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Abstract 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)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

Summary: 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. The mass resolution for membrane protein complexes is similar to that achievable with soluble proteins owing to the low carrier heterogeneity. Using the ABC transporter BtuCD, we show that MP can quantify interactions between peptidisc-reconstituted membrane protein receptors and their soluble protein binding partners. Using the BAM complex, we further show that MP reveals interactions between a membrane protein receptor and a bactericidal antibody. Our results highlight the utility of peptidiscs for membrane protein characterization in detergent-free solution and provide a rapid and powerful platform for quantifying membrane protein interactions.

The Main protease of SARS-CoV-2 is key for viral replication, but its maturation process is still not completely understood. Here, the authors not only reveal unique details from the first step of maturation, but also demonstrate how different classes of inhibitor can block this step.

Voltage-gated Ca2+ channel subtype Cav2.3 represents a potential drug target for neurological diseases. Here, authors report cryo-EM structures of Cav2.3 and its mutant to reveal the crosstalk of intracellular segments, which may facilitate future drug discovery.

Bacterial cell wall enzymes and their precursors are critical targets for antibiotic development. Here, the authors investigate several biosynthetic enzymes with their substrates and show that the passage of substrates and products in the pathway is controlled by their relative binding affinities.

The Kir potassium channels are known to operate and gate without a major conformational change. Here, the authors identify the permeation gate of Kir channels as a steric plug within the conduction pathway, describing how tightly associated anionic lipids pushing into fenestrations in the pore walls engage with the plug to operate the gate.

E. coli MukBEF is an SMC complex that plays key roles in chromosome organization-segregation. Here the authors show that the interaction between MukBEF and the Acyl Carrier Protein (AcpP) is essential for MukBEF activity in vitro (ATPase) and in vivo.

The Wzc–Wza complex forms part of the bacterial extracellular polysaccharides synthesis machinery, where cycling of the Wzc between phosphorylation states is crucial to both synthesis and export. Here the authors report the structure of the Wzc octamer and provide insight into its regulation through phosphorylation.

Bacteria can secrete diffusible protein toxins that kill competing bacteria. Here, the authors use biochemical, biophysical and structural analyses to show how one of these toxins exploits TolC (a major antibiotic efflux channel) to transport itself across the outer membrane of target cells.

In Bacteroidetes, SusCD complexes mediate uptake of large nutrients across the outer membrane. SusCD structures in the apo state and in complex with β2,6 fructo-oligosaccharides reveal several substrate molecules in the binding cavity and suggest details of the pedal bin mechanism employed in glycan import.

Archaerhodopsin-3 (AR3) mutants are commonly used in optogenetics for neuron silencing and membrane voltage sensing. High-resolution crystal structures show that desensitization of the AR3 photoreceptor occurs when internal hydrogen-bonded water networks are modified in response to changes in chromophore isomerization.

The transition between conducting and non-conducting states of K+ channels has been explained by conformational changes at the intracellular entrance to the conduction pathway. Here authors demonstrate that control over K+ currents in Kir channels is not explained by the canonical pore-gating model, as conduction is not impaired by a constricted inner helix bundle.

F1Fo ATP synthase consists of two coupled rotary molecular motors: the soluble ATPase F1 and the transmembrane Fo. Here, the authors present cryo-EM structures of E. coli ATP synthase in four discrete rotational sub-states at 3.1-3.4 Å resolution and observe a rotary sub-step of the Fo motor cring that reveals the mechanism of elastic coupling between the two rotary motors, which is essential for effective ATP synthesis.

Detergents are indispensable reagents in membrane protein structural biology. Here, L. H. Urner and co-workers introduce oligoglycerol detergents (OGDs) and use native mass spectrometry to show how interactions of membrane proteins with native membrane lipids can be preserved during purification.

Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains.

The Ups/PRELI family mediates intramitochondrial lipid distribution and synthesis by shuttling phospholipids between both mitochondrial membranes. Here the authors combine X-ray crystallography, MD simulations and yeast genetic screens and identify residues that are important for Ups/PRELI lipid specificity and reveal a dual lipid recognition mechanism in Ups/PRELI family members.

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.

ABSTRACT Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

Centriole duplication is tightly regulated in vivo, but the underlying molecular mechanisms are incompletely understood. Here the authors use high-resolution structural and imaging methods to show that CEP85 directly interacts with STIL and mediates efficient centriolar targeting of STIL, PLK4 activation and centriole assembly.

Gram-negative bacteria assemble biofilms from amyloid fibres, which translocate across the outer membrane as unfolded amyloid precursors through a secretion system. Here, the authors characterise the structural details of the amyloid transporter FapF in Pseudomonas.

The properties of many transmembrane or aggregation-prone proteins make them difficult to recombinantly express. Here the authors use a modified N-terminal domain of a spider silk protein to express and purify several difficult to express proteins at levels considerably higher than with conventional tags.

Na+/H+antiporters transport sodium or lithium ions across the membrane in exchange for protons. Here the authors combine ion mobility mass spectrometry and molecular dynamics simulations to uncover a facilitating role for lipids in the transport mechanism.

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.

Targeting of the CD36 scavenger receptor by the malaria parasite effector PfEMP1 prevents splenic clearance of infected erythrocytes. Here, the authors propose that diverse PfEMP1 achieve this by binding to a conserved phenylalanine residue in CD36 that is also required for lipoprotein binding.

Counteracting oxidative stress is essential in all organisms. Here, the authors outline a mechanism used by actinomycete bacteria in which oxidation of zinc-binding RsrA blocks its interaction with σR by sequestering hydrophobic residues used to bind σRwithin its own core.

Pore-forming toxins act by forming oligomeric pores in lipid membranes. Here the authors report the crystal structure of the lysenin pore, providing insights into the assembly and function of the pore in addition to suggesting that its properties make lysenin potentially well-suited for nanopore sensing applications.

The prolactin receptor consists of a folded extracellular domain, a transmembrane domain and an intracellular intrinsically disordered domain. Here the authors use a combined experimental and computational approach to obtain a structure of a class I cytokine receptor, the human prolactin receptor.

FLRT proteins are known to interact with Lphns and Unc5s, mediating cell adhesion and repulsion respectively. Here the authors use crystallography, native mass spectrometry, molecular dynamics simulations and cell-based assays to show that these three proteins form large super-complexes with functions distinct from their smaller subcomplexes.

Summary: Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3′ end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain. : Timely and efficient transcription termination is essential for release of functional mRNAs as well as for Pol II recycling. Kecman et al. demonstrate that the conserved PP1 phosphatase Dis2 regulates transcription termination in fission yeast by mediating elongation to termination factor exchange and by dephosphorylating Pol II C-terminal domain. Keywords: RNA polymerase II, C-terminal domain, CTD, PP1 phosphatase, transcription termination, CTD phosphorylation, Spt5, CTD interacting domain, CID

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR–ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.