Structural Insights into the Lipid A Transport Pathway in MsbA.

MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile.

Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory.

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity.

Chemically Stable Lipids for Membrane Protein Crystallization.

Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs.

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

P-glycoprotein (P-gp) is a transporter of great clinical and pharmacological significance. Several structural studies of P-gp and its homologs have provided insights into its transport cycle, but questions remain regarding how P-gp recognizes diverse substrates and how substrate binding is coupled to ATP hydrolysis. Here, four new P-gp co-crystal structures with a series of rationally designed ligands are presented.

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.

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.

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.

Dynamic adhesive force measurements under vertical and horizontal motions of interacting rough surfaces.

An instrument to measure dynamic adhesive forces between interacting rough surfaces has been developed. It consists of four parts, namely, main instrument body, vertical positioning system with both micrometer and nanometer positioning accuracies, horizontal positioning system with nanometer positioning accuracy, and custom-built high-resolution, and high dynamic bandwidth capacitive force transducer. The vertical piezoelectric actuator (PZT) controls the vertical (approaching and retracting) motion of the upper specimen, while the horizontal PZT controls the horizontal (reciprocal) motion of the lower specimen.

Role of phospholipase D2 in anti-apoptotic signaling through increased expressions of Bcl-2 and Bcl-xL.

We have previously reported that Fas-resistant A20 cells (FasR) have phospholipase D (PLD) activity upregulated by endogenous PLD2 overexpression. In the present study, we investigated how overexpressed PLD2 in FasR could generate survival signals by regulating the protein levels of anti-apoptotic Bcl-2 and Bcl-xL. To confirm the effect of PLD2 on Bcl-2 protein levels, we transfected PLD2 into wild-type murine B lymphoma A20 cells.

Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC.

Gap-junctional channels, permeable to large hydrophilic solutes of up to M(r) approximately 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits.

Membrane transport proteins with complete replacement of transmembrane helices with polyalanine sequences remain functional.

Approximately 25% of all genome coding sequences correspond to membrane proteins, which perform varied and essential functions in cells. Eukaryotic integral membrane proteins are predominantly alpha-helical proteins that span the membrane several times. The most frequent approach to identifying transmembrane-helix amino acids essential for function is to substitute native residues, one at a time, with Cys or Ala (Cys- and Ala-scanning mutagenesis).

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide.

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD).

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells.

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells.