Probing the Pharmacological Binding Sites of P-Glycoprotein Using Umbrella Sampling Simulations.

The human multidrug transporter P-glycoprotein (P-gp) transports over 200 chemically diverse substrates, influencing their bioavailability and tissue distribution. Pharmacological studies have identified both competitive and noncompetitive P-gp substrates, but neither the precise location of the substrate binding sites, nor the basis of competitive and noncompetitive interactions has been fully characterized. Here, potential of mean force (PMF) calculations are used to identify the transport-competent minimum free energy binding locations of five compounds, Hoechst 33342, Rhodamine 123, paclitaxel, tariquidar, and verapamil to P-gp. Unrestrained molecular dynamics simulations were also performed to confirm the substrates were stable in the energy wells determined using the PMF calculations. All compounds had energy minima within the P-gp transmembrane (TM) pore. For Hoechst 33342 and Rhodamine 123, a second minimum outside the TM pore was also identified. Based on this and previous studies of nicardipine and morphine [ Subramanian et al. J. Chem. Inf. Model. 2015 , 55 , 1202 ], a general scheme that accounts for the observed noncompetitive and competitive substrate interactions with P-gp is proposed.

Impact of Detergents on Membrane Protein Complex Isolation.

Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that ß-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij-35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

Understanding the accumulation of P-glycoprotein substrates within cells: The effect of cholesterol on membrane partitioning.

The apparent activity of the multidrug transporter P-glycoprotein (P-gp) is enhanced by the presence of cholesterol. Whether this is due to the direct effect of cholesterol on the activity of P-gp, its effect on the local concentration of substrate in the membrane, or its effect on the rate of entry of the drug into the cell, is unknown. In this study, molecular dynamics simulation techniques coupled with potential of mean force calculations have been used to investigate the role of cholesterol in the movement of four P-gp substrates across a POPC bilayer in the presence or absence of 10% cholesterol. The simulations suggest that the presence of cholesterol lowers the free energy associated with entering the middle of the bilayer in a substrate-specific manner. These findings suggest that P-gp substrates may preferentially accumulate in cholesterol-rich regions of the membrane, which may explain its enhanced transport activity.

Molecular basis for the interaction of the mammalian amino acid transporters B0AT1 and B0AT3 with their ancillary protein collectrin.

Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B(0)AT1/3 fusion constructs, we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B(0)AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.

Identification of Possible Binding Sites for Morphine and Nicardipine on the Multidrug Transporter P-Glycoprotein Using Umbrella Sampling Techniques.

The multidrug transporter P-glycoprotein (P-gp) is central to the development of multidrug resistance in cancer. While residues essential for transport and binding have been identified, the location, composition, and specificity of potential drug binding sites are uncertain. Here molecular dynamics simulations are used to calculate the free energy profile for the binding of morphine and nicardipine to P-gp. We show that morphine and nicardipine primarily interact with key residues implicated in binding and transport from mutational studies, binding at different but overlapping sites within the transmembrane pore. Their permeation pathways were distinct but involved overlapping sets of residues. The results indicate that the binding location and permeation pathways of morphine and nicardipine are not well separated and cannot be considered as unique. This has important implications for our understanding of substrate uptake and transport by P-gp. Our results are independent of the choice of starting structure and consistent with a range of experimental studies.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment.

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the statistical significance of any results obtained in simulations of large flexible proteins, especially where the initial structure is uncertain.

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuTAa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuTAa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

Structural and dynamic perspectives on the promiscuous transport activity of P-glycoprotein.

The multidrug transporter P-glycoprotein (P-gp) is expressed in the blood-brain barrier endothelium where it effluxes a range of drug substrates, preventing their accumulation within the brain. P-gp has been studied extensively for 40 years because of its crucial role in the absorption, distribution, metabolism and elimination of a range of pharmaceutical compounds. Despite this, many aspects of the structure-function mechanism of P-gp are unresolved. Here we review the emerging role of molecular dynamics simulation techniques in our understanding of the membrane-embedded conformation of P-gp. We discuss its conformational plasticity in the presence and absence of ATP, and recent efforts to characterize the drug binding sites and uptake pathways.

New insights into the dimerization and site-specific cooperative interaction of Azure B with model transport proteins by spectroscopic and computational studies.

In the present study, the interaction of model transport proteins Human serum albumin (HSA) and Bovine serum albumin (BSA) with a photoactive dye, Azure B (AZB) were studied by spectroscopic and in silico methods. The absorption spectral behavior of AZB in the presence of varying concentrations of serum albumins (HSA and BSA) revealed the formation of dye aggregates within the protein cavity. The binding parameters computed from the emission quenching data showed that AZB bind to HSA and BSA with significant affinity and it was revealed that both the serum proteins (HSA and BSA) can bind AZB at more than one binding sites having at least one high-affinity binding site with different affinities (non-independent). The existence of static quenching mechanism was further evidenced from the time-resolved fluorescence spectroscopic analysis. Site-competitive replacement experiments with specific site markers showed that AZB binds to site I of HSA and BSA. AutoDock based blind docking approach and molecular dynamics simulation studies were used to analyze the most probable binding location of AZB in HSA and BSA. The AZB induced unfolding of HSA and BSA was established by using absorption, circular dichroism and FT-IR spectral studies. The influence of AZB complexation on the biological function of HSA and BSA was evaluated by probing the hydrolysis of p-nitrophenyl acetate.

Correction: Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

[This corrects the article DOI: 10.1371/journal.pone.0157583.].

Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

InPACdb--Indian plant anticancer compounds database.

Indian Plant Anticancer Compounds Database (InPACdb) is a web-based open access database of phytochemicals. The objective of this initiative is to project the potential of anticancer phytochemicals from Indian pharmacopoeia in an integrated environment. This database is unique in providing comprehensive information covering cancer type, molecular target, 3D Stereochemical structures (tautomers, stereoisomers, conformers and resonance structures) and Chemical descriptors etc. for each entry, enabling effective cheminformatics analysis. The complete dataset of InPACdb encompasses 32 descriptive fields for each entry, and is freely available for download at http://www.inpacdb.org.