Membrane Protein Structures in Lipid Bilayers; Small-Angle Neutron Scattering With Contrast-Matched Bicontinuous Cubic Phases

This perspective describes advances in determining membrane protein structures in lipid bilayers using small-angle neutron scattering (SANS). Differentially labeled detergents with a homogeneous scattering length density facilitate contrast matching of detergent micelles; this has previously been used successfully to obtain the structures of membrane proteins. However, detergent micelles do not mimic the lipid bilayer environment of the cell membrane in vivo. Deuterated vesicles can be used to obtain the radius of gyration of membrane proteins, but protein-protein interference effects within the vesicles severely limits this method such that the protein structure cannot be modeled. We show herein that different membrane protein conformations can be distinguished within the lipid bilayer of the bicontinuous cubic phase using contrast-matching. Time-resolved studies performed using SANS illustrate the complex phase behavior in lyotropic liquid crystalline systems and emphasize the importance of this development. We believe that studying membrane protein structures and phase behavior in contrast-matched lipid bilayers will advance both biological and pharmaceutical applications of membrane-associated proteins, biosensors and food science.

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

<div> <div> <div><div><div><p>Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional charac- terization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.</p></div></div></div> </div> </div>

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

<div> <div> <div><div><div><p>Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional charac- terization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.</p></div></div></div> </div> </div>

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

ABSTRACTCLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

Active site competition is the mechanism for the inhibition of lipoprotein-associated phospholipase A2 by detergent micelles or lipoproteins and for the efficacy reduction of darapladib

Lipoprotein associated phospholipase A2 (Lp-PLA2) has been characterized for its interfacial activation as well as inhibition by detergent micelles and lipoprotein particles. The enzyme has been shown to bind on the surfaces of hydrophobic aggregates, such as detergent micelles, lipoprotein particles and even polystyrene latex nanobeads. Binding to hydrophobic aggregates stimulates the activity of Lp-PLA2 but may not be the necessary step for catalysis. However, at higher concentrations, detergent micelles, latex nanobeads or lipoprotein particles inhibit Lp-PLA2 possibly by blocking the access of substrates to the active site. The competition mechanism also blocks inhibitors such as darapladib binding to Lp-PLA2 and reduces the efficacy of the drug. Darapladib has very low solubility and mainly exists in solutions as complexes with detergents or lipoprotein particles. The inhibition of Lp-PLA2 by darapladib is dependent on many factors such as concentrations of detergents or lipoproteins, incubation time, as well as the order of mixing reaction components. The in vitro Lp-PLA2 activity assays used in clinical studies may not accurately reflect the residual Lp-PLA2 activity in vivo. Darapladib has been found mainly bound on HDL and albumin when it is incubated with human serum. However, Lp-PLA2 is more sensitive to darapladib when bound on LDL and relatively resistant to darapladib when bound on HDL. Therefore, high cholesterol levels may decrease the efficacy of darapladip and cause the drug to be less effective in high risk patients. Our study will help to design better inhibitors for Lp-PLA2. The discoveries also contribute to understanding the mechanism of interfacial activation and inhibition for Lp-PLA2 and provide a new concept for researchers in building better kinetic model for interfacial enzymes.

Drug binding sites in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs

MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-β-D-maltopyranoside (DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the If (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the If structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM.Statement of SignificanceMdfA is multidrug transporter from E. coli, which exhibits multidrug efflux activities with an unusually broad spectrum of drug specificities. While it has been established that solute transport by similar transporters is coupled to significant conformational changes, previous studies raised the possibility that this is not the case for MdfA. Moreover, it is not clear how MdfA functionally accommodates chemically dissimilar substrates. Towards resolving these open questions, we used double-electron electron resonance distance measurements to determine the binding site of a spin labeled drug analog within available crystal structures of MdfA and to examine how MdfA responds conformationally to drug binding. Moreover, we explored how these two are affected by the media, detergent micelles vs lipid nanodiscs.

Structure Basis of Cav1.1 Modulation by Dihydropyridine Compounds

Abstract1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. Recent structural elucidation of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remains elusive. Here, we report five cryo-EM structures of nanodisc-embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that the agonistic effect of (S)-(-)-Bay K8644 might be transient. The similarity of these structures to that obtained in detergent micelles alleviates the concerns about potential structural perturbation by detergents.

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

<div> <div> <div> <p>Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamel- lar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these com- plexes remains challenging because of their chemical composition, the hydrophobicity and structural instabil- ity of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid su- pramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework- 8 (ZIF-8) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF-8 bio- composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embed- ded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization. </p> </div> </div> </div>

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+channels in nanodisc

NaChBac, the first bacterial voltage-gated Na+(Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure–function relationship of Navchannels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Navchannels in lipid bilayers. To study GMTs in mammalian Navchannels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV–bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Navchannels in a membrane environment.