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The last decade has witnessed the rise of an extremely threatening healthcare-associated multidrug-resistant non-albicans Candida (NAC) species, Candida auris. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the major facilitator superfamily (MFS) of efflux pumps, drug/H + antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against most of the tested substrates, the ortholog when overexpressed in a minimal background Saccharomyces cerevisiae strain, AD1-8u - , showed significant enhancement in the minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms. KEY POINTS: • Fourteen putative DHA1 transporters are encoded in the Candida auris genome. • Deletion of the CauMDR1 gene does not lead to major changes in drug resistance. • CauMdr1 recognizes and effluxes numerous xenobiotics, including prominent azoles., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

V H H, i.e., VH domains of camelid single-chain antibodies, are very promising therapeutic agents due to their significant physicochemical advantages compared to classical mammalian antibodies. The number of experimentally solved V H H structures has significantly improved recently, which is of great help, because it offers the ability to directly work on 3D structures to humanise or improve them. Unfortunately, most V H Hs do not have 3D structures. Thus, it is essential to find alternative ways to get structural information. The methods of structure prediction from the primary amino acid sequence appear essential to bypass this limitation. This review presents the most extensive overview of structure prediction methods applied for the 3D modelling of a given V H H sequence (a total of 21). Besides the historical overview, it aims at showing how model software programs have been shaping the structural predictions of V H Hs. A brief explanation of each methodology is supplied, and pertinent examples of their usage are provided. Finally, we present a structure prediction case study of a recently solved V H H structure. According to some recent studies and the present analysis, AlphaFold 2 and NanoNet appear to be the best tools to predict a structural model of V H H from its sequence.

Antifungal resistance mediated by overexpression of ABC transporters is one of the primary roadblocks to effective therapy against Candida infections. Thus, identification and characterization of the ABC transporter repertoire in Candida species are of high relevance. The method described in the chapter is based on our previously developed bioinformatic pipeline for identification of ABC proteins in Candida species. The methodology essentially involves the utilization of a hidden Markov model (HMM) profile of the nucleotide-binding domain (NBD) of ABC proteins to mine these proteins from the proteome of Candida species. Further, a widely used tool to predict membrane protein topology is exploited to identify the true transporter candidates out of the ABC proteins. Even though the chapter specifically focuses on a method to identify ABC transporters in Candida auris , the same can also be applied to any other Candida species., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In view of the multiple clinical and physiological implications of ABC transporter proteins, there is a considerable interest among researchers to characterize them functionally. However, such characterizations are based on the premise that ABC proteins are accurately identified in the proteome of an organism, and their topology is correctly predicted. With this objective, we have developed ABC-finder, i.e., a Docker-based package for the identification of ABC proteins in all organisms, and visualization of the topology of ABC proteins using a web browser. ABC-finder is built and deployed in a Linux container, making it scalable for many concurrent users on our servers and enabling users to download and run it locally. Overall, ABC-finder is a convenient, portable, and platform-independent tool for the identification and topology prediction of ABC proteins. ABC-finder is accessible at http://abc-finder.osdd.jnu.ac.in., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Schizosaccharomyces pombe RNA polymerase II comprises twelve different subunits. Its Rpb9 subunit comprises 113 amino acids, and is the only non-essential subunit of S. pombe RNA polymerase II. However, its functions have not been studied in S. pombe. The results presented in this study demonstrate that Rpb9 is involved in regulating growth under optimum and certain stress conditions in S. pombe. To further address the role (s) of various domains of this subunit in regulating these phenotypes, deletion mutant analysis was done. We observed that the region spanning 1-74 amino acids, encompassing the amino-terminal zinc finger domain and the linker region of Rpb9 was able to rescue the phenotypes associated with rpb9 + deletion. We also demonstrate that the functions of this subunit are only partially conserved among yeast and humans. Our computational biology approaches provide a structural basis for the differential role of various Rpb9 domains in S. pombe. Furthermore, using these tools we show that there has been a co-evolution of the interaction residues between the Rpb9 subunit and the two largest subunits of RNA polymerase II, allowing for a more stringent organism-specific packing. Taken together, our results have provided functional and structural insights into the Rpb9 subunit of S. pombe., (Copyright © 2021 Elsevier B.V. All rights reserved.)

COVID-19, caused by Severe Acute Respiratory Syndrome Corona Virus 2, is declared a Global Pandemic by WHO in early 2020. In the present situation, though more than 180 vaccine candidates with some already approved for emergency use, are currently in development against SARS-CoV-2, their safety and efficacy data is still in a very preliminary stage to recognize them as a new treatment, which demands an utmost emergency for the development of an alternative anti-COVID-19 drug sine qua non for a COVID-19 free world. Since RNA-dependent RNA polymerase (RdRp) is an essential protein involved in replicating the virus, it can be held as a potential drug target. We were keen to explore the plant-based product against RdRp and analyze its inhibitory potential to treat COVID-19. A unique collection of 248 plant compounds were selected based on their antiviral activity published in previous literature and were subjected to molecular docking analysis against the catalytic sub-unit of RdRp. The docking study was followed by a pharmacokinetics analysis and molecular dynamics simulation study of the selected best-docked compounds. Tellimagrandin I, SaikosaponinB2, Hesperidin and (-)-Epigallocatechin Gallate were the most prominent ones that showed strong binding affinity toward RdRp. All the compounds mentioned showed satisfactory pharmacokinetics properties and remained stabilized at their respective binding sites during the Molecular dynamics simulation. Additionally, we calculated the free-binding energy/the binding properties of RdRp-ligand complexes with the connection of MM/GBSA. Interestingly, we observe that SaikosaponinB2 gives the best binding affinity (∆G binding = -42.43 kcal/mol) in the MM/GBSA assay. Whereas, least activity is observed for Hesperidin (∆G binding = -22.72 kcal/mol). Overall our study unveiled the feasibility of the SaikosaponinB2 to serve as potential molecules for developing an effective therapy against COVID-19 by inhibiting one of its most crucial replication proteins, RdRp., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saha, Nandi, Vishwakarma, Prakash and Kumar.)

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys - mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR3 1-145 from multidrug resistance protein into SNAP-23 Cys - mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys - and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys - -MDR3 1-145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris , a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris . Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR_02725 orthologous to CDR1 , a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris . Homologs of other ABC transporter genes, such as CDR4 , CDR6 , and SNQ2 , also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris .

ABC transporter proteins are involved in active transport, both in prokaryotes and eukaryotes. Sequence analysis of nucleotide binding domains (NBDs) of ABC proteins from all taxa revealed a well-conserved new motif having the signature: xT/ShxE/DNhxF, located between Q-loop and ABC signature sequence. A recent structure of an ABC transporter, ABCG5/G8 highlighted the motif as an essential structural determinant of inter-domain crosstalk and termed it as E-helix. We carried out an extensive computational analysis to unravel important structural entities alongside E-helix which plausibly play role in the interlocking mechanism of NBD with TMD. We identified E-helix to be a central structural moiety which interacts with three helices and an intracellular loop that leads to the transmembrane domain. Considering its wide occurrence, we examined the importance of this motif in one representative multidrug ABC transporter of Candida albicans, Cdr1p. The motif residues were replaced by alanines both individually as well as in combinations. The GFP-tagged versions of mutant proteins were overexpressed in Saccharomyces cerevisiae. Overall, our mutational data suggested that this motif plays a role in the maintenance of proper structural fold and/or inter-domain contacts in Cdr1p. We, thus, unveil an essential structural motif in ABC superfamily transporters., (Copyright © 2019. Published by Elsevier B.V.)

ABC transporters are membrane-bound pumps composed of two major domains: the transmembrane domain(s) (TMDs) and the nucleotide-binding domain(s) (NBDs). Sequence analyses of the NBDs of key ABC exporters revealed a residue position within the H-loop to be differentially conserved in the ABCG family, wherein there lies glutamine instead of positively charged arginine/lysine as in non-ABCG members. Consequently, contrasting NBD sequences of fungal Pleiotropic Drug Resistance transporters (PDR/ABCG) with that of Cholesterol/Phospholipid and Retinal (CPR/ABCA) Flippase family revealed a high Cumulative Relative Entropy (CRE) score of this residue position implying its family-specific functional significance. Further, substitution of the glutamine by arginine in both the NBDs of a representative PDR/ABCG member, (Candida drug resistance 1 protein) Cdr1p led to selective susceptibility of the Saccharomyces cerevisiae strains overexpressing the corresponding mutant proteins (Q362R and Q1060R) towards antifungal substrates without any impact on the ATPase activity. Consistent with the findings from previous studies on H-loop motif of fungal PDR transporters, the current report points towards a role of the glutamine residue within both canonical and divergent H-loop of Cdr1p in conferring substrate selection in a precisely identical manner., (Copyright © 2019 Elsevier Inc. All rights reserved.)

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Major facilitator superfamily is one of the largest superfamily of secondary transporters present across the kingdom of life. Considering the physiological and clinical importance of MFS proteins, we attempted to explore the phylogenetic and structural aspects of the superfamily. To achieve the objectives, we performed global sequence-based analyses of MFS proteins encompassing multiple taxa. Notably, phylogenetic analysis of MFS proteins resulted in the clustering of MFS proteins based on their function, rather than lineage of the respective organisms. Additionally, we employed information theoretic measures, Relative entropy (RE) and Cumulative relative entropy (CRE) to decipher fold-specific and function-specific residues, respectively, in the MFS proteins. The residues with high RE score when mapped on to the 3D-structure of MFS transporter LacY, were found to be distributed throughout the tertiary structure of the protein. On the other hand, CRE calculation was employed to contrast two subfamilies Drug H + antiporter 1 and 3 (DHA1 and DHA3). The particular analysis unveiled certain differentially conserved residues in DHA1 as compared to DHA3 highlighting family-specific importance of them. Remarkably, a number of high scoring CRE residues have already established functional roles, for instance, the arginine residue present in TMH4. Altogether, the current study apart from providing an insight into the functional clustering of MFS proteins also identifies residues with established or plausible roles in the transport mechanism. Thus, the study lays a platform for future structure-function studies of these proteins., Competing Interests: Compliance with ethical standardsThe authors would like to declare that no conflicts of interest exist.

ATP-binding cassette (ABC) is one of the two major superfamilies of transporters present across the evolutionary scale. ABC superfamily members came to prominence due to their ability to extrude broad spectrum of substrates and to confer multi drug resistance (MDR). Overexpression of some ABC transporters in clinical isolates of Candida species was attributed to the development of MDR phenotypes. Among Candida species, Candida glabrata is an emerging drug resistant species in human fungal infections. A comprehensive analysis of such proteins in C. glabrata is required to untangle their role not only in MDR but also in other biological processes. Bioinformatic analysis of proteins encoded by genome of human pathogenic yeast C. glabrata identified 25 putative ABC protein coding genes. On the basis of phylogenetic analysis, domain organization and nomenclature adopted by the Human Genome Organization (HUGO) scheme, these proteins were categorized into six subfamilies such as Pleiotropic Drug Resistance (PDR)/ABCG, Multi Drug Resistance (MDR)/ABCB, Multi Drug Resistance associated Protein (MRP)/ABCC, Adrenoleukodystrophy protein (ALDp)/ABCD, RNase L Inhibitor (RLI)/ABCE and Elongation Factor 3 (EF3)/ABCF. Among these, only 18 ABC proteins contained transmembrane domains (TMDs) and were grouped as membrane proteins, predominantly belonging to PDR, MDR, MRP, and ALDp subfamilies. A comparative phylogenetic analysis of these ABC proteins with other yeast species revealed their orthologous relationship and pointed towards their conserved functions. Quantitative real time PCR (qRT-PCR) analysis of putative membrane localized ABC protein encoding genes of C. glabrata confirmed their basal expression and showed variable transcriptional response towards antimycotic drugs. This study presents first comprehensive overview of ABC superfamily proteins of a human fungal pathogen C. glabrata, which is expected to provide an important platform for in depth analysis of their physiological relevance in cellular processes and drug resistance., Competing Interests: The authors have declared that no competing interests exist.

Misfolding and aggregation of Cu, Zn Superoxide Dismutase (SOD1) is often found in amyotrophic lateral sclerosis (ALS) patients. The central apo SOD1 barrel was involved in protein maturation and pathological aggregation in ALS. In this work, we employed atomistic molecular dynamics (MD) simulations to study the conformational dynamics of SOD1 barrel monomer in different concentrations of trifluoroethanol (TFE). We find concentration dependence unusual structural and dynamical features, characterized by the local unfolding of SOD1 barrel . This partially unfolded structure is characterized by the exposure of hydrophobic core, is highly dynamic in nature, and is the precursor of aggregation seen in SOD1 barrel . Our computational studies supports the hypothesis of the formation of aggregation 'building blocks' by means of local unfolding of apo monomer as the mechanism of SOD1 fibrillar aggregation. The non-monotonic TFE concentration dependence of protein conformational changes was explored through simulation studies. Our results suggest that altered protein conformation and dynamics within its structure may underlie the aggregation of SOD1 in ALS.

Proteins of the SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) family play a significant role in all vesicular fusion events involved in endocytic and exocytic pathways. These proteins act as molecular machines that assemble into tight four-helix bundle complex, bridging the opposing membranes into close proximity forming membrane fusion. Almost all SNARE proteins share a 53 amino acid coiled-coil domain, which is mostly linked to the transmembrane domain at the C-terminal end. Despite significant variations between SNARE sequences across species, the SNARE mediated membrane fusion is evolutionary conserved in all eukaryotes. It is of interest to compare the functional divergence of SNARE proteins across various eukaryotic groups during evolution. Here, we report an exhaustive phylogeny of the SNARE proteins retrieved from SNARE database including plants, animals, fungi and protists. The Initial phylogeny segregated SNARE protein sequences into five well-supported clades Qa, Qb, Qc, Qbc and R reflective of their positions in the four-helix SNARE complex. Further to improve resolution the Qa, Qb, Qc and R family specific trees were reconstructed, each of these were further segregated into organelle specific clades at first and later diverged into lineage specific subgroups. This revealed that most of the SNARE orthologs are conserved at subcellular locations or at trafficking pathways across various species during eukaryotic evolution. The paralogous expansion in SNARE repertoire was observed at metazoans (animals) and plants independently during eukaryotic evolution. However, results also show that the multi-cellular and saprophytic fungi have limited SNAREs.

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0-4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located β-sheets. The reduction of 60-65% tertiary contacts in 7.0-8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.