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Protein tertiary structure mimetics are valuable tools to target large protein–protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein–protein interaction through target dimerization.

Antibiotic resistance is a growing global health concern. Colistin is one of the last-resort antibiotics that treats multidrug-resistant (MDR) Gram-negative bacterial infection. However, bacteria resistant to colistin have become increasingly prevalent. Using a bacterial whole-cell screen of a fragment-based library, one compound was discovered to resensitize MDR

The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations (SHM) of 15.2%. 44m neutralized 79% of HIV-1 heterologous viruses tested, with a geometric mean IC50titer of 0.36 μg/ml. The cryoEM structure of 44m Fab in complex with fully-cleaved glycosylated native-like BG505.SOSIP envelope trimer at 4.4 Å resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01 bnAb. This study improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

DNA-binding protein under starvation (Dps), is a miniature ferritin complex which plays a vital role in protecting bacterial DNA during starvation for maintaining the integrity of bacteria from hostile conditions.Mycobacterium smegmatisis one such bacteria that express MsDps2, which binds DNA to protect it under oxidative and nutritional stress conditions. Several approaches, including cryo-electron tomography (Cryo-ET), were implemented to identify the structure of the Dps protein that is bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to be able to identify the DNA binding residues. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps protein. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA and identify that the region close to N-terminal confers the DNA binding property. Based on cryo-EM data, we performed mutations of several arginine residues proximal to DNA binding region, which dramatically reduced the MsDps2-DNA interaction. In addition, we demonstrated the proposed model for DNA compaction during lattice formation. We also pinpointed arginine residues, which are responsible for DNA binding in lattice arrangement of MsDps2. We performed single-molecule imaging experiments of MsDps2-DNA interactions that corroborate well with our structural studies.

We present new 3-mm continuum and molecular lines observations from the ATOMS survey towards the massive protostellar clump, MM1, located in the filamentary infrared dark cloud (IRDC), G034.43+00.24 (G34). The lines observed are the tracers of either dense gas (e.g. HCO+/H13CO+ J = 1-0) or outflows (e.g. CS J = 2-1). The most complete picture to date of seven cores in MM1 is revealed by dust continuum emission. These cores are found to be gravitationally bound, with virial parameter, $\alpha_{vir}, Comment: 14 pages with 6 figures, and in press

The structures of a cyanophage small heat shock protein (sHSP) were determined as octahedrons of 24-mers and 48-mers and as icosahedrons of 60-mers. An N-terminal deletion construct of an 18 kDa sHSP of Synechococcus sp. phage S-ShM2 crystallized as a 24-mer and its structure was determined at a resolution of 7 Å. The negative stain electron microscopy (EM) images showed that the full-length protein is a mixture of a major population of larger and a minor population of smaller cage-like particles. Their structures have been determined by electron cryomicroscopy 3D image reconstruction at a resolution of 8 Å. The larger particles are 60-mers with icosahedral symmetry and the smaller ones are 48-mers with octahedral symmetry. These structures are the first of the viral/phage origin and the 60-mer is the largest and the first icosahedral assembly to be reported for sHSPs.

While vaccines have by large been found to effective against the evolving SARS-CoV-2 variants, the profound and rapid effectivity of monoclonal antibodies (mAbs) in significantly reducing hospitalization to severe disease outcomes have also been demonstrated. In the present study, by high resolution cryo-electron microscopy (cryo-EM), we examined the structural insights of two trimeric spike (S) protein bound mAbs isolated from an Indian convalescent individual infected with ancestral SARS-CoV-2 which we recently reported to potently neutralize SARS-CoV-2 from its ancestral form through highly virulent Delta form however different in their ability to neutralize Omicron variants. Our findings showed binding and conformational heterogeneities of both the mAbs (THSC20.HVTR04 and THSC20.HVTR26) bound to S trimer in its apo and hACE-2 bound forms. Additionally, cryo-EM resolved structure assisted modeling highlighted key residues associated with the ability of these two mAbs to neutralize Omicron variants. Our findings highlighted key interacting features modulating antigen-antibody interacting that can further aid in structure guided antibody engineering to enhance their breadth and potency.HighlightsTwo potent human mAbs obtained from a single donor differ binding to Omicron spikesPattern of binding and conformation of these mAbs bound to full length spike differsAntibody binding alters the conformational states of S trimer in its apo and hACE-2 bound forms.Cryo-EM structure guided modeling highlighted correlates of interacting residues associated with resistance and sensitivity of BA.1, BA.2, BA.4/BA.5 resistance and sensitivity against these mAbs.

Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.

γ-hemolysin (γ-HL) is a hemolytic and leukotoxic bicomponent β-pore-forming toxin (β-PFT), a potent virulence factor from Staphylococcus aureus Newman strain. In this study, we performed single particle cryo-EM of γ-HL in a lipid environment. We observed clustering and square lattice packing of octameric HlgAB pores upon membrane bilayer, and an octahedral superassembly of octameric pore complexes, that we resolved at resolution 3.5 Å. Our atomic model further demonstrated the key residues involved in hydrophobic zipping between the rim domains of adjacent octameric pore complexes, thus providing first evidence of additional structural stability in PFTs upon membrane lysis. We also observed lipid densities at the octahedral and octameric interfaces, providing critical insights into the lipid-binding residues involved for both HlgA and HlgB components. Furthermore, the hitherto elusive N-terminal region of HlgA has also been resolved in our cryo-EM map and an overall mechanism of pore formation for bicomponent β-PFTs is proposed.

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.

Methionine and cysteine metabolisms are important for the survival and pathogenesis ofMycobacterium tuberculosis(Mtb). The transsulfuration pathway converts methionine to cysteine and represents an important link between antioxidant and methylation metabolism in diverse organisms. Using a combination of biochemistry and cryo–electron microscopy, we characterized the first enzyme of the transsulfuration pathway, cystathionine β-synthase (MtbCbs) inMtb. We demonstrated thatMtbCbs is a heme-less, pyridoxal-5′-phosphate–containing enzyme, allosterically activated byS-adenosylmethionine (SAM). The atomic model ofMtbCbs in its native and SAM-bound conformations revealed a unique mode of SAM-dependent allosteric activation. Further, SAM stabilizedMtbCbs by sterically occluding proteasomal degradation, which was crucial for supporting methionine and redox metabolism inMtb. Genetic deficiency ofMtbCbs reducedMtbsurvival upon homocysteine overload in vitro, inside macrophages, and in mice coinfected with HIV. Thus, theMtbCbs-SAM axis constitutes an important mechanism of coordinating sulfur metabolism inMtb.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Multidrug-resistant (MDR) Gram-negative bacteria are an urgent and rapidly spreading threat to human health with limited treatment options. Previously, we discovered a novel [1,2,5]oxadiazolo[3,4-b]pyrazine-containing compound (1) that selectively re-sensitized a variety of MDR Gram-negative bacteria to colistin, one of the last-resort antibiotic. Herein, we report the structure–activity relationship studies of compound 1 that led to the discovery of several more potent and/or less toxic resistance-modifying agents (RMAs). Further evaluation of these RMAs showed that they were effective in a wide range of MDR bacteria. These results demonstrated these compounds as a novel class of RMAs and may be further developed as therapeutic agents.

β-barrel pore-forming toxins perforate cell membranes by forming oligomeric β-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane β-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during β-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the β-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related β-barrel pore-forming toxins.

Stabilization of the metastable envelope glycoprotein (Env) of HIV-1 is hypothesized to improve induction of broadly neutralizing antibodies. We improved the expression yield and stability of the HIV-1 envelope glycoprotein BG505SOSIP.664 gp140 by means of a previously described automated sequence and structure-guided computational thermostabilization approach, PROSS. This combines sequence conservation information with computational assessment of mutant stabilization, thus taking advantage of the extensive natural sequence variation present in HIV-1 Env. PROSS is used to design three gp140 variants with 17-45 mutations relative to the parental construct. One of the designs is experimentally observed to have a fourfold improvement in yield and a 4 °C increment in thermostability. In addition, the designed immunogens have similar antigenicity profiles to the native flexible linker version of wild type, BG505SOSIP.664 gp140 (NFL Wt) to major epitopes targeted by broadly neutralizing antibodies. PROSS eliminates the laborious process of screening many variants for stability and functionality, providing a proof of principle of the method for stabilization and improvement of yield without compromising antigenicity for next generation complex, highly glycosylated vaccine candidates.

We have conducted a line survey towards Orion KL using the Q-band receiver of Tianma 65 m radio telescope (TMRT), covering 34.8--50 GHz with a velocity resolution between 0.79 km s$^{-1}$ and 0.55 km s$^{-1}$ respectively. The observations reach a sensitivity on the level of 1-8 mK, proving that the TMRT is sensitive for conducting deep line surveys. In total, 597 Gaussian features are extracted. Among them, 177 radio recombination lines (RRLs) are identified, including 126, 40 and 11 RRLs of hydrogen, helium and carbon, with a maximum $\Delta n$ of 16, 7, and 3, respectively. The carbon RRLs are confirmed to originate from photodissociation regions with a $V_{\rm LSR}\sim$9 km s$^{-1}$. In addition, 371 molecular transitions of 53 molecular species are identified. Twenty-one molecular species of this survey were not firmly detected in the Q band by Rizzo et al. (2017), including species such as H$_2$CS, HCOOH, C$_2$H$_5$OH, H$_2^{13}$CO, H$_2$CCO, CH$_3$CHO, CH$_2$OCH$_2$, HCN $v_2=1$, and CH$_3$OCHO $v_t=1$. In particular, the vibrationally excited states of ethyl cyanide (C$_2$H$_5$CN $v$13/$v$21) are for the first time firmly detected in the Q band. NH$_3$ (15,15) and (16,16) are identified, and they are so far the highest transitions of the NH$_3$ inversion lines detected towards Orion KL. All the identified lines can be reproduced by a radiative transfer model., Comment: 51 pages, 18 figures, accepted by ApJS

We present the four-year survey results of monthly submillimeter monitoring of eight nearby ($< 500 $pc) star-forming regions by the JCMT Transient Survey. We apply the Lomb-Scargle Periodogram technique to search for and characterize variability on 295 submillimeter peaks brighter than 0.14 Jy beam$^{-1}$, including 22 disk sources (Class II), 83 protostars (Class 0/I), and 190 starless sources. We uncover 18 secular variables, all of them protostars. No single-epoch burst or drop events and no inherently stochastic sources are observed. We classify the secular variables by their timescales into three groups: Periodic, Curved, and Linear. For the Curved and Periodic cases, the detectable fractional amplitude, with respect to mean peak brightness, is $\sim4$ % for sources brighter than $\sim$ 0.5 Jy beam$^{-1}$. Limiting our sample to only these bright sources, the observed variable fraction is 37 % (16 out of 43). Considering source evolution, we find a similar fraction of bright variables for both Class 0 and Class I. Using an empirically motivated conversion from submillimeter variability to variation in mass accretion rate, six sources (7 % of our full sample) are predicted to have years-long accretion events during which the excess mass accreted reaches more than 40 % above the total quiescently accreted mass: two previously known eruptive Class I sources, V1647 Ori and EC 53 (V371 Ser), and four Class 0 sources, HOPS 356, HOPS 373, HOPS 383, and West 40. Considering the full protostellar ensemble, the importance of episodic accretion on few years timescale is negligible, only a few percent of the assembled mass. However, given that this accretion is dominated by events of order the observing time-window, it remains uncertain as to whether the importance of episodic events will continue to rise with decades-long monitoring., Accepted for publication in the Astrophysical Journal

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 °C and were stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼ three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core β-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in the previously determined crystal structure of TDH. Moreover, the functional implication of NTR for the membrane-damaging action of TDH remains unknown. In the present study, we have explored the implications of NTR for the structure-function mechanism of TDH. Our data show that the presence of NTR modulates the physicochemical property of TDH in terms of augmenting the amyloidogenic propensity of the protein. Deletion of NTR compromises the binding of TDH toward target cell membranes and drastically affects the membrane-damaging cytolytic/cytotoxic activity of the toxin. Mutations of aromatic/hydrophobic residues within NTR also confer compromised cell-killing activity. Moreover, covalent trapping of NTR, via an engineered disulfide bond, against the core β-sandwich domain also abrogates the cytolytic/cytotoxic activity of TDH. This observation suggests that an unrestrained configuration of NTR is crucial for the membrane-damaging action of TDH. On the basis of our study, we propose a model explaining the role of NTR in the membrane-damaging function of TDH.

Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.