Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen Acinetobacter baumannii.

Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-EM reconstructions of Acinetobacter baumannii TolQ in apo and TolR- bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a trans-membrane funnel leading towards a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton non-permeable stance, characterized by the TolQ pentamer's flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers' interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope. Teaser: Apo TolQ and TolQ-TolR structures depict structural rearrangements required for cell envelope organization in bacterial cell division.

Genome-wide association study identifies novel loci and candidate genes for rust resistance in wheat (Triticum aestivum L.).

BACKGROUND: Wheat rusts are important biotic stresses, development of rust resistant cultivars through molecular approaches is both economical and sustainable. Extensive phenotyping of large mapping populations under diverse production conditions and high-density genotyping would be the ideal strategy to identify major genomic regions for rust resistance in wheat. The genome-wide association study (GWAS) population of 280 genotypes was genotyped using a 35 K Axiom single nucleotide polymorphism (SNP) array and phenotyped at eight, 10, and, 10 environments, respectively for stem/black rust (SR), stripe/yellow rust (YR), and leaf/brown rust (LR). RESULTS: Forty-one Bonferroni corrected marker-trait associations (MTAs) were identified, including 17 for SR and 24 for YR. Ten stable MTAs and their best combinations were also identified. For YR, AX-94990952 on 1A + AX-95203560 on 4A + AX-94723806 on 3D + AX-95172478 on 1A showed the best combination with an average co-efficient of infection (ACI) score of 1.36. Similarly, for SR, AX-94883961 on 7B + AX-94843704 on 1B and AX-94883961 on 7B + AX-94580041 on 3D + AX-94843704 on 1B showed the best combination with an ACI score of around 9.0. The genotype PBW827 have the best MTA combinations for both YR and SR resistance. In silico study identifies key prospective candidate genes that are located within MTA regions. Further, the expression analysis revealed that 18 transcripts were upregulated to the tune of more than 1.5 folds including 19.36 folds (TraesCS3D02G519600) and 7.23 folds (TraesCS2D02G038900) under stress conditions compared to the control conditions. Furthermore, highly expressed genes in silico under stress conditions were analyzed to find out the potential links to the rust phenotype, and all four genes were found to be associated with the rust phenotype. CONCLUSION: The identified novel MTAs, particularly stable and highly expressed MTAs are valuable for further validation and subsequent application in wheat rust resistance breeding. The genotypes with favorable MTA combinations can be used as prospective donors to develop elite cultivars with YR and SR resistance.

In-vivo anti-diabetic and anti-hyperlipidemic effects of natural metabolites from resin of Commiphora mukul and their in-silico to in-vitro target fishing.

Diabetes mellitus is a rapidly spreading global metabolic disorder that has serious social, health, and economic consequences. Herein, we have evaluated in vivo antidiabetic and antihyperlipidemic effects of myrrhanone-B and myrrhanol-B (isolated from Commiphora mukul Hook). We observed that treatment with myrrhanone-B and myrrhanol-B at a dose of 5 and 10 mg/kg body weight for 21 days significantly improved body weight loss, water consumption, and the concentration of blood glucose level (BGL) in alloxan (120 mg/kg) induced diabetic mice, which indicates that the compounds possess strong anti-diabetic activities. In the biochemical analysis, these compounds improved an abnormal level of total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein cholesterol (LDL-C) to a normal level and increased the high-density lipoprotein cholesterol level (HDLC). Later, drug target of compounds was predicted through in-silico docking which shows that these compounds nicely fit in the active site of α-glucosidase enzyme and mediates excellent interactions with the catalytic residues, Asp214 and Asp349. The in-silico results were confirmed by in-vitro testing of myrrhanone-B and myrrhanol-B against α-glucosidase where both the compounds exhibited excellent inhibitory potency with IC50 values of 19.50 ± 0.71, and 16.11 ± 0.69 µM, respectively. Furthermore, mechanistic study was conducted to observe their binding mechanism, which reflect that myrrhanol-B has mixed type of inhibition (ki = 12.33 ± 0.030 µM), while myrrhanone-B demonstrates competitive type of inhibition (ki =14.53 ± 0.040 µM).

Virulence and genetic analysis of Puccinia graminis tritici in the Indian sub-continent from 2016 to 2022 and evaluation of wheat varieties for stem rust resistance.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), has re-emerged as one of the major concerns for global wheat production since the evolution of Ug99 and other virulent pathotypes of Pgt from East Africa, Europe, Central Asia, and other regions. Host resistance is the most effective, economic, and eco-friendly approach for managing stem rust. Understanding the virulence nature, genetic diversity, origin, distribution, and evolutionary pattern of Pgt pathotypes over time and space is a prerequisite for effectively managing newly emerging Pgt isolates through host resistance. In the present study, we monitored the occurrence of stem rust of wheat in India and neighboring countries from 2016 to 2022, collected 620 single-pustule isolates of Pgt from six states of India and Nepal, analyzed them on Indian stem rust differentials, and determined their virulence phenotypes and molecular genotypes. The Ug99 type of pathotypes did not occur in India. Pathotypes 11 and 40A were most predominant during these years. Virulence phenotyping of these isolates identified 14 Pgt pathotypes, which were genotyped using 37 Puccinia spp.-specific polymorphic microsatellites, followed by additional phylogenetic analyses using DARwin. These analyses identified three major molecular groups, demonstrating fewer lineages, clonality, and long-distance migration of Pgt isolates in India. Fourteen of the 40 recently released Indian wheat varieties exhibited complete resistance to all 23 Pgt pathotypes at the seedling stage. Twelve Sr genes were postulated in 39 varieties based on their seedling response to Pgt pathotypes. The values of slow rusting parameters i.e. coefficient of infection, area under disease progress curve, and infection rates, assessed at adult plant stage at five geographically different locations during two crop seasons, indicated the slow rusting behavior of several varieties. Six Sr genes (Sr2, Sr57, Sr58, Sr24, Sr31, and Sr38) were identified in 24 wheat varieties using molecular markers closely linked to these genes. These findings will guide future breeding programs toward more effective management of wheat stem rust.

Harnessing High Yield Potential in Wheat (Triticum aestivum L.) under Climate Change Scenario.

Wheat is a major staple food crop for food security in India and South Asia. The current rate (0.8-1.2%) of genetic gain in wheat is significantly shorter than the 2.4% needed to meet future demand. The changing climate and increased yield loss due to factors such as terminal heat stress necessitate the need for climate-resilient practices to sustain wheat production. At ICAR-Indian Institute of Wheat and Barley Research in Karnal, Haryana, India, a new High Yield Potential Trial (HYPT) was conceptualized and subsequently conducted at six locations in the highly productive North Western Plain Zone (NWPZ). An attempt was made to harness higher wheat yields through the best pipeline genotypes suitable for early sowing and modified agronomic practices to explore the feasibility of a new approach that is profitable to farmers. The modified agronomic practices included like early sowing, application of 150% recommended dose of fertilizers, and two sprays of growth regulators (Chlormaquate chloride and Tebuconazole) to prevent lodging. The mean yield in the HYPT was 19.4% superior compared to the best trials conducted during the normal sowing time. A highly positive and significant correlation of grain yield with grain filling duration (0.51), biomass (0.73), harvest index (0.75), normalized difference vegetation Index (0.27), chlorophyll content index (0.32), and 1000-grain weight (0.62) was observed. An increased return of USD 201.95/ha was realized in the HYPT when compared to normal sowing conditions. This study proves that new integrated practices have the potential to provide the best profitable yields in wheat in the context of climate change.

Genome-Wide Association Study for Grain Protein, Thousand Kernel Weight, and Normalized Difference Vegetation Index in Bread Wheat (Triticum aestivum L.).

Genomic regions governing grain protein content (GPC), 1000 kernel weight (TKW), and normalized difference vegetation index (NDVI) were studied in a set of 280 bread wheat genotypes. The genome-wide association (GWAS) panel was genotyped using a 35K Axiom array and phenotyped in three environments. A total of 26 marker-trait associations (MTAs) were detected on 18 chromosomes covering the A, B, and D subgenomes of bread wheat. The GPC showed the maximum MTAs (16), followed by NDVI (6), and TKW (4). A maximum of 10 MTAs was located on the B subgenome, whereas, 8 MTAs each were mapped on the A and D subgenomes. In silico analysis suggest that the SNPs were located on important putative candidate genes such as NAC domain superfamily, zinc finger RING-H2-type, aspartic peptidase domain, folylpolyglutamate synthase, serine/threonine-protein kinase LRK10, pentatricopeptide repeat, protein kinase-like domain superfamily, cytochrome P450, and expansin. These candidate genes were found to have different roles including regulation of stress tolerance, nutrient remobilization, protein accumulation, nitrogen utilization, photosynthesis, grain filling, mitochondrial function, and kernel development. The effects of newly identified MTAs will be validated in different genetic backgrounds for further utilization in marker-aided breeding.

Expression of CD22 in Triple-Negative Breast Cancer: A Novel Prognostic Biomarker and Potential Target for CAR Therapy.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Due to the lack of expression of well-known molecular targets [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)], there is a need for more alternative treatment approaches in TNBC. Chimeric antigen receptor (CAR)-T cell-based immunotherapy treatment is one of the latest treatment technologies with outstanding therapeutic advances in the past decade, especially in the treatment of hematologic malignancies, but the therapeutic effects of CAR-T cells against solid tumors have not yet shown significant clinical benefits. Identification of highly specific CAR-T targets in solid tumors is also crucial for its successful treatment. CD22 is reported to be a multifunctional receptor that is mainly expressed on the surface of mature B-cells (lymphocytes) and is also highly expressed in most B-cell malignancies. This study aimed to investigate the expression of CD22 in TNBC. Bioinformatic analysis was performed to evaluate the expression of CD22 in breast carcinoma and normal tissues. RNA-seq data of normal and breast carcinoma patients were downloaded from The Cancer Genome Atlas (TCGA), and differential gene expression was performed using R language. Additionally, online bioinformatics web tools (GEPIA and TNM plot) were used to evaluate the expression of CD22 in breast carcinoma and normal tissues. Western blot (WB) analysis and immunofluorescence (IF) were performed to characterize the expression of CD22 in TNBC cell lines. Immunohistochemical (IHC) staining was performed on tumor specimens from 97 TNBC patients for CD22 expression. Moreover, statistical analysis was performed to analyze the association of clinical pathological parameters with CD22 expression. Correlation analysis between overall survival data of TNBC patients and CD22 expression was also performed. Differential gene expression analysis of TCGA data revealed that CD22 is among the upregulated differentially expressed genes (DEGs) with high expression in breast cancer, as compared to normal breast tissues. WB and IF analysis revealed high expression of CD22 in TNBC cell lines. IHC results also showed that approximately 62.89% (61/97) of TNBC specimens were stained positive for CD22. Cell membrane expression of CD22 was evident in 23.71% (23/97) of TNBC specimens, and 39.18% (38/97) of TNBC specimens showed cytoplasmic/membrane expression, while 37.11% (36/97) specimens were negative for CD22. Furthermore, significant associations were found between the size of tumors in TNBC patients and CD22 expression, which unveils its potential as a prognostic biomarker. No significant correlation was found between the overall survival of TNBC patients and CD22 expression. In conclusion, we demonstrated for the first time that CD22 is highly expressed in TNBC. Based on our findings, we anticipated that CD22 could be used as a prognostic biomarker in TNBC, and it might be a potential CAR-T target in TNBC for whom few therapeutic options exist. However, more large-scale studies and clinical trials will ensure its potential usefulness as a CAR-T target in TNBC.

CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Genome-wide association mapping for component traits of drought and heat tolerance in wheat.

Identification of marker trait association is a prerequisite for marker-assisted breeding. To find markers linked with traits under heat and drought stress in bread wheat (Triticum aestivum L.), we performed a genome-wide association study (GWAS). GWAS mapping panel used in this study consists of advanced breeding lines from the IARI stress breeding programme produced by pairwise and complex crosses. Phenotyping was done at multi locations namely New Delhi, Karnal, Indore, Jharkhand and Pune with augmented-RCBD design under different moisture and heat stress regimes, namely timely sown irrigated (IR), timely sown restricted irrigated (RI) and late sown (LS) conditions. Yield and its component traits, viz., Days to Heading (DH), Days to Maturity (DM), Normalized Difference Vegetation Index (NDVI), Chlorophyll Content (SPAD), Canopy temperature (CT), Plant Height (PH), Thousand grain weight (TGW), Grain weight per spike (GWPS), Plot Yield (PLTY) and Biomass (BMS) were phenotyped. Analysis of variance and descriptive statistics revealed significant differences among the studied traits. Genotyping was done using the 35k SNP Wheat Breeder's Genotyping Array. Population structure and diversity analysis using filtered 10,546 markers revealed two subpopulations with sufficient diversity. A large whole genome LD block size of 7.15 MB was obtained at half LD decay value. Genome-wide association search identified 57 unique markers associated with various traits across the locations. Twenty-three markers were identified to be stable, among them nine pleiotropic markers were also identified. In silico search of the identified markers against the IWGSC ref genome revealed the presence of a majority of the SNPs at or near the gene coding region. These SNPs can be used for marker-assisted transfer of genes/QTLs after validation to develop climate-resilient cultivars.

Genome-wide association study for grain yield and component traits in bread wheat (Triticum aestivum L.).

Genomic regions governing days to heading (DH), grain filling duration (GFD), grain number per spike (GNPS), grain weight per spike (GWPS), plant height (PH), and grain yield (GY) were investigated in a set of 280 diverse bread wheat genotypes. The genome-wide association studies (GWAS) panel was genotyped using a 35K Axiom Array and phenotyped in five environments. The GWAS analysis showed a total of 27 Bonferroni-corrected marker-trait associations (MTAs) on 15 chromosomes representing all three wheat subgenomes. The GFD showed the highest MTAs (8), followed by GWPS (7), GY (4), GNPS (3), PH (3), and DH (2). Furthermore, 20 MTAs were identified with more than 10% phenotypic variation. A total of five stable MTAs (AX-95024590, AX-94425015, AX-95210025 AX-94539354, and AX-94978133) were identified in more than one environment and associated with the expression of DH, GFD, GNPS, and GY. Similarly, two novel pleiotropic genomic regions with associated MTAs i.e. AX-94978133 (4D) and AX-94539354 (6A) harboring co-localized QTLs governing two or more traits were also identified. In silico analysis revealed that the SNPs were located on important putative candidate genes such as F-box-like domain superfamily, Lateral organ boundaries, LOB, Thioredoxin-like superfamily Glutathione S-transferase, RNA-binding domain superfamily, UDP-glycosyltransferase family, Serine/threonine-protein kinase, Expansin, Patatin, Exocyst complex component Exo70, DUF1618 domain, Protein kinase domain involved in the regulation of grain size, grain number, growth and development, grain filling duration, and abiotic stress tolerance. The identified novel MTAs will be validated to estimate their effects in different genetic backgrounds for subsequent use in marker-assisted selection (MAS).

Genetic dissection of grain iron and zinc, and thousand kernel weight in wheat (Triticum aestivum L.) using genome-wide association study.

Genetic biofortification is recognized as a cost-effective and sustainable strategy to reduce micronutrient malnutrition. Genomic regions governing grain iron concentration (GFeC), grain zinc concentration (GZnC), and thousand kernel weight (TKW) were investigated in a set of 280 diverse bread wheat genotypes. The genome-wide association (GWAS) panel was genotyped using 35 K Axiom Array and phenotyped in five environments. The GWAS analysis showed a total of 17 Bonferroni-corrected marker-trait associations (MTAs) in nine chromosomes representing all the three wheat subgenomes. The TKW showed the highest MTAs (7), followed by GZnC (5) and GFeC (5). Furthermore, 14 MTAs were identified with more than 10% phenotypic variation. One stable MTA i.e. AX-95025823 was identified for TKW in both E4 and E5 environments along with pooled data, which is located at 68.9 Mb on 6A chromosome. In silico analysis revealed that the SNPs were located on important putative candidate genes such as Multi antimicrobial extrusion protein, F-box domain, Late embryogenesis abundant protein, LEA-18, Leucine-rich repeat domain superfamily, and C3H4 type zinc finger protein, involved in iron translocation, iron and zinc homeostasis, and grain size modifications. The identified novel MTAs will be validated to estimate their effects in different genetic backgrounds for subsequent use in marker-assisted selection. The identified SNPs will be valuable in the rapid development of biofortified wheat varieties to ameliorate the malnutrition problems.

Recent Advances in Syndactyly: Basis, Current Status and Future Perspectives.

A comprehensive summary of recent knowledge in syndactyly (SD) is important for understanding the genetic etiology of SD and disease management. Thus, this review article provides background information on SD, as well as insights into phenotypic and genetic heterogeneity, newly identified gene mutations in various SD types, the role of HOXD13 in limb deformities, and recently introduced modern surgical techniques for SD. This article also proposes a procedure for genetic analysis to obtain a clearer genotype-phenotype correlation for SD in the future. We briefly describe the classification of non-syndromic SD based on variable phenotypes to explain different phenotypic features and mutations in the various genes responsible for the pathogenesis of different types of SD. We describe how different types of mutation in HOXD13 cause various types of SD, and how a mutation in HOXD13 could affect its interaction with other genes, which may be one of the reasons behind the differential phenotypes and incomplete penetrance. Furthermore, we also discuss some recently introduced modern surgical techniques, such as free skin grafting, improved flap techniques, and dermal fat grafting in combination with the Z-method incision, which have been successfully practiced clinically with no post-operative complications.

Wheat spike blast: genetic interventions for effective management.

The fundamental concepts of the genetics, race classification and epidemiology of the Wheat spike blast causing fungus Magnaporthe oryzae pathotype Triticum (MoT) are still evolving despite of its discovery in 1985 in Brazil for the first time. The fungus seems to defy the research progress that is being made globally by continuously evolving into pathotypes which have already overcome the much celebrated 2NS resistance in wheat lines as well as few of the initially effective fungicides. The compartmentalized i.e. two speed genome of the MoT, conferring the fungus an evolutionary advantage, has emerged as a challenge for the wheat spike blast researchers complicating its already difficult management. The airborne fungus with a range of alternative hosts is finding new geographical niches situated on different continents and is a matter of great apprehension among the nations whose food security is primarily dependent on wheat. The wheat blast outbreak in Bangladesh during 2016 was attributed to an isolate from Latin America escaping through a seed import consignment while the latest Zambian outbreak is still to be studied in detail regarding its origin and entry. The challenges in dealing wheat spike blast are not only on the level of genetics and epidemiology alone but also on the levels of policy making regarding international seed movement and research collaborations. The present review deals with these issues mainly concerning the effective management and controlling the international spread of this deadly disease of wheat, with a particular reference to India. We describe the origin, taxonomy, epidemiology and symptomology of MoT and briefly highlight its impact and management practices from different countries. We also discuss the advances in genomics and genome editing technologies that can be used to develop elite wheat genotypes resistant against different stains of wheat spike blast.

Pollen viability as a potential trait for screening heat-tolerant wheat (Triticum aestivum L.).

High temperature during reproductive stage of winter crops causes sterility of pollen grains and reduced yield. It is essential to find the genotypes with higher pollen viability, as it is most sensitive to temperature extremes. A field study was conducted with wheat (Triticum aestivum L.) genotypes to understand the effect of high temperature on pollen viability and grain yield for 2years under timely (TS) and late sown (LS) conditions. A strong correlation was observed between higher pollen viability and higher grain yield under heat stress condition. Genotypes like K7903, HD2932, WH730 and RAJ3765 showed higher pollen viability, whereas DBW17, HUW468, RAJ4014 and UP2425 had lower pollen viability under LS condition. Further, the quantification of antioxidant enzymes activity mainly, Super oxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) and Glutathione peroxidase (GPX) has showed significant variation among study genotypes. Thus, the identified high pollen viability genotypes can serve as a potential source for trait based breeding under heat stress in wheat. The present study is a first of its kind to assess more number of wheat genotypes for pollen viability and antioxidants activity under field condition. It also confirms that pollen viability can be used as a potential trait to screen genotypes for heat stress tolerance in wheat.

Comparative genome sequence and phylogenetic analysis of chloroplast for evolutionary relationship among Pinus species.

Genus Pinus is a widely dispersed genus of conifer plants in the Northern Hemisphere. However, the inadequate accessibility of genomic knowledge limits our understanding of molecular phylogeny and evolution of Pinus species. In this study, the evolutionary features of complete plastid genome and the phylogeny of the Pinus genus were studied. A total of thirteen divergent hotspot regions (trnk-UUU, matK, trnQ-UUG, atpF, atpH, rpoC1, rpoC2, rpoB, ycf2, ycf1, trnD-GUC, trnY-GUA, and trnH-GUG) were identified that would be utilized as possible genetic markers for determination of phylogeny and population genetics analysis of Pinus species. Furthermore, seven genes (petD, psaI, psaM, matK, rps18, ycf1, and ycf2) with positive selection site in Pinus species were identified. Based on the whole genome this phylogenetic study showed that twenty-four Pinus species form a significant genealogical clade. Divergence time showed that the Pinus species originated about 100 million years ago (MYA) (95% HPD, 101.76.35-109.79 MYA), in lateral stages of Cretaceous. Moreover, two of the subgenera are consequently originated in 85.05 MYA (95% HPD, 81.04-88.02 MYA). This study provides a phylogenetic relationship and a chronological framework for the future study of the molecular evolution of the Pinus species.

Standard Binding Free Energy and Membrane Desorption Mechanism for a Phospholipase C.

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signaling, lipid metabolism, and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in an aqueous solution. At the theoretical level, calculation of the standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion, and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2 ± 1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6 ± 0.2 kcal/mol). In light of the 2.3-µs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage.

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the ß clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in ß clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_ßIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the ß clade, St_ßIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_ßIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and ß clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.