking, and 3D QSAR to describe the binding preferences of the proteins. The pharmacophore overlays between dual and/or highly selective inhibitors point to binding sites of different topology and physiochemical properties for MRP1 and P-gp. Docking of selective inhibitors into the P-gp binding cavity by the use of a structural model based on the recently resolved P-gp structure confirms the P-gp pharmacophore features identified, and reveals the interactions of some functional groups and atoms in the structures with particular protein residues. The 3D QSAR analysis of the dual-effect inhibitors allows satisfactory prediction of the selectivity index of the compounds and outlines electrostatics as most important for selectivity. The results from the combined modeling approach complement each other and could improve our understanding of the protein-ligand interactions involved, and could aid in the development of highly selective and potent inhibitors of P-gp and MRP1.

Secondary amphiphilicity is inherent to the secondary structural elements of proteins. By forming energetically favorable contacts with each other these amphiphilic building blocks give rise to the formation of a tertiary structure. Small proteins and peptides, on the other hand, are usually too short to form multiple structural elements and cannot stabilize them internally. Therefore, these molecules are often found to be structurally ambiguous up to the point of a large degree of intrinsic disorder in solution. Consequently, their conformational preference is particularly susceptible to environmental conditions such as pH, salts, or presence of interfaces. In this study we use molecular dynamics simulations to analyze the conformational behavior of two synthetic peptides, LKKLLKLLKKLLKL (LK) and EAALAEALAEALAE (EALA), with built-in secondary amphiphilicity upon forming an alpha-helix. We use these model peptides to systematically study their aggregation and the influence of macroscopic and molecular interfaces on their conformational preferences. We show that the peptides are neither random coils in bulk water nor fully formed alpha helices, but adopt multiple conformations and secondary structure elements with short lifetimes. These provide a basis for conformation-selection and population-shift upon environmental changes. Differences in these peptides’ response to macroscopic and molecular interfaces (presented by an aggregation partner) can be linked to their inherent alpha-helical tendencies in bulk water. We find that the peptides’ aggregation behavior is also strongly affected by presence or absence of an interface, and rather subtly depends on their surface charge and hydrophobicity.

The direct structure-function relationships of P-glycoprotein (P-gp) are presently unknown. In this paper two P-gp models are described: a homology model based on the Escherichia coli MsbA lipid transporter and a model based on the cross-linking results of Loo and Clarke. The pharmacophore pattern for the H-site (Hoechst 33342) is derived and binding sites on the transmembrane domains TM5 and TM11 are identified. Binding sites of rhodamines are also proposed on TM6 and TM12 in accordance with the published data. Location of the binding sites is opposite in both models, suggesting that TMs undergo rotation exposing the substrate bound from the membrane to the pore. It has been concluded that the models derived represent two different functional states of P-gp corresponding to nucleotide-free and nucleotide-bound P-gp. A qualitative correspondence to the P-gp crystallographic structure at 20 A resolution is found. A hypothesis is proposed about rearrangement of TMs upon state transition.