Structural insights into the high basal activity and inverse agonism of the orphan receptor GPR6 implicated in Parkinson's disease.

GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate G heterotrimer.

Coupling and Activation of the β1 Adrenergic Receptor - The Role of the Third Intracellular Loop.

G protein-coupled receptors (GPCRs) belong to the most diverse group of membrane receptors with a conserved structure of seven transmembrane (TM) α-helices connected by intracellular and extracellular loops. Intracellular loop 3 (ICL3) connects TM5 and TM6, the two helices shown to play significant roles in receptor activation. Herein, we investigate the activation and signaling of the β adrenergic receptor (βAR) using mass spectrometry (MS) with a particular focus on the ICL3 loop.

Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway.

Intracellular accumulation of misfolded proteins causes serious human proteinopathies. The transmembrane emp24 domain 9 (TMED9) cargo receptor promotes a general mechanism of cytotoxicity by entrapping misfolded protein cargos in the early secretory pathway. However, the molecular basis for this TMED9-mediated cargo retention remains elusive.

Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters.

The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up 'omics' approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes.

How Clavulanic Acid Inhibits Serine β-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS).

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc).

Phage defence system CBASS is regulated by a prokaryotic E2 enzyme that imitates the ubiquitin pathway.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2.

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear.

ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT). Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS).

Phospholipids Differentially Regulate Ca Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap.

Connecting single-nucleotide polymorphisms, glycosylation status, and interactions of plasma serine protease inhibitors.

Understanding the combined impacts of genetic variances and post-translational modifications requires new approaches. Here, we delineate proteoforms of plasma serine protease inhibitors and relate specific proteoforms to their interactions in complexes through the use of native mass spectrometry (MS). First, we dissect the proteoform repertoire of an acute-phase plasma protein, serine protease inhibitor A1 (SERPINA1), resolving four SERPINA1 variants (M1V, M1A, M2, and M3) with common single-nucleotide polymorphisms (SNPs).

Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics.

The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB.

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.

Constitutive activation mechanism of a class C GPCR.

Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156-G signaling contributes to loss of hearing.

Characterization of membrane protein interactions by peptidisc-mediated mass photometry.

Membrane proteins perform numerous critical functions in the cell, making many of them primary drug targets. However, their preference for a lipid environment makes them challenging to study using established solution-based methods. Here, we show that peptidiscs, a recently developed membrane mimetic, provide an ideal platform to study membrane proteins and their interactions with mass photometry (MP) in detergent-free conditions.

Structural characterization of human urea transporters UT-A and UT-B and their inhibition.

In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTB-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis.

Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1.

Developing rights-based standards for children having tests, treatments, examinations and interventions: using a collaborative, multi-phased, multi-method and multi-stakeholder approach to build consensus.

Children continue to experience harm when undergoing clinical procedures despite increased evidence of the need to improve the provision of child-centred care. The international ISupport collaboration aimed to develop standards to outline and explain good procedural practice and the rights of children within the context of a clinical procedure. The rights-based standards for children undergoing tests, treatments, investigations, examinations and interventions were developed using an iterative, multi-phased, multi-method and multi-stakeholder consensus building approach.

The solute carrier SPNS2 recruits PI(4,5)P to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data.