P-glycoprotein (ABCB1) - weak dipolar interactions provide the key to understanding allocrite recognition, binding, and transport.

P-glycoprotein (ABCB1) is the first discovered mammalian member of the large family of ATP binding cassette (ABC) transporters. It facilitates the movement of compounds (called allocrites) across membranes, using the energy of ATP binding and hydrolysis. Here, we review the thermodynamics of allocrite binding and the kinetics of ATP hydrolysis by ABCB1. In combination with our previous molecular dynamics simulations, these data lead to a new model for allocrite transport by ABCB1. In contrast to previous models, we take into account that the transporter was evolutionarily optimized to operate within a membrane, which dictates the nature of interactions. Hydrophobic interactions drive lipid-water partitioning of allocrites, the transport process's first step. Weak dipolar interactions (including hydrogen bonding, π-π stacking, and π-cation interactions) drive allocrite recognition, binding, and transport by ABCB1 within the membrane. Increasing the lateral membrane packing density reduces allocrite partitioning but enhances dipolar interactions between allocrites and ABCB1. Allocrite flopping (or reorientation of the polar part towards the extracellular aqueous phase) occurs after hydrolysis of one ATP molecule and opening of ABCB1 at the extracellular side. Rebinding of ATP re-closes the transporter at the extracellular side and expels the potentially remaining allocrite into the membrane. The high sensitivity of the steady-state ATP hydrolysis rate to the nature and number of dipolar interactions, as well as to the dielectric constant of the membrane, points to a flopping process, which occurs to a large extent at the membrane-transporter interface. The proposed unidirectional ABCB1 transport cycle, driven by weak dipolar interactions, is consistent with membrane biophysics.

A Familiar Protein-Ligand Interaction Revisited with Multiple Methods.

The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been well-characterized by biophysical methods and structural biology. In this chapter, we present a series of experiments designed to detect and quantify that interaction using several commonly available biophysical methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of representative data from several years of practical courses, we are able to demonstrate the robustness of the protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the degree of consistency between those methods when applied to a simple system in a single location by different experimenters.

Systematic and Quantitative Structure-Property Relationships of Polymeric Medical Nanomaterials: From Systematic Synthesis and Characterization to Computer Modeling and Nano-Bio Interaction and Toxicity.

Nanomaterials allow designing targeted therapies, facilitate molecular diagnostics, and are therefore enabling platforms for personalized medicine. A systematic science and a predictive understanding of molecular/supramolecular structure relationships and nanoparticle structure/biological property relationships are needed for rational design and clinical progress but are hampered by the anecdotal nature, nonsystematic and nonrepresentative nanomaterial assortment, and oligo-disciplinary approach of many publications. Here, we find that a systematic and comprehensive multidisciplinary approach to production and exploration of molecular-structure/nanostructure relationship and nano-bio structure/function relationship of medical nanomaterials can be achieved by combining systematic chemical synthesis, thorough physicochemical analysis, computer modeling, and biological experiments, as shown in a nanomaterial family of amphiphilic, micelle-forming oxazoline/siloxane block copolymers suited for the clinical application. This comprehensive interdisciplinary approach leads to improved understanding of nanomaterial structures, allows good insights into binding modes for the nanomaterial protein corona, induces the design of minimal cell-binding materials, and yields rational strategies to avoid toxicity. Thus, this work contributes to a systematic and scientific basis for rational design of medical nanomaterials.

Thermal and Chemical Unfolding of Lysozyme. Multistate Zimm-Bragg Theory Versus Two-State Model.

Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein unfolding with the multistate Zimm-Bragg theory. The two-state model is shown to be the noncooperative limit of the Zimm-Bragg theory. In particular, the Zimm-Bragg theory provides a molecular interpretation of the empirical linear extrapolation method (LEM) of the two-state model. Differential scanning calorimetry (DSC) experiments reported in the literature are analyzed with both methods. Lysozyme unfolding is associated with a large endothermic enthalpy that decreases significantly upon addition of guanidine HCl. In contrast, the Gibbs free energy of unfolding is small, negative, and independent of the guanidine HCl concentration, contradicting, in part, the conclusions of the LEM. The unfolding enthalpy is compensated by an even larger entropy term. The multistate Zimm-Bragg theory predicts a larger conformational enthalpy and a smaller Gibbs free energy than the two-state model. The Zimm-Bragg theory provides the protein cooperativity parameter, the average length of independently folding protein domains, and the Gibbs free energy of unfolding of individual amino acid residues. Guanidine HCl binding to lysozyme is exothermic and counteracts the endothermic unfolding enthalpy. The number of bound denaturant molecules is determined from the decrease in enthalpy and is extrapolated to the guanidine HCl-to-amino acid stoichiometry at complete lysozyme unfolding. Chemical unfolding isotherms measured with circular dichroism (CD) spectroscopy are analyzed with both models. The chemical Zimm-Bragg theory is a cooperative molecular model, yielding the guanidine HCl binding constant and the protein cooperativity parameter. It allows a quantitative comparison between thermal and chemical protein unfolding. The two reactions have almost identical changes in Gibbs free energy. However, thermal unfolding is significantly more cooperative than chemical unfolding. Finally, distinct differences are observed in thermal unfolding between DSC and CD spectroscopy.

Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides.

The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of Escherichia coli is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. In vitro characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCE Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

Predicting Activators and Inhibitors of the Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1) Based on Mechanistic Considerations.

Colocalized in membrane barriers, the ABC transporters ABCB1 and ABCG2 strongly contribute to multidrug resistance (MDR). Here we investigate the as yet unknown mechanisms of activation and inhibition of ABCG2. For this purpose we measured the ATPase activity of ABCG2 and ABCB1 as a function of allocrite concentration using a calibration set of 30 diverse compounds and a validation set of 23 compounds. We demonstrate that ABCG2 is activated at low and inhibited at high allocrite concentrations, yielding bell-shaped activity curves. With an ATP regeneration assay we prove that the inhibitory part is indeed due to a decrease in activity because of high allocrite load in the transporter. However, inhibition is only observed if the membrane solubility of allocrites is sufficiently high. The concentrations of half-maximum activation and inhibition are at least 10-fold lower for ABCG2 than for ABCB1. Because ABCG2 binds its allocrites with higher affinity than ABCB1, it can extract hydrophilic, nonamphiphilic, and highly charged compounds out of the lipid membrane, typically exhibiting low lipid-water partition coefficients, but is inhibited by hydrophobic, amphiphilic, and moderately charged compounds, with high lipid-water partition coefficients. In contrast, ABCB1 is barely interacting with hydrophilic compounds, but is activated by hydrophobic compounds. We show that hydrophobicity, amphiphilicity, and charge have a dual role; they predict, on the one hand, allocrites' lipid-water partition coefficient and, on the other hand, the transporters' preference for the chemical nature of allocrites. Parameters reflecting hydrophobicity, amphiphilicity, and charge are therefore sufficient for differentiating between allocrites, activators, and inhibitors of ABCB1 and ABCG2.

Allocrite Sensing and Binding by the Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1).

The ATP binding cassette (ABC) transporters ABCG2 and ABCB1 perform ATP hydrolysis-dependent efflux of structurally highly diverse compounds, collectively called allocrites. Whereas much is known about allocrite-ABCB1 interactions, the chemical nature and strength of ABCG2-allocrite interactions have not yet been assessed. We quantified and characterized interactions of allocrite with ABCG2 and ABCB1 using a set of 39 diverse compounds. We also investigated potential allocrite binding sites based on available transporter structures and structural models. We demonstrate that ABCG2 binds its allocrites from the lipid membrane, despite their hydrophilicity. Hence, binding of allocrite to both transporters is a two-step process, starting with a lipid-water partitioning step, driven mainly by hydrophobic interactions, followed by a transporter binding step in the lipid membrane. We show that binding of allocrite to both transporters increases with the number of hydrogen bond acceptors in allocrites. Scrutinizing the transporter translocation pathways revealed ample hydrogen bond donors for allocrite binding. Importantly, the hydrogen bond donor strength is, on average, higher in ABCG2 than in ABCB1, which explains the higher measured affinity of allocrite for ABCG2. π-π stacking and π-cation interactions play additional roles in binding of allocrite to ABCG2 and ABCB1. With this analysis, we demonstrate that these membrane-mediated weak electrostatic interactions between transporters and allocrites allow for transporter promiscuity toward allocrites. The different sensitivities of the transporters to allocrites' charge and amphiphilicity provide transporter specificity. In addition, we show that the different hydrogen bond donor strengths in the two transporters allow for affinity tuning.

Sav1866 from Staphylococcus aureus and P-glycoprotein: similarities and differences in ATPase activity assessed with detergents as allocrites.

The ATP-binding cassette exporters Sav1866 from Staphylococcus aureus and P-glycoprotein are known to share a certain sequence similarity and disposition for cationic allocrites. Conversely, the two ATPases react very differently to neutral detergents that have previously been shown to be inhibitory allocrites for P-glycoprotein. To gain insight into the functional differences of the two proteins, we compared their basal and detergent-stimulated ATPase activity. P-Glycoprotein was investigated in NIH-MDR1-G185 plasma membrane vesicles and Sav1866 in lipid vesicles exhibiting a membrane packing density and a surface potential similar to those of the plasma membrane vesicles. Under basal conditions, Sav1866 revealed a lower catalytic efficiency and concomitantly a more pronounced sodium chloride and pH dependence than P-glycoprotein. As expected, the cationic allocrites (alkyltrimethylammonium chlorides) induced similar bell-shaped activity curves as a function of concentration for both exporters, suggesting stimulation upon binding of the first and inhibition upon binding of the second allocrite molecule. However, the neutral allocrites (n-alkyl-ß-d-maltosides and n-ethylene glycol monododecyl ethers) reduced P-glycoprotein's ATPase activity at concentrations well below their critical micelle concentration (CMC) but strongly enhanced Sav1866's ATPase activity even at concentrations above their CMC. The lack of ATPase inhibition at high concentrations of neutral of detergents could be explained by their comparatively low binding affinity for the transmembrane domains of Sav1866, which seems to prevent binding of a second inhibitory molecule. The high ATPase activity in the presence of hydrophobic, long chain detergents moreover revealed that Sav1866, despite its lower basal catalytic efficiency, is a more efficient floppase for lipidlike amphiphiles than P-glycoprotein.

The brain entry of HIV-1 protease inhibitors is facilitated when used in combination.

One hypothesis for persisting HIV-associated neurocognitive disorders (HAND) in effectively treated individuals is the limited permeation of antiretroviral agents (ARV) across the blood-brain barrier (BBB). However, the physicochemical factors limiting the brain entry of a given ARV and the mutual interactions of combined drugs on their brain entry have not been properly characterized. Using transporter kinetic measurements, we show that large lipophilic drugs such as protease inhibitors (PI) have strong binding affinities to drug efflux transporters expressed at the BBB and thus are prevented from entering the brain. However, when combined, the PI with the highest binding affinity (i.e., boosting ritonavir) will occupy a large proportion of the transporter binding sites and thus slow down the efflux rate of the coadministered PI thereby facilitating its brain entry. Furthermore, using thermodynamic measurements and computational modeling, we show that ARV with small cross-sectional areas (AD < 70 Å(2)) and octanol-water distribution coefficients (-1 < log D <5) such as most nucleoside analogues have a high passive influx and cross the BBB despite interactions with drug transporters. These data indicate that HIV therapies combining small diffusing molecules with large lipophilic molecules are better suited for brain entry and should be preferred for HAND. This work highlights the role of PI as modulators of drugs' brain entry.

P-glycoprotein-ATPase modulation: the molecular mechanisms.

P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and cationic detergents with different hydrophobic and hydrophilic characteristics. Measurement of the P-glycoprotein-ATPase activity as a function of concentration showed that seven detergents activated the ATPase as expected, whereas 27 closely related detergents reduced it significantly. Assessment of the free energy of detergent partitioning into the lipid membrane and the free energy of detergent binding from the membrane to the transporter revealed that the ratio, q, of the two free energies of binding determined the rate of ATP hydrolysis. Neutral (cationic) detergents with a ratio of q = 2.7 ± 0.2 (q > 3) followed the aforementioned exponential dependence. Small deviations from the optimal ratio strongly reduced the rates of ATP hydrolysis and flopping, respectively, whereas larger deviations led to an absence of interaction with the transporter. P-glycoprotein-ATPase inhibition due to membrane disordering by detergents could be fully excluded using (2)H-NMR-spectroscopy. Similar principles apply to modulating drugs.

On the interaction of ionic detergents with lipid membranes. Thermodynamic comparison of n-alkyl-+N(CH3)3 and n-alkyl-SO4⁻.

Ionic detergents find widespread commercial applications as disinfectants, fungicides, or excipients in drug formulations and cosmetics. One mode of action is their ease of insertion into biological membranes. Very little quantitative information on this membrane-binding process is available to date. Using isothermal titration calorimetry (ITC) and dynamic light scattering (DLS), we have made a systematic comparison of the binding of cationic and anionic detergents to neutral and negatively charged lipid membranes. The detergents investigated were n-alkyl chains carrying either the trimethylammonium chloride (-(+)N(CH3)3Cl⁻) or the sodium sulfate (-SO4⁻Na(+)) headgroup with chain lengths of n = 10-16. The titration of lipid vesicles into detergent solutions provided the binding enthalpy and the binding isotherm in a model-independent manner. At 25 °C the membrane binding enthalpies, ΔH(mem)(0), were small (-0.4 to -4.2 kcal/mol) and showed little correlation with the length of the alkyl chains. The ITC binding isotherms were analyzed in terms of a surface partition model. To this purpose, the surface concentration, cM, of detergent immediately above the plane of binding was calculated with the Gouy-Chapman theory. The surface concentration corrects for electrostatic attraction or repulsion and can be larger or smaller than the bulk detergent concentration, c(eq), at equilibrium. The analysis provides the chemical or hydrophobic binding constant, K(D)(0), of the detergent and the corresponding free energy. The free energies of binding, ΔG(mem)(0), vary between -4 and -10 kcal/mol. They show a linear dependence on the chain length, which can be used to separate the contributions of the polar group and the hydrocarbon tail in membrane binding. The neutral maltose and the cationic (+)N(CH3)3 headgroup show steric repulsion energies of about 2.5 kcal/mol counteracting the hydrophobic binding of the alkyl tail, whereas the anionic SO4⁻ headgroup makes almost no contribution to membrane binding. The chemical nature of the headgroup influences the packing density of the hydrocarbon chains in the lipid bilayer with (+)N(CH3)3 eliciting the weakest chain-chain interaction. The minimum repulsive interaction of the SO4⁻ polar group makes the sodium n-alkyl-sulfates much stronger detergents than the nonionic or cationic counterparts, the binding constants, K(D)(0), being 10-50 times larger than those of the corresponding n-alkyl-trimethylammonium chlorides. The membrane insertion was further compared with micelle formation of the same detergent. A cooperative aggregation model which includes all possible aggregation states is proposed to analyze micelle formation. The partition function can be defined in closed form, and it is straightforward to predict the thermodynamic properties of the micellar system. When aggregated in micelles, the detergent polar groups are in direct interaction and are not separated by lipid molecules. Under these conditions the SO4⁻ group exhibits a strong electrostatic repulsive effect of 3.2 kcal/mol, while the contributions of the maltose and (+)N(CH3)3 headgroups are very similar to those in the lipid bilayer.

Exploring the P-glycoprotein binding cavity with polyoxyethylene alkyl ethers.

P-glycoprotein (ABCB1) moves allocrits from the cytosolic to the extracellular membrane leaflet, preventing their intrusion into the cytosol. It is generally accepted that allocrit binding from water to the cavity lined by the transmembrane domains occurs in two steps, a lipid-water partitioning step, and a cavity-binding step in the lipid membrane, whereby hydrogen-bond (i.e., weak electrostatic) interactions play a crucial role. The remaining key question was whether hydrophobic interactions also play a role for allocrit binding to the cavity. To answer this question, we chose polyoxyethylene alkyl ethers, C(m)EO(n), varying in the number of methylene and ethoxyl residues as model allocrits. Using isothermal titration calorimetry, we showed that the lipid-water partitioning step was purely hydrophobic, increasing linearly with the number of methylene, and decreasing with the number of ethoxyl residues, respectively. Using, in addition, ATPase activity measurements, we demonstrated that allocrit binding to the cavity required minimally two ethoxyl residues and increased linearly with the number of ethoxyl residues. The analysis provides the first direct evidence, to our knowledge, that allocrit binding to the cavity is purely electrostatic, apparently without any hydrophobic contribution. While the polar part of allocrits forms weak electrostatic interactions with the cavity, the hydrophobic part seems to remain associated with the lipid membrane. The interplay between the two types of interactions is most likely essential for allocrit flipping.

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.

Detergents as intrinsic P-glycoprotein substrates and inhibitors.

We assessed the interaction of three electrically neutral detergents (Triton X-100, C(12)EO(8), and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, DeltaG(tl)(0), that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately DeltaG(EO)(0)=-1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.

Halogenation of drugs enhances membrane binding and permeation.

Halogenation of drugs is commonly used to enhance membrane binding and permeation. We quantify the effect of replacing a hydrogen residue by a chlorine or a trifluoromethyl residue in position C-2 of promazine, perazine, and perphenazine analogues. Moreover, we investigate the influence of the position (C-6 and C-7) of residue CF(3) in benzopyranols. The twelve drugs are characterized by surface activity measurements, which yield the cross-sectional area, the air-water partition coefficient, and the critical micelle concentration. By using the first two parameters (A(D) and K(aw)) and the appropriate membrane packing density, the lipid-water partition coefficients, are calculated in excellent agreement with the lipid-water partition coefficients measured by means of isothermal titration calorimetry for small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Replacement of a hydrogen residue by a chlorine and a trifluoromethyl residue enhances the free energy of partitioning into the lipid membrane, on average by deltaG(lw) approximately -1.3 or -4.5 kJ mol(-1), respectively, and the permeability coefficient by a factor of approximately 2 or approximately 9, respectively. Despite exhibiting practically identical hydrophobicities, the two benzopyranol analogues differ in their permeability coefficients by almost an order of magnitude; this is due to their different cross-sectional areas at the air-water and lipid-water interfaces.