Tumor-acquired somatic mutation affects conformation to abolish ABCG2-mediated drug resistance.

ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears "locked" in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.

Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

Differential dynamics and direct interaction of bound ligands with lipids in multidrug transporter ABCG2.

ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). µs-scale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter.

Structures of the TMC-1 complex illuminate mechanosensory transduction.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Proton-driven alternating access in a spinster lipid transporter.

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.

Biodiversity and Spatial Distribution of Mosquitoes (Diptera: Culicidae) in Kurdistan Province, Western Iran.

Background: Mosquitoes (Diptera: Culicidae) have always been considered as the vector/s of viral and parasitic diseases. This study aimed to conduct a comprehensive survey on the species composition, spatial distribution, and biodiversity indices of mosquitoes in Kurdistan Province, western Iran. Methods: This study was carried out in 10 counties of Kurdistan Province. The immature stages of mosquitoes were collected monthly from June to September. ArcGIS software was used to spatial analysis and create maps. Alpha diversity indices were calculated using the related formula. Results: Totally, 5831 larvae belonging to the family Culicidae were collected. Twelve species were identified including: Anopheles claviger, An. maculipennis s.l, An. superpictus s.l, Culiseta. longiareolata, Cs. subochrea, Culex hortensis, Cx. mimeticus, Cx. perexiguus, Cx. pipiens, Cx. theileri, Cx. modestus and Cx. territans. Based on this analysis, the high-risk areas of the province are determined as Anopheles in the west, Culex in the north, and the Culiseta in the south of the province. Analyzing the Alpha biodiversity indices showed Baneh and Sarabad had the maximum and Bijar had the minimum mosquito biodiversity. Conclusion: The western counties of the province are regarded as the hotspots for anopheline mosquitos. Moreover, reporting of malaria cases in the past, bordering with Iraq and the high traffic of travelers have made these areas as potential foci for malaria transmission. So that, routine entomological inspections are proposed to detect any suspicious vector or case entrance.

Cation-π Interactions and their Functional Roles in Membrane Proteins.

Cation-π interactions arise as a result of strong attractive forces between positively charged entities and the π-electron cloud of aromatic groups. The physicochemical characteristics of cation-π interactions are particularly well-suited to the dual hydrophobic/hydrophilic environment of membrane proteins. As high-resolution structural data of membrane proteins bring molecular features into increasingly sharper view, cation-π interactions are gaining traction as essential contributors to membrane protein chemistry, function, and pharmacology. Here we review the physicochemical properties of cation-π interactions and present several prominent examples which demonstrate significant roles for this specialized biological chemistry.

Mechanical properties of ester- and ether-DPhPC bilayers: A molecular dynamics study.

In addition to its biological importance, DPhPC lipid bilayers are widely used in droplet bilayers, study of integral membrane proteins, drug delivery systems as well as patch-clamp electrophysiology of ion channels, yet their mechanical properties are not fully measured. Herein, we examined the effect of the ether linkage on the mechanical properties of ester- and ether-DPhPC lipid bilayers using all-atom molecular dynamics simulation. The values of area per lipid, thickness, intrinsic lateral pressure profile, order parameter, and elasticity moduli were estimated using various computational frameworks and were compared with available experimental values. Overall, a good agreement was observed between the two. The global properties of the two lipid bilayers are vastly different, with ether bilayer being stiffer, less ordered, and thicker than ester bilayer. Moreover, ether linkage decreased the area per lipid in the ether lipid bilayer. Our computational framework and output demonstrate how ether modification changes the mechano-chemical properties of DPhPC bilayers.

Comparison of the Effects of Iron Oxide, as a New Form of Iron Supplement, and Ferrous Sulfate on the Blood Levels of Iron and Total Iron-Binding Globulin in the Rabbit.

Iron oxide is an important biological agent that has a key role in medical processes; however, the mechanism whereby it provides iron for human and animal cells and its biological uses remains unclear. We aimed to evaluate the effects of oral iron oxide on serum iron status and compare the results with those of iron sulfate as a reference salt. Fifteen adult rabbits were divided into 3 groups of 5 each: control group, iron sulfate group, and iron oxide group. The groups received doses of 3.3, 10, and 33 mg/kg in 3 experiments. Venous blood samples were obtained just before the oral administration of iron sulfate and iron oxide (3.3 mg/kg). More blood samples were taken 3 times at the time points of 1, 6, and 12 hours after the administration of the solutions. Serum was separated for the measurement of iron (Fe) and total iron-binding globulin (TIBG) with routine methods. One week later, the same experiment was repeated with 10 mg/kg of iron sulfate and iron oxide; and 1 week later after the second experiment, again the same experiment was repeated with 33 mg/kg of iron sulfate and iron oxide. The results showed that 33 mg/kg of iron sulfate 1 hour after treatment caused a significant difference in the Fe and TIBG levels between all the groups (P=0.014 for Fe and P=0.027 for TIBG). Our data showed that the absorption of iron oxide was similar to that of ferrous sulfate and in high doses was as useful as iron supplement.

Simulation of blood oxygenation in capillary membrane oxygenators using modified sulfite solution.

Blood oxygenation is the main performance characteristic of capillary membrane oxygenators (CMOs). Handling of natural blood in in vitro investigations of CMOs is quite complex and time-consuming. Since the conventional blood analog fluids (e.g. water/glycerol) lack a substance with an affinity to capture oxygen comparable to hemoglobin's affinity, in this study a novel approach using modified sulfite solution is proposed to address this challenge. The solution comprises sodium sulfite as a component, simulating the role of hemoglobin in blood oxygenation. This approach is validated by OTR (oxygen transfer rate) measured using native porcine blood, in two types of commercially available CMOs. Consequently, the number of complicated natural blood investigations in the evolution procedure of newly developed oxygenators would considerably decrease. Moreover, the reassessing of failed devices, in clinics, would be performed more precisely using a modified sulfite solution than simple water/glycerol testing.