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Nucleic acids from bacteria or viruses induce potent immune responses in infected cells 1 – 4 . The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses 5 , 6 . Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor 7 , 8 . It catalyses the synthesis of cyclic GMP-AMP (cGAMP) 9 – 12 , which stimulates the induction of type I interferons through the STING–TBK1–IRF-3 signalling axis 13 – 15 . STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase 8 , 16 . The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK1 8 , 17 – 20 . Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons 21 . However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNβ after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders. A molecular model of STING-mediated signalling is proposed, as structural analysis identifies a crucial motif for the binding of TBK1 to STING, and a separate model involved in IRF-3 binding.

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H + /Fe 2+ antiporter so that transport of each Fe 2+ is coupled to transport of two H + in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H + and Fe 2+ . These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases. Ferroportin is an iron exporter essential for releasing cellular iron into circulation and is inhibited by a peptide hormone, hepcidin. Here authors present cryo-EM structures of the ferroportin from the primate Philippine tarsier (TsFpn) with and without hepcidin and show that TsFpn is an electroneutral H +  /Fe 2+ antiporter.

The diverse lipid environment of the biological membrane can modulate the structure and function of membrane proteins. However, little is known about the role that lipids play in modulating protein–protein interactions. Here we employed native mass spectrometry (MS) to determine how individual lipid-binding events to the ammonia channel (AmtB) modulate its interaction with the regulatory protein, GlnK. The thermodynamic signature of AmtB–GlnK in the absence of lipids indicates conformational dynamics. A small number of lipids bound to AmtB is sufficient to modulate the interaction with GlnK, and lipids with different headgroups display a range of allosteric modulation. We also find that lipid chain length and stereochemistry can affect the degree of allosteric modulation, indicating an unforeseen selectivity of membrane proteins toward the chemistry of lipid tails. These results demonstrate that individual lipid-binding events can allosterically modulate the interactions of integral membrane and soluble proteins. Native mass spectrometry (MS) is a technique that preserves non-covalent interactions in the mass spectrometer. Here the authors use native MS to study integral membrane proteins, and find that lipids with different headgroups and tails can allosterically modulate protein-protein interactions in different fashions.

G-protein-gated inward rectifying potassium channels (GIRKs) require G βγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by G βγ and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to G βγ -binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions. G-protein-gated inward rectifying potassium channels (GIRKs) require G βγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Here authors use native ion mobility mass spectrometry to monitor small molecule binding events to GIRK2 and shed light on the selectivity of GIRK2 towards PIPs.

Mass spectrometry (MS) of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes, where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here we describe a protocol for MS of membrane protein complexes. The protocol begins with the preparation of the membrane protein complex, enabling not only the direct assessment of stoichiometry, delipidation and quality of the target complex but also the evaluation of the purification strategy. A detailed list of compatible nonionic detergents is included, along with a protocol for screening detergents to find an optimal one for MS, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a quadrupole time-of-flight (Q-TOF) mass spectrometer after the introduction of complexes from gold-coated nanoflow capillaries.

Pendrin (SLC26A4) is an anion exchanger that mediates bicarbonate (HCO 3 − ) exchange for chloride (Cl − ) and is crucial for maintaining pH and salt homeostasis in the kidney, lung, and cochlea. Pendrin also exports iodide (I − ) in the thyroid gland. Pendrin mutations in humans lead to Pendred syndrome, causing hearing loss and goiter. Inhibition of pendrin is a validated approach for attenuating airway hyperresponsiveness in asthma and for treating hypertension. However, the mechanism of anion exchange and its inhibition by drugs remains poorly understood. We applied cryo-electron microscopy to determine structures of pendrin from Sus scrofa in the presence of either Cl − , I − , HCO 3 − or in the apo-state. The structures reveal two anion-binding sites in each protomer, and functional analyses show both sites are involved in anion exchange. The structures also show interactions between the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) and transmembrane domains, and mutational studies suggest a regulatory role. We also determine the structure of pendrin in a complex with niflumic acid (NFA), which uncovers a mechanism of inhibition by competing with anion binding and impeding the structural changes necessary for anion exchange. These results reveal directions for understanding the mechanisms of anion selectivity and exchange and their regulations by the STAS domain. This work also establishes a foundation for analyzing the pathophysiology of mutations associated with Pendred syndrome. Here the authors report structures of pendrin, an anion exchanger, in complex with its substrate Cl − , I − , or HCO 3 − , which reveal two anion binding sites in each protomer. The authors also identify binding sites of a pendrin inhibitor, niflumic acid.

Most proteins assemble into multisubunit complexes 1 . The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work 2 . In many complexes, however, multimerization does not enable any known function 3 . An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function 3 – 7 . Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous. Accumulation of hydrophobic residues at the interface between monomers may favour the maintenance of multimeric protein states during evolution, even if multimerization confers no functional advantage.

Diacylglycerol O -acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans 1 . DGAT1 belongs to the membrane-bound O -acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins 2 , 3 . How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity. The structure of human diacylglycerol O -acyltransferase 1, a membrane protein that synthesizes triacylglycerides, is solved with cryo-electron microscopy, providing insight into its function and mechanism of enzymatic activity.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D- manno -oct-2-ulosonic acid (Kdo) 2 -lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5’-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as –100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

TRAAK is an ion channel from the two-pore domain potassium (K 2P ) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K 2P 4.1 toward lipids remains poorly understood. Here we show the two isoforms of K 2P 4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN 1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K 2P 4.1. Liposome potassium flux assays with K 2P 4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K 2P 4.1. Native ion mobility mass spectrometry reveals two isoforms of the two-pore domain K + channel K2P4.1 have distinct binding preferences for lipids and show a relationship between the strength of individual lipid binding events and channel activity.