Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

Antimicrobial Activity of Snake β-Defensins and Derived Peptides

β-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several β-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from β-defensins, were designed applying the bioisosterism strategy. The linear β-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7–14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake β-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

GM-CSF Nitration Is a New Driver of Myeloid Suppressor Cell Activity in Tumors

Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer.

The tertiary structure of the human Xkr8–Basigin complex that scrambles phospholipids at plasma membranes

Xkr8–Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8–Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8–Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.

Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation–π interaction

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

Agaricales Mushroom Lignin Peroxidase: From Structure–Function to Degradative Capabilities

Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.

TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells

TMEM120A, a member of the Transmembrane protein 120 (TMEM120) family, has pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells, and have solved cryo-EM structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing or metabolism.

Structures of ABCB4 provide insight into phosphatidylcholine translocation

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.