•A 2.2-Å structure of transhydrogenase proton channel was determined in lipids•The structures solved at two different pHs show conformational flexibility•Water molecules and key interacting residues were revealed in the proton channel•Molecular dynamics provides insights into the mechanism of channel gatingThe nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic “dry” region in the middle of the membrane channel, with key residues His42α2 (chain A) being protonated and Thr214β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.Download high-res image (163KB)Download full-size image

We describe cholate-based cage amphiphiles with a unique architecture that combines elements of structural rigidity and flexibility. The cage compounds are built by extending and bridging three polar chains underneath the concave steroid rings of cholate and capping with another rigid, symmetrically trifunctionalized cyanuric acid moiety. The connecting chains are varied to include, for instance, oligo(ethylene glycol) or chains containing 1,2,3-triazole units to present flexibility in the chemical and structural space and potentially deliver functional molecules for molecular recognition applications.

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.Keywords: cyclic peptides; cyclitive cleavage; solid-phase synthesis

Amphiphile selection is a critical step for structural studies of membrane proteins (MPs). We have developed a family of steroid-based facial amphiphiles (FAs) that are structurally distinct from conventional detergents and previously developed FAs. The unique FAs stabilize MPs and form relatively small protein–detergent complexes (PDCs), a property considered favorable for MP crystallization. We attempted to crystallize several MPs belonging to different protein families, including the human gap junction channel protein connexin 26, the ATP binding cassette transporter MsbA, the seven-transmembrane G protein-coupled receptor-like bacteriorhodopsin, and cytochrome P450s (peripheral MPs). Using FAs alone or mixed with other detergents or lipids, we obtained 3D crystals of the above proteins suitable for X-ray crystallographic analysis. The fact that FAs enhance MP crystallizability compared with traditional detergents can be attributed to several properties, including increased protein stability, formation of small PDCs, decreased PDC surface flexibility, and potential to mediate crystal lattice contacts.

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.

A challenging requirement for structural studies of integral membrane proteins (IMPs) is the use of amphiphiles that replicate the hydrophobic environment of membranes. Progress has been impeded by the limited number of useful detergents and the need for a deeper understanding of their structure−activity relationships. To this end, we designed a family of detergents containing short, branched alkyl chains at the interface between the polar head and the apolar tail. This design mimics the second aliphatic chain of lipid molecules and reduces water penetration, thereby increasing the hydrophobicity within the interior of the micelle. To compare with the popular straight-chained maltoside detergents, the branch-chained β-d-maltosides were synthesized efficiently in pure anomeric form. The branch-chained maltosides form smaller micelles by having shorter main chains, while having comparable hydrophobicity to the detergents with only straight chains. Selected branch-chained and straight-chained maltoside detergents were examined for their ability to solubilize, stabilize, and aid the crystallization of human connexin 26, an α-helical IMP that forms hexamers. We showed that the branch-chained maltosides with optimized micellar properties performed as well as or better than the straight-chained analogues and enabled crystallization in different space groups.

•New tools enabled EM visualization of dynamic transporter conformations in lipids•Distinct conformational spectra of two homologous ABC transporters were revealed•P-gp prevails in inward-facing conformations under ATP hydrolysis conditions•MsbA with ATP displays both outward and a continuum of inward-facing conformationsATP-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.Download high-res image (159KB)Download full-size image