Amanda J. Rice

The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas–FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5–7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas–FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs

Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under nondenaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.

Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility

Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the proteins functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.

Tuning the Membrane Selectivity of Antimicrobial Peptides by Using Multivalent Design

Rapid emergence of antibacterial drug resistance poses a critical problem for the treatment of infectious diseases. The number of new classes of antibiotics approved by the FDA has declined in recent years. Since their discovery in the early 1980s, antimicrobial peptides (AMPs, also called host defense peptides) have stimulated interest as prospective antibiotic agents because they rapidly inactivate a wide range of microorganisms including Gram-positive and negative bacteria, fungi, and some viruses. In many cases they are indifferent to current multidrug-resistant (MDR) strains. Naturally occurring AMPs span a wide range of size, sequence, and structure. They generally share only amphiphilicity and positive charge. Different chemical approaches have been pursued in efforts to increase the effectiveness of AMPs, including incorporation of unnatural dor b-amino acids, cyclization of peptides, and screening of synthetic AMP combinatorial libraries. Despite recent progress, the diversity and lack of structural homology among known AMPs make it difficult to predict the activity of a peptide or to design AMPs with desired activities in vivo. In a recent study, a linguistic model was applied to the rational design of AMPs. To date, classical drug R&D approaches have not been successfully applied to derive novel antibiotics from AMPs. The amino acids Arg (R) and Trp (W) occur in natural AMPs that span a range of sizes and secondary structures, including indolicidin and tritrpticin, which have a broad spectrum of antibacterial activity but lyse erythrocytes. QSAR analysis of Rand W-rich peptides suggested that the order of amino acids is not crucial whereas R and W content correlates most strongly with activity. Strom et al. proposed that simple combinations of R and W side chains constitute one type of pharmacophore for AMPs. We recently investigated the length requirement for antimicrobial activity of RW peptides. Even short repeats of RW are active, providing a starting point for de novo design of AMPs. The hypothesis we test in this work is that multi/polyvalent RW peptide display can enhance the effectiveness of antibacterials against MDR bacterial strains. Application of multivalency to cell surface receptors has been demonstrated previously. Our thinking was originally based on the two-state model of AMP action proposed by Huang, which is broadly consistent with other current ideas. The model assumes that AMPs act at the level of the cell membrane via electrostatic interactions with the negatively charged head groups of bacterial lipids. At peptide-to-lipid ratios (P/L) above a threshold value, the peptides form clusters that lead to permeation of the cell membrane. The susceptibility of a cell to an AMP depends on the value of P/L determined by the lipid composition of the cell membrane. Huang ascribes this to an elastic perturbation of the surface. However alternative physical mechanisms can be invoked. A consequence of assembly models is that prenucleating AMP monomers might reduce the critical concentration for cluster formation as AMPs bind to the bacterial membrane. Initial attachment of AMPs to bacterial surfaces or membranes is salt sensitive, consistent with electrostatic interactions. General arguments suggest that covalently tethering a number of weakly interacting ligands can enhance overall avidity for targets such as cell surfaces. We recently applied this strategy to create new antimicrobial agents by linking tetrapeptides RWRW and RRWW to a polydisperse polymer scaffold. These constructs enhanced antimicrobial activity, with an increase of roughly tenfold in potency against both Gram-negative and positive strains. At the same time the hemolytic activity of the polymers increased, but the ratio of antimicrobial to hemolytic activity remained roughly constant. To further explore multivalent AMP designs, we have synthesized a variety of dendrimeric AMPs containing multiple RW dipeptides. Of these the most successful is (RW)4D (Scheme 1). A detailed account of the constructs based on alternative scaffolds will be presented elsewhere. Dendrimeric peptide displays were first developed in the 1980s as multiple antigenic peptides. They consist of a core formed by radially branched lysines or other residues. Peptide sequences are grafted to the core by standard solid-phase chemistry. Herein, we report the antibacterial and hemolytic activity of (RW)4D compared to the natural AMP indolicidin (ILPWKWPWWPWRR-NH2) from bovine

The Structure of the Trimer of Human 4-1BB Ligand Is Unique among Members of the Tumor Necrosis Factor Superfamily

Binding of the 4-1BB ligand (4-1BBL) to its receptor, 4-1BB, provides the T lymphocyte with co-stimulatory signals for survival, proliferation, and differentiation. Importantly, the 4-1BB-4-1BBL pathway is a well known target for anti-cancer immunotherapy. Here we present the 2.3-Å crystal structure of the extracellular domain of human 4-1BBL. The ectodomain forms a homotrimer with an extended, three-bladed propeller structure that differs from trimers formed by other members of the tumor necrosis factor (TNF) superfamily. Based on the 4-1BBL structure, we modeled its complex with 4-1BB, which was consistent with images obtained by electron microscopy, and verified the binding site by site-directed mutagenesis. This structural information will facilitate the development of immunotherapeutics targeting 4-1BB.

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo-phosphorylated states.

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca2+‐ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation‐mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation‐mimicking mutation does not perturb the pentamer structure. However, 15N R1 and R2 relaxation rates and 15N(1H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.

Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae.

Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.