Nuclear hormone receptors (NHRs) control numerous physiological processes through the regulation of gene expression. The present study provides a structural basis for understanding the role of DNA in the spatial organization of NHR heterodimers in complexes with coactivators such as Med1 and SRC-1. We have used SAXS, SANS and FRET to determine the solution structures of three heterodimer NHR complexes (RXR-RAR, PPAR-RXR and RXR-VDR) coupled with the NHR interacting domains of coactivators bound to their cognate direct repeat elements. The structures show an extended asymmetric shape and point to the important role played by the hinge domains in establishing and maintaining the integrity of the structures. The results reveal two additional features: the conserved position of the ligand-binding domains at the 5' ends of the target DNAs and the binding of only one coactivator molecule per heterodimer, to RXR's partner.

High density lipoprotein (HDL), the carrier of so-called "good" cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity. High density lipoprotein (HDL)2 functions in removal of cholesterol from peripheral tissues, such as within the artery wall, for delivery to the liver and ultimate excretion as biliary cholesterol within the intestinal lumen, a process called reverse cholesterol transport (1, 2). Plasma levels of HDL cholesterol and apolipoprotein AI (apoA-I), the major protein component of HDL, are inversely related to the risk of developing coronary artery disease (3-5). Moreover, genetic alterations that induce changes in apoA-I levels in both animals and humans alter susceptibility for development of atherosclerotic heart disease (3-6). Thus, numerous interventions aimed at enhancing reverse cholesterol transport are being examined as potential novel therapeutic interventions for the prevention and treatment of cardiovascular disease (7,8). Examples include methods for generating new HDL particles through enhanced production or delivery of either intact apoA-I (9, 10) or peptide mimetics of apoA-I (11), as well as modulating interactions between nascent HDL and proteins involved in HDL particle maturation and remodeling for potential therapeutic benefit (12-14). Structural elucidation often serves as the "Rosetta Stone" for enhanced understanding of function. It is thus remarkable that despite its importance to numerous biological and biomedical functions and its current prominent role as a target for therapeutic interventions, to date, the structures of neither the protein nor lipid components of nascent HDL have been directly visualized, and the high resolution structure of the particl...

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniquesquartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane.