Houchao Tao

Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump

Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

Engineered nanostructured β-sheet peptides protect membrane proteins.

We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP–β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

Snapshots of ligand entry, malleable binding and induced helical movement in P-glycoprotein

Co-crystal structures of P-glycoprotein with a series of engineered ligands reveal multiple ligand-binding modes, a ligand-binding site on the outer surface of the transporter and a conformational change that may couple to ATP hydrolysis.

New amphiphiles for membrane protein structural biology

A challenging requirement for X-ray crystallography or NMR structure determination of membrane proteins (MPs), in contrast to soluble proteins, is the necessary use of amphiphiles to mimic the hydrophobic environment of membranes. A number of new detergents, lipids and non-detergent-like amphiphiles have been developed that stabilize MPs, and these have contributed to increased success in MP structural determinations in recent years. Despite some successes, currently available reagents are inadequate and there remains a pressing need for new amphiphiles. Literature examples and some new developments are selected here as a framework for discussing desirable properties of new amphiphiles for MP structural biology.

Distinct Conformational Spectrum of Homologous Multidrug ABC Transporters

ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes.

Understanding polyspecificity within the substrate‐binding cavity of the human multidrug resistance P‐glycoprotein

Human P‐glycoprotein (P‐gp) controls drugs bioavailability by pumping structurally unrelated drugs out of cells. The X‐ray structure of the mouse P‐gp ortholog has been solved, with two SSS enantiomers or one RRR enantiomer of the selenohexapeptide inhibitor QZ59, found within the putative drug‐binding pocket (Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL et al. (2009). Science 323, 1718–1722). This offered the first opportunity to localize the well‐known H and R drug‐binding sites with respect to the QZ59 inhibition mechanisms of Hoechst 33342 and daunorubicin transports, characterized here in cellulo. We found that QZ59‐SSS competes efficiently with both substrates, with KI,app values of 0.15 and 0.3 μm, which are 13 and 2 times lower, respectively, than the corresponding Km,app values. In contrast, QZ59‐RRR non‐competitively inhibited daunorubicin transport with moderate efficacy (KI,app = 1.9 μm); it also displayed a mixed‐type inhibition of the Hoechst 33342 transport, resulting from a main non‐competitive tendency (Ki2,app = 1.6 μm) and a limited competitive tendency (Ki1,app = 5 μm). These results suggest a positional overlap of QZ59 and drugs binding sites: full for the SSS enantiomer and partial for the RRR enantiomer. Crystal structure analysis suggests that the H site overlaps both QZ59‐SSS locations while the R site overlaps the most embedded location.

Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

Design and synthesis of Selenazole-containing peptides for cocrystallization with P-glycoprotein.

Over-expression of P-glycoprotein (Pgp) in some cancer cells is a primary cause of multidrug resistance (MDR) that results in chemotherapy failure. As a consequence, Pgp has been a long-sought target for drug development in the hope of circumventing MDR in clinical oncology.[1,2] More than three decades of biochemical studies have indicated that Pgp might possess several distinct substrate-binding sites within a large, flexible region located between the two transmembrane domains; these sites recognize hundreds of chemically unrelated compounds, including a majority of traditional anti-cancer drugs.[3–6] These compounds can be exported from cells by Pgp using energy released by self-catalyzed ATP hydrolysis in conjunction with substantial conformational changes. Our groups recently determined a structure of mouse Pgp at 3.8 A resolution by X-ray diffraction.[7] We also solved the cocrystal structures of Pgp with a pair of enantiomeric cyclic peptides, QZ59Se-RRR and QZ59Se-SSS (Scheme 1), in which the two enantiomers bind to Pgp with different stoichiometries and at distinct sites within a transmembrane portal open to the cytoplasmic side. These structures provided the first glimpse of the polyspecific drug-binding site of Pgp in atomic detail.

Synthesis and Properties of Dodecyl Trehaloside Detergents for Membrane Protein Studies

Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization, and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carbon alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC), and size of micelles, were evaluated and compared with the most popular maltoside analogue, β-D-dodecyl maltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive, given their interesting and unique chemical-physical properties and potential interactions with membrane lipids.

Synthesis of azole-enriched cyclic peptides by a clean solid-phase-based cyclization-cleavage strategy.

Naturally occurring azole-enriched cyclic peptides have broad biological and pharmacological activities. Previous synthetic efforts have mainly concentrated on the preparation of individual target molecules in solution phase. A solid-phase-based cyclitive cleavage strategy was deployed here for efficient library synthesis of azole cyclopeptide derivatives, which is part of our continuous efforts for the characterization of potent modulators of multidrug resistance efflux proteins. Procedures were optimized to afford the azole cyclopeptides at high yield and purity, eliminating the need for any chromatographic purification steps. This development is ideal for high throughput library synthesis and screening and will facilitate the ultimate discovery of novel azole cyclopeptides with potent biological activities.