Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis.

The cytochrome P450 enzyme CYP121A1 endogenously catalyzes the formation of a carbon-carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of 20 CYP enzymes in Mtb, CYP121A1 continues to garner significant interest as a potential drug target. The accompanying reports the use of 19F NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that were theorized to stabilize a static active site water network. The active site residue Asn-85, whose hydrogen bonds with the diketopiperazine ring of cYY contributes to a contiguous active site water network in the absence of cYY, was mutated to a serine (N85S) and to a glutamine (N85Q). These conservative changes in the hydrogen bond donor side chain result in inactivation of the enzyme. Moreover, the N85S mutation induces reverse type-I binding as measured by absorbance difference spectra. NMR spectra monitoring the ligand-adaptive FG-loop and the active site Trp-182 side chain confirm that disruption of the active site water network also significantly alters the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrate that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. These findings implicate a slowly exchanging water network as a critical factor in CYP121A1 function and a likely contributor to the unusual rigidity of the structure.

CoVe-Tracker: An Interactive SARS-CoV-2 Pan Proteome Evolution Tracker.

SARS-CoV-2 has significantly mutated its genome during the past 3 years, leading to the periodic emergence of several variants. Some of the variants possess enhanced fitness advantage, transmissibility, and pathogenicity and can also reduce vaccine efficacy. Thus, it is important to track the viral evolution to prevent and protect the mankind from SARS-CoV-2 infection. To this end, an interactive web-GUI platform, namely, CoVe-tracker (SARS-CoV-2 evolution tracker), is developed to track its pan proteome evolutionary dynamics (https://project.iith.ac.in/cove-tracker/). CoVe-tracker provides an opportunity for the user to fetch the country-wise and protein-wise amino acid mutations (currently, 44139) of SARS-CoV-2 and their month-wise distribution. It also provides position-wise evolution observed in the SARS-CoV-2 proteome. Importantly, CoVe-tracker provides month- and country-wise distributions of 2065 phylogenetic assignment of named global outbreak (PANGO) lineages and their 177564 variants. It further provides periodic updates on SARS-CoV-2 variant(s) evolution. CoVe-tracker provides the results in a user-friendly interactive fashion by projecting the results onto the world map (for country-wise distribution) and protein 3D structure (for protein-wise mutation). The application of CoVe-tracker in tracking the closest cousin(s) of a variant is demonstrated by considering BA.4 and BA.5 PANGO lineages as test cases. Thus, CoVe-tracker would be useful in the quick surveillance of newly emerging mutations/variants/lineages to facilitate the understanding of viral evolution, transmission, and disease epidemiology.

Sequence dependent influence of an A A mismatch in a DNA duplex: An insight into the recognition by hZαADAR1 protein.

Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A…A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A…A mismatch in the midst of 5'CAG/3'GAC, 5'GAC/3'CAG and 5'CAA/3'GAT (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A…A mismatch in the midst of 5'GAA/3'CAT, 5'GAG/3'CAC, 5'AAC/3'TAG, 5'AAG/3'TAC, 5'TAA/3'AAT, 5'TAT/3'AAA and 5'AAT/3'TAA sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZαADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZαADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5'GAC/3'CAG upon titrating with hZαADAR1. Intriguingly, 5'AAC/3'TAG, 5'AAG/3'TAC and 5'GAA/3'CAT exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5'CAA/3'GAT. These indicate that although A…A mismatch could induce a local B-Z junction transiently, hZαADAR1 requires the presence of a G…C/C…G base pair adjacent to the A…A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZαADAR1 in the presence of the A…A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A…A mismatch towards the B-Z junction.

A B-Z junction induced by an A A mismatch in GAC repeats in the gene for cartilage oligomeric matrix protein promotes binding with the hZαADAR1 protein.

GAC repeat expansion from five to seven in the exonic region of the gene for cartilage oligomeric matrix protein (COMP) leads to pseudoachondroplasia, a skeletal abnormality. However, the molecular mechanism by which GAC expansions in the COMP gene lead to skeletal dysplasias is poorly understood. Here we used molecular dynamics simulations, which indicate that an A … A mismatch in a d(GAC)6·d(GAC)6 duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)7·d(GAC)7 with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZαADAR1), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZαADAR1 recognizes the zigzag backbone of d(GAC)7·d(GAC)7 at the B-Z junction and subsequently converts it into Z-DNA via the so-called passive mechanism. Molecular dynamics simulations carried out for the modeled hZαADAR1-d(GAC)6d(GAC)6 complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZαADAR1 binding with the GAC hairpin stem in COMP can lead to a non-genetic, RNA editing-mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.

EK3D: an E. coli K antigen 3-dimensional structure database.

A very high rate of multidrug resistance (MDR) seen among Gram-negative bacteria such as Escherichia, Klebsiella, Salmonella, Shigella, etc. is a major threat to public health and safety. One of the major virulent determinants of Gram-negative bacteria is capsular polysaccharide or K antigen located on the bacterial outer membrane surface, which is a potential drug & vaccine target. It plays a key role in host-pathogen interactions as well as host immune evasion and thus, mandates detailed structural information. Nonetheless, acquiring structural information of K antigens is not straightforward due to their innate enormous conformational flexibility. Here, we have developed a manually curated database of K antigens corresponding to various E. coli serotypes, which differ from each other in their monosaccharide composition, linkage between the monosaccharides and their stereoisomeric forms. Subsequently, we have modeled their 3D structures and developed an organized repository, namely EK3D that can be accessed through www.iith.ac.in/EK3D/. Such a database would facilitate the development of antibacterial drugs to combat E. coli infections as it has evolved resistance against 2 major drugs namely, third-generation cephalosporins and fluoroquinolones. EK3D also enables the generation of polymeric K antigens of varying lengths and thus, provides comprehensive information about E. coli K antigens.

Twisting right to left: A A mismatch in a CAG trinucleotide repeat overexpansion provokes left-handed Z-DNA conformation.

Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders that arise from overexpansion of trinucleotide repeats located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington's disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A…A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A…A mismatches in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A…A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.

Entangled World of DNA Quadruplex Folds.

DNA quadruplexes participate in many biological functions. It takes up a variety of folds based on the sequence and environment. Here, a meticulous analysis of experimentally determined 437 quadruplex structures (433 PDBs) deposited in the PDB is carried out. The analysis reveals the modular representation of the quadruplex folds. Forty-eight unique quadruplex motifs (whose diversity arises out of the propeller, bulge, diagonal, and lateral loops that connect the quartets) are identified, leading to simple to complex inter/intramolecular quadruplex folds. The two-layered structural motifs are further classified into 33 continuous and 15 discontinuous motifs. While the continuous motifs can directly be extended to a quadruplex fold, the discontinuous motif requires an additional loop(s) to complete a fold, as illustrated here with examples. Similarly, higher-order quadruplex folds can also be represented by continuous or discontinuous motifs or their combinations. Such a modular representation of the quadruplex folds may assist in custom engineering of quadruplexes, designing motif-based drugs, and the prediction of the quadruplex structure. Furthermore, it could facilitate understanding of the role of quadruplexes in biological functions and diseases.

G-quadruplex landscape and its regulation revealed by a new antibody capture method.

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

SSP: An In Silico Tool for Salmonella Species Serotyping Using the Sequences of O-Antigen Biosynthesis Proteins and H-Antigen Filament Proteins.

Over 2500 Salmonella species (alternatively, serovars) encompassing different combinations of O-, H1- and H2-antigens are present in nature and cause millions of deaths worldwide every year. Since conventional serotyping is time-consuming, a user-friendly Salmonellaspecies serotyping (SSP) web tool (https://project.iith.ac.in/SSP/) is developed here to predict the serotypes using Salmonella protein(s) or whole proteome sequences. Prior to SSP implementation, a detailed analysis of protein sequences involved in O-antigen biosynthesis and H-antigen formation is carried out to assess their serotype specificity. Intriguingly, the results indicate that the initializing transferases WbaP, WecA and GNE can efficiently distinguish the O-antigens, which have Gal, GlcNAc and GalNAc as initial sugars respectively. Rigorous analysis shows that Wzx and Wzy are sufficient to distinguish the O-types. Exceptionally, some situations warrant additional proteins. Thus, 150 additional transferases, RfbE for O2, O9 and O9,46 types, Orf17.4 for O3,10 and O1,3,19 types, WecB, WbbE and WbbF for O54 and, Wzm and Wzt for O67 are utilized in serotyping. An in-depth analysis of 302 reference datasets representing 56 H1- and 20 H2-types leads to the identification and utilization of 61 unique sequence patterns of FliC and FljB in H-typing. A test dataset of 2136 whole proteome sequences covering 740 Salmonella serovars, including 13 new species are successfully predicted with 99.72% accuracy. Prior to this, all the O-, H1- and H2-antigens are predicted accurately when tested independently. Indeed, SSP also identifies wrongly annotated Salmonella species; hence, it can easily identify new species that emerge with any combination of O-, H1- and H2-antigens. Thus, SSP can act as a valuable tool in the surveillance of Salmonella species.

Structural diversity among Acinetobacter baumannii K-antigens and its implication in the in silico serotyping.

Acinetobacter baumannii is an emerging opportunistic pathogen. It exhibits multi-, extreme-, and pan-drug resistance against several classes of antibiotics. Capsular polysaccharide (CPS or K-antigen) is one of the major virulence factors which aids A. baumannii in evading the host immune system. K-antigens of A. baumannii exploit the Wzx/Wzy-dependent pathway that involves 13 different proteins for its assembly and transport onto the outer membrane. A total of 64 (out of 237 K-locus(KL) types) known K-antigen sugar repeating structures are discussed here and are classified into seven groups based on their initial sugars, QuiNAc4NAc, GalNAc, GlcNAc, Gal, QuiNAc/FucNAc, FucNAc, and GlcNAc along with Leg5Ac7Ac/Leg5Ac7R. Thus, the corresponding seven initializing glycosyltransferases (ItrA1, ItrA2, ItrA3, ItrA4, ItrB1, ItrB3, and ItrA3 along with ItrB2) exhibit serotype specificity. The modeled 3D-structural repository of the 64 K-antigens can be accessed at https://project.iith.ac.in/ABSD/k_antigen.html. The topology of K-antigens further reveals the presence of 2-6 and 0-4 sugar monomers in the main and side chains, respectively. The presence of negatively (predominant) or neutrally charged K-antigens is observed in A. baumannii. Such diversity in the K-antigen sugar composition provides the K-typing specificity (viz., 18-69% in terms of reliability) for Wza, Wzb, Wzc, Wzx, and Wzy proteins involved in the Wzx/Wzy-dependent pathway. Interestingly, the degree of uniqueness of these proteins among different K-types is estimated to be 76.79%, considering the 237 reference sequences. This article summarizes the A. baumannii K-antigen structural diversity and creation of a K-antigen digital repository and provides a systematic analysis of the K-antigen assembly and transportation marker proteins.

Concentration and time-dependent amyloidogenic characteristics of intrinsically disordered N-terminal region of Saccharomyces cerevisiae Stm1.

Saccharomyces cerevisiae Stm1 protein is a ribosomal association factor, which plays an important role in preserving ribosomes in a nutrition-deprived environment. It is also shown to take part in apoptosis-like cell death. Stm1 N-terminal region (Stm1_N1-113) is shown to recognize purine motif DNA triplex and G-quadruplex. Circular dichroism (CD) spectra of Stm1_N1-113 (enriched in positively-charged Lysine and Arginine; negatively-charged Aspartate; polar-uncharged Threonine, Asparagine, Proline and Serine; hydrophobic Alanine, Valine, and Glycine) collected after 0 and 24 h indicate that the protein assumes beta-sheet conformation at the higher concentrations in contrast to intrinsically disordered conformation seen for its monomeric form found in the crystal structure. Thioflavin-T kinetics experiments indicate that the lag phase is influenced by the salt concentration. Atomic force microscopy (AFM) images collected for a variety of Stm1_N1-113 concentrations (in the range of 1-400 µM) in the presence of 150 mM NaCl at 0, 24, and 48 h indicate a threshold concentration requirement to observe the time-dependent amyloid formation. This is prominent seen at the physiological salt concentration of 150 mM NaCl with the fibrillation observed for 400 µM concentration at 48 h, whereas oligomerization or proto-fibrillation is seen for the other concentrations. Such concentration-dependent fibrillation of Stm1_N1-113 explains that amyloid fibrils formed during the overexpression of Stm1_N1-113 may act as a molecular device to trigger apoptosis-like cell death.

Destabilizing Effect of Organo Ru(II) Salts on the Intermolecular Parallel CGG Repeat DNA Quadruplex Associated with Neurodegenerative/Neuromuscular Diseases.

The cationic organo ruthenium(II) salts ([Ru(p-cymene)(ipit)(Cl)](Cl) (RuS), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-thione (ipit) and [Ru(p-cymene)(ipis)(Cl)](Cl) (RuSe), 1-isopropyl-3-(pyridin-2-yl)-imidazol-2-selenone (ipis)) are isolated, and their binding efficacy with d(CGG)15 quadruplex is investigated. Circular dichroism (CD) wavelength scan titration experiments of RuS and RuSe compounds with the intermolecular parallel quadruplex formed by d(CGG)15 (associated with neurodegenerative/neuromuscular/neuronal intranuclear inclusion disorders like FXTAS, OPMD, OPDM types 1-4, and OPML as well as FXPOI) and with the control d(CGG)15·d(CCG)15 duplex indicate their specificity toward the former. Electrophoretic mobility shift titration experiments also confirm the binding of the ligands with d(CGG)15. CD thermal denaturation experiments indicate that both RuS and RuSe destabilize the quadruplex, specifically at 10 mM concentration of the ligands. This is further confirmed by 1D 1H NMR experiments. Such a destabilizing effect of these ligands on the d(CGG)15 quadruplex indicates that RuS and RuSe chalcogen complexes can act as a template for the design of novel molecules for the diagnostics and/or therapeutics of CGG repeat expansion-associated diseases.

Active Site Aromatic Residues Play a Dual Role in the Substrate Interaction and Protein Structure in Functional Dimers of CYP121A1 of Mycobacterium tuberculosis.

The essential enzyme CYP121A1 of Mycobacterium tuberculosis forms a functional dimer, which when disrupted results in a decrease of activity and substrate specificity. The crystal structure of CYP121A1 in complex with its substrate di-cyclotyrosine (cYY) indicates that the aromatic side chains of Phe-168 and Trp-182 form stabilizing π-π interactions with a tyrosyl ring of cYY. In the enclosed study, we utilize targeted 19F labeling of aromatic residues to label CYP121A1 for detection by nuclear magnetic resonance (NMR) spectroscopy. 19F-NMR spectra and functional characterization of mutations to Phe-168 and Trp-182 are combined with all-atom molecular dynamics simulations of substrate-bound and substrate-free CYP121A1. This study shows that these aromatic residues interact with cYY predominantly through π-π stacking. In addition to playing an essential role in substrate binding, these active site residues also stabilize the tertiary and quaternary structures of CYP121A1. An additional unexpected finding was the presence of cYY-induced long-range allostery that affects residues located near the homodimer interface. Taken together, this study highlights a structural relationship between the active site environment of this essential enzyme with its global structure that was previously unknown.

Characterization of the interaction between the Salmonella type III secretion system tip protein SipD and the needle protein PrgI by paramagnetic relaxation enhancement.

Many Gram-negative bacteria that cause major diseases and mortality worldwide require the type III secretion system (T3SS) to inject virulence proteins into their hosts and cause infections. A structural component of the T3SS is the needle apparatus, which consists of a base, an external needle, and a tip complex. In Salmonella typhimurium, the external needle is assembled by the polymerization of the needle protein PrgI. On top of this needle sits a tip complex, which is partly formed by the tip protein SipD. How SipD interacts with PrgI during the assembly of the T3SS needle apparatus remains unknown. The central region of PrgI forms an α-helical hairpin, whereas SipD has a long central coiled-coil, which is a defining structural feature of other T3SS tip proteins as well. Using NMR paramagnetic relaxation enhancement, we have identified a specific region on the SipD coiled-coil that interacts directly with PrgI. We present a model of how SipD might dock at the tip of the needle based on our paramagnetic relaxation enhancement results, thus offering new insight about the mechanism of assembly of the T3SS needle apparatus.

STRIDER: Steric hindrance and metal coordination identifier.

Biomolecular structural knowledge is important to understand the biological processes and the mechanisms underlying human diseases. In silico modeling plays a vital role in de novo design and docking of biomacromolecules as well as in exploring their conformational dynamics. Additionally, it has a major role in acquiring the structural insights using the parameters derived from experimental techniques such as cryo-electron microscopy. Steric hindrance is one of the important measures to validate the accuracy of the modeled biomolecular structures. A web user interface (WUI), namely, STRIDER (steric hindrance and metal coordination identifier) (www.iith.ac.in/strider/) estimates and reports pairwise inter- and intra- molecular steric hindrances using the van der Waals radius of 117 elements. STRIDER identifies and reports the coordination pattern of 64 metals in an interactive mode. It can provide conformer wise interaction pattern(s) of an ensemble of conformers which is needed in circumventing sampling issue in flexible docking, protein folding and structure based virtual screening. Further, it generates a pymol session file which highlights the aforementioned interaction for an offline analysis. Since STRIDER simply requires the Cartesian coordinates of a molecule in PDB format, any chemical structure can be given as an input. Functionality of STRIDER is illustrated here by considering several examples.

Geographical distribution of SARS-CoV-2 amino acids mutations and the concomitant evolution of seven distinct clades in non-human hosts.

Since its first emergence in December 2019, the world has witnessed the eruption of mutations in the SARS-CoV-2 genome that have led to increased viral transmissibility and pathogenicity due to sustained local viral transmission. Zooanthroponotic and zoonotic transmissions have further raised concerns as they could result in the emergence of viral variants with a novel antigenicity and transmissibility that could jeopardize the vaccine efficacy. To understand the viral evolution during such transmissions, 1016 whole-genome sequences (deposited in GISAID as of March 7, 2022) (from 18 countries) corresponding to mink, cat, deer, dog, hyena, tiger, lion, gorilla, Syrian hamster, leopard cat, fishing cat, bear cat, coati, ferret, snow leopard and green monkey have been analysed here. Intriguingly, phyloproteome analysis indicate that Nsp2:R218C, Nsp2:D268-(deletion), Spike:D614G, Nsp12:P323L, Nsp2:A192V, ORF3a protein:Q57H, N protein:R203K and N protein:G204R/L, Spike:A222V, ORF10 protein:V30L and N protein:A220V are moderate or high recurring and clade decisive mutations, leading to 6 primary clades during the early stage of pandemic. Most interestingly, the human evolved delta variant having a combination of 26 (clade decisive) mutations defines the seventh clade and transmits to non-human hosts across the globe without exhibiting any country-specific mutation(s). Nonetheless, Spike:D614G and Nsp12:P323L together with (i)N protein:R203K,N protein:G204R/L,Spike:V70-, Spike:H69-, Nsp12:T739I, and Nsp1:M85-, (ii)Nsp2:A192V, Nsp3:D178Y, (iii)Nsp2:T85I, N protein:P67S and ORF3a protein:Q57H and (iv)Spike:A222V, ORF10 protein:V30L, N protein:A220V and Spike:F486I are specific to Denmark, Netherlands, USA and Latvia respectively and, (v)Nsp2:D268- and Nsp13:R292C that are devoid of Spike:D614G and Nsp12:P323L is specific to Netherlands. SARS-CoV-2 variants consisting of these mutations are also seen in the human SARS-CoV-2 sequences from the same country. Independent country-specific SARS-CoV-2 variant evolution further indicates distinct epidemiological dynamics during zooanthroponotic and zoonotic transmissions. Thus, the results presented here indicate the need for the surveillance of viral evolution in non-human hosts also during the future pandemic.

SARS-CoV-2 whole-proteome sequences from environment as an indicator of community viral distribution, evolution and epidemiological dynamics: A cohort analysis of Austria.

Several investigations have been carried out to detect SARS-CoV-2 samples from the environment such as sewage waters and surface swabs. Whole-proteome sequence analysis of 847 SARS-CoV-2 genome sequences collected from the environment in Austria during 2021 and deposited in GISAID indicates that alpha and delta are two dominant variants, coinciding with the human clinical samples with a Pearson correlation coefficient in the range of 0.58 (alpha variant) to 0.82 (delta variant). Both environmental and human samples show that Austrian SARS-CoV-2 alpha variant is found to possess N protein R203K and G204R/P mutations, whereas they are absent in the delta variant. SARS-CoV-2 delta variant is continuously seen in both the environmental and human clinical samples from the month of September 2021 and it spiked in November 2021, which is directly reflected in the increase of the number of SARS-CoV-2 infections and deaths in Austria during November 2021. Thus, the results presented here indicate that the environmental SARS-CoV-2 whole-genome sequences collected from Austria reflect the community viral distribution, evolution and the concomitant epidemiological dynamics. Since SARS-CoV-2 keeps evolving, the results presented here further suggest the need to monitor the environment for the early detection of SARS-CoV-2 variants to take appropriate precautionary measures.

Biophysical interaction between lanthanum chloride and (CG)n or (GC)n repeats: A reversible B-to-Z DNA transition.

The transition from right-handed to left-handed DNA is not only acts as the controlling factor for switching gene expression but also has equal importance in designing nanomechanical devices. The (CG)n and (GC)n repeat sequences are well known model molecules to study B-Z transition in the presence of higher concentration of monovalent cations. In this communication, we report a cyclic transition in (CG)6 DNA using millimolar concentration of trivalent lanthanide salt LaCl3. The controlled and reversible transition was seen in (CG)12, and (GC)12 DNA employing CD spectroscopy. While LaCl3 failed to induce B-Z transition in shorter oligonucleotides such as (CG)3 and (GC)3, a smooth B-Z transition was recorded for (CG)6, (CG)12 and (GC)12 sequences. Interestingly, the phenomenon was reversible (Z-B transition) with addition of EDTA. Particularly, two rounds of cyclic transition (B-Z-B-Z-B) have been noticed in (CG)6 DNA in presence of LaCl3 and EDTA which strongly suggest that B-Z transition is reversible in short repeat sequences. Thermal melting and annealing behaviour of B-DNA are reversible while the thermal melting of LaCl3-induced Z-DNA is irreversible which suggest a stronger binding of LaCl3 to the phosphate backbone of Z-DNA. This was further supported by isothermal titration calorimetric study. Molecular dynamics (MD) simulation indicates that the mode of binding of La3+ (of LaCl3) with d(CG)8.d(CG)8 is through the minor groove, wherein, 3 out of 11 La3+ bridge the anionic oxygens of the complementary strands. Such a tight coordination of La3+ with the anionic oxygens at the minor groove surface may be the reason for the experimentally observed irreversibility of LaCl3-induced Z-DNA seen in longer DNA fragments. Thus, these results indicate LaCl3 can easily be adopted as an inducer of left-handed DNA in other short oligonucleotides sequences to facilitate the understanding of the molecular mechanism of B-Z transition.

Conformational distortions induced by periodically recurring A A in d(CAG).d(CAG) provide stereochemical rationale for the trapping of MSH2.MSH3 in polyQ disorders.

CAG repeat instability causes a number of neurodegenerative disorders. The unusual hairpin stem structure formed by the CAG repeats in DNA traps the human mismatch repair MSH2.MSH3 (Mutsß) complex. To understand the mechanism behind the abnormal binding of Mutsß with the imperfect hairpin stem structure formed by CAG repeats, molecular dynamics simulations have been carried out for Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 (1 A…A mismatch) and Mutsß-d(CAG)5.d(CAG)5 (5 mismatches, wherein, A…A occurs periodically) complexes. The interaction of MSH3 residue Tyr245 at the minor groove side of A…A, an essential interaction responsible for the recognition by Mutsß, are retained in both the cases. Nevertheless, the periodic unwinding caused by the nonisostericity of A…A with the flanking canonical base pairs in d(CAG)5.d(CAG)5 distorts the regular B-form geometry. Such an unwinding exposes one of the A…A mismatches (that interacts with Tyr245) at the major groove side and also facilitates the on and off hydrogen bonding interaction with Lys546 sidechain (MSH2-domain-IV). In contrast, kinking of the DNA towards the major groove in Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 doesn't facilitate such an exposure of the bases at the major groove. Further, the unwinding of the helix in d(CAG)5.d(CAG)5 enhances the tighter binding between MSH2-domain-I and d(CAG)5.d(CAG)5 at the major groove side as well as between MSH3-domain-I and MSH3-domain-IV. Markedly, such enhanced interactions are absent in Mutsß-d(CAG)2(CAG)(CAG)2.d(CTG)2(CAG)(CTG)2 that has a single A…A mismatch. Thus, the above-mentioned enhancement in intra- and inter- molecular interactions in Mutsß-d(CAG)5.d(CAG)5 provide the stereochemical rationale for the trapping of Mutsß in CAG repeat expansion disorders.

CD-NuSS: A Web Server for the Automated Secondary Structural Characterization of the Nucleic Acids from Circular Dichroism Spectra Using Extreme Gradient Boosting Decision-Tree, Neural Network and Kohonen Algorithms.

Nucleic acids exhibit a repertoire of conformational preference depending on the sequence and environment. Circular dichroism (CD) is an essential and valuable tool for monitoring such secondary structural conformations of nucleic acids. Nonetheless, the CD spectral diversity associated with these structures poses a challenge in obtaining the quantitative information about the secondary structural content of a given CD spectrum. To this end, the competence of the extreme gradient boosting decision-tree (XGBoost), Kohonen and neural network (nnet) algorithms have been exploited here to predict the diverse secondary structures of nucleic acids. A curated library of 450 CD spectra corresponding to 16 different secondary structures of nucleic acids has been created and used as a training dataset. The hyper-parameters corresponding to the aforementioned algorithms have been optimized using holdout and k-fold (here, k = 5) cross-validation methods. For a test dataset of 150 CD spectra, both the nnet and XGBoost algorithms have exhibited nearly similar prediction accuracy in the range of 85% and 87% (the latter exhibited a slightly higher prediction accuracy). Thus, the nnet and XGBoost algorithms tested here can be employed for predicting the hybrid nucleic acid topologies in future. For the sake of accessibility, the entire process has been automated and implemented as a webserver, called CD-NuSS (CD to nucleic acids secondary structure) and is freely accessible at https://project.iith.ac.in/cdnuss/.