Small isoprenoid diphosphates, such as ()-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (STAA or TA) investigated, confirm that the backbone amide of at least one Thr (Thr), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear Magnetic Resonance is one such high resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, Integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodiscs through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like SMA lipid discs and Saposin-A lipoprotein discs.

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Despite arduous efforts and recent technological developments structural investigation of integral membrane proteins remains a challenge. The primary deterrents include difficulties with their expression, low inherent solubility and various problems associated with existing membrane mimicking systems. A relatively new class of membrane mimetics, nanodiscs, has been developed as a promising alternative. Although nanodiscs have been proven successful for several biophysical applications, they yet remain to become the system of preferred choice for structure determination. We have hereby made nanodiscs more suitable for solution NMR applications by reducing the diameter of the self-assembly complex to its potential limit. We achieved a noticeable improvement in the quality of NMR spectra obtained for the transmembrane and cytoplasmic domains of integrin αIIb incorporated into these smaller discs rendering them susceptible for a thorough structural investigation. We also present an on-column method for a rapid, efficient, single step preparation of protein incorporated nanodiscs at high concentrations. These discs have been fully characterized by transmission electron microscopy, dynamic light scattering and differential scanning calorimetry.

Edited by Karen G. Fleming A third of the genes in prokaryotic and eukaryotic genomes encode membrane proteins that are either essential for signal transduction and solute transport or function as scaffold structures. Unlike many of their soluble counterparts, the overall structural and functional organization of membrane proteins is sparingly understood. Recent advances in X-ray crystallography, cryo-EM, and nuclear magnetic resonance (NMR) are closing this gap by enabling an in-depth view of these ever-elusive proteins at atomic resolution. Despite substantial technological advancements, however, the overall proportion of membrane protein entries in the Protein Data Bank (PDB) remains <4%. This paucity is mainly attributed to difficulties associated with their expression and purification, propensity to form large multisubunit complexes, and challenges pertinent to identification of an ideal detergent, lipid, or detergent/lipid mixture that closely mimic their native environment. NMR is a powerful technique to obtain atomic-resolution and dynamic details of a protein in solution. This is accomplished through an assortment of isotopic labeling schemes designed to acquire multiple spectra that facilitate deduction of the final protein structure. In this review, we discuss current approaches and technological developments in the determination of membrane protein structures by solution NMR and highlight recent structural and mechanistic insights gained with this technique. We also discuss strategies for overcoming size limitations in NMR applications, and we explore a plethora of membrane mimetics available for the structural and mechanistic understanding of these essential cellular proteins.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family.

PLIC, Protein Linking IAP (CD47) to Cytoskeleton, have long since been implicated in connecting the extracellular membrane to the intracellular cell cytoskeleton. This phenomenon is supposedly achieved by bridging a receptor protein CD47 to vimentin, an intermediate filament, which in turn regulates integrin dependent cell spreading. Since the discovery of these proteins, the molecular details of the above-mentioned interactions and the underlying complexes are yet to be characterized. Several independent studies have together emphasized PLIC/Ubiquilin’s role in the proteasomal degradation pathway. This seems to be in contrast to the purported initial discovery of PLIC as a cytoskeletal adaptor protein. In an effort to reconcile the different roles associated with the ubiquitous PLIC proteins, we tested the involvement of PLIC-2 both in the proteasomal degradation pathway and as a protein linking the cell cytoskeleton to the cytoplasmic tail of CD47. This was achieved thorough an in vitro investigation of their binding interface using a combination of biophysical techniques. Our results show that the two terminal domains of PLIC-2 interact weakly with each other, while the C-terminal UBA domain interacts strongly with ubiquitin. Interestingly, no perceptible interaction was observed for PLIC-2 with the cytoplasmic tail of CD47 questioning its role as a “PLIC” protein linking the cell membrane to the cytoskeleton.