@article {152902, title = {Conformational diversity in class C GPCR positive allosteric modulation.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Jan 13}, pages = {619}, abstract = {

The metabotropic glutamate receptors (mGlus) are class C G protein-coupled receptors (GPCR) that form obligate dimers activated by the major excitatory neurotransmitter L-glutamate. The architecture of mGlu receptor comprises an extracellular Venus-Fly Trap domain (VFT) connected to the transmembrane domain (7TM) through a Cysteine-Rich Domain (CRD). The binding of L-glutamate in the VFTs and subsequent conformational change results in the signal being transmitted to the 7TM inducing G protein binding and activation. The mGlu receptors signal transduction can be allosterically potentiated by positive allosteric modulators (PAMs) binding to the 7TMs, which are of therapeutic interest in various neurological disorders. Here, we report the cryoEM structures of metabotropic glutamate receptor 5 (mGlu) purified with three chemically and pharmacologically distinct PAMs. We find that the PAMs modulate the receptor equilibrium through their different binding modes, revealing how their interactions in the 7TMs impact the mGlu receptor conformational landscape and function. In addition, we identified a PAM-free but agonist-bound intermediate state that also reveals interactions mediated by intracellular loop 2. The activation of mGlu receptor is a multi-step process in which the binding of the PAMs in the 7TM modulates the equilibrium towards the active state.

}, keywords = {Allosteric Regulation, Animals, Cryoelectron Microscopy, Glutamic Acid, HEK293 Cells, Humans, Models, Molecular, Protein Binding, Protein Conformation, Protein Domains, Receptor, Metabotropic Glutamate 5, Receptors, Metabotropic Glutamate, Signal Transduction}, issn = {2041-1723}, doi = {10.1038/s41467-024-55439-9}, author = {Cannone, Giuseppe and Berto, Ludovic and Malhaire, Fanny and Ferguson, Gavin and Fouillen, Aurelien and Balor, St{\'e}phanie and Font-Ingles, Joan and Llebaria, Amadeu and Goudet, Cyril and Kotecha, Abhay and Vinothkumar, Kutti R and Lebon, Guillaume} } @article {159720, title = {Distinct filament morphology and membrane tethering features of the dual FtsZ paralogs in Odinarchaeota.}, journal = {EMBO J}, year = {2025}, month = {2025 Aug 08}, abstract = {

The Asgard phylum has emerged as a model to study eukaryogenesis because of their close relatedness with the eukaryotes. In this study, we use FtsZ proteins from a member of the class Odinarchaeia as representatives to investigate the probable origin, evolution, and assembly of the FtsZ/tubulin protein superfamily in Asgard archaea. We performed a comparative analysis of the biochemical properties and cytoskeletal assembly of FtsZ1 and FtsZ2, the two FtsZ isoforms in the Odinarchaeota metagenome. Our electron microscopy analysis reveals that OdinFtsZ1 assembles into curved single protofilaments, while OdinFtsZ2 forms stacked spiral ring-like structures. Upon sequence analysis, we identified an N-terminal amphipathic helix in OdinFtsZ1, which mediates direct membrane tethering. In contrast, OdinFtsZ2 is recruited to the membrane by the anchor OdinSepF via OdinFtsZ2{\textquoteright}s C-terminal tail. Overall, we report the presence of two distant evolutionary paralogs of FtsZ in Odinarchaeota, with distinct filament assemblies and differing modes of membrane targeting. Our findings highlight the diversity of FtsZ proteins in the archaeal phylum Asgardarchaeota, providing valuable insights into the evolution and differentiation of tubulin-family proteins.

}, issn = {1460-2075}, doi = {10.1038/s44318-025-00529-7}, author = {Kumari, Jayanti and Uthaman, Akhilesh and Bose, Sucharita and Kundu, Ananya and Sharma, Vaibhav and Dutta, Soumyajit and Dhar, Anubhav and Roy, Srijita and Srinivasan, Ramanujam and Pande, Samay and Vinothkumar, Kutti R and Gayathri, Pananghat and Palani, Saravanan} } @article {162694, title = {Somatic hypermutation patterns are shaped by both motif position and sequence grammar.}, journal = {EMBO J}, year = {2025}, month = {2025 Dec 11}, abstract = {

Somatic hypermutation (SHM) in variable regions of immunoglobulin genes by activation-induced deaminase (AID) is essential for the maturation of protective antibodies against pathogen and vaccine antigens. AID preferentially mutates cytosines within WRCH motifs (wherein W = A/T, R = A/G, and H = A/C/T) in single-stranded DNA, yet these motifs show large but reproducible variation in mutation frequency, suggesting a crucial role for sequences flanking the WRCH motifs (i.e., a sequence grammar) in determining mutational outcomes. However, the nature of this sequence grammar is poorly understood. Here, we demonstrate that identical sequence contexts can exert significantly varying effects on the mutagenesis of different WRCH motifs. Molecular dynamics simulations reveal that both the sequence context and the specific WRCH motif modulate AID activity by altering the mode and strength of AID{\textquoteright}s interactions with single-stranded DNA. Repositioning a motif and its context within the variable region significantly alters its mutability. Therefore, the mutability of AID target cytosines is determined by a motif-specific sequence grammar that determines, in part, how activation-induced deaminase binds single-stranded DNA, as well as the motif position.

}, issn = {1460-2075}, doi = {10.1038/s44318-025-00640-9}, author = {Bartl, Bianca and Schoeberl, Ursula E and Valieris, Renan and Fitz, Johanna and Roeder, Konstantin and Vinothkumar, Kutti R and Gundinger, Benjamin and Tojal Da Silva, Israel and Pavri, Rushad} } @article {140645, title = {Exploring advances in single particle CryoEM with apoferritin: From blobs to true atomic resolution.}, journal = {Int J Biochem Cell Biol}, volume = {169}, year = {2024}, month = {2024 Feb 01}, pages = {106536}, abstract = {

Deciphering the three-dimensional structures of macromolecules is of paramount importance for gaining insights into their functions and roles in human health and disease. Single particle cryoEM has emerged as a powerful technique that enables direct visualization of macromolecules and their complexes, and through subsequent averaging, achieve near atomic-level resolution. A major breakthrough was recently achieved with the determination of the apoferritin structure at true atomic resolution. In this review, we discuss the latest technological innovations across the entire single-particle workflow, which have been instrumental in driving the resolution revolution and in transforming cryoEM as a mainstream technique in structural biology. We illustrate these advancements using apoferritin as an example that has served as an excellent benchmark sample for assessing emerging technologies. We further explore whether the existing technology can routinely generate atomic structures of dynamic macromolecules that more accurately represent real-world samples, the limitations in the workflow, and the current approaches employed to overcome them.

}, issn = {1878-5875}, doi = {10.1016/j.biocel.2024.106536}, author = {Premageetha, Gowtham ThambraRajan and Vinothkumar, Kutti R and Bose, Sucharita} } @article {145428, title = {Factors affecting macromolecule orientations in thin films formed in cryo-EM.}, journal = {Acta Crystallogr D Struct Biol}, volume = {80}, year = {2024}, month = {2024 Jul 01}, pages = {535-550}, abstract = {

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

}, keywords = {Animals, Cryoelectron Microscopy, Freezing, Macromolecular Substances, Proteins, Vitrification}, issn = {2059-7983}, doi = {10.1107/S2059798324005229}, author = {Yadav, Swati and Vinothkumar, Kutti R} } @article {147287, title = {Modeling Ligands into Maps Derived from Electron Cryomicroscopy.}, journal = {J Vis Exp}, year = {2024}, month = {2024 Jul 19}, abstract = {

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu5, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied β-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.

}, keywords = {Cryoelectron Microscopy, Ligands, Models, Molecular}, issn = {1940-087X}, doi = {10.3791/66310}, author = {Jha, Shaileshanand and Bose, Sucharita and Vinothkumar, Kutti R} } @article {123128, title = {Regulatory mechanisms of c-di-AMP synthase (MsDisA) protein from Mycobacterium smegmatis revealed by a structure - function analysis.}, journal = {Protein Sci}, year = {2023}, month = {2023 Jan 20}, pages = {e4568}, abstract = {

Cyclic-di-nucleotide based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although, c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous B. subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Further, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of T. maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.4568}, author = {Gautam, Sudhanshu and Mahapa, Avisek and Yeramala, Lahari and Gandhi, Apoorv and Krishnan, Sushma and Vinothkumar, Kutti R and Chatterji, Dipankar} } @article {122345, title = {Structural heterogeneity of the Rieske iron-sulfur protein in a yeast complex III.}, journal = {IUCrJ}, volume = {10}, year = {2023}, month = {2023 Jan 01}, pages = {1-3}, abstract = {

Cryo-EM has been used to analyze the positions of the hydrophilic domain of the Rieske iron{\textendash}sulfur protein in a yeast complex III under various redox and substrate/inhibitor conditions.

}, keywords = {Electron Transport Complex III, Iron-Sulfur Proteins, Mitochondrial Membranes, Saccharomyces cerevisiae}, issn = {2052-2525}, doi = {10.1107/S2052252522012003}, author = {Vinothkumar, Kutti R} } @article {135860, title = {Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.}, journal = {IUCrJ}, volume = {10}, year = {2023}, month = {2023 Nov 01}, pages = {738-753}, abstract = {

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

}, keywords = {Catalysis, Models, Molecular, Mycobacterium tuberculosis, Phosphopyruvate Hydratase, Protein Conformation}, issn = {2052-2525}, doi = {10.1107/S2052252523008485}, author = {Ahmad, Mohammed and Jha, Bhavya and Bose, Sucharita and Tiwari, Satish and Dwivedy, Abhisek and Kar, Deepshikha and Pal, Ravikant and Mariadasse, Richard and Parish, Tanya and Jeyakanthan, Jeyaraman and Vinothkumar, Kutti R and Biswal, Bichitra Kumar} } @article {108402, title = {Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methyltransferases.}, journal = {ACS Chem Biol}, year = {2022}, month = {2022 Mar 22}, abstract = {

Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of the 30S-KsgA complex by cryo-electron microscopy, a state was captured, where KsgA juxtaposes between helices h44 and h45 of the 30S ribosome, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27, were mutated and evaluated for their methylation efficiency revealing factors that direct the enzyme to its cognate site with high fidelity. The biochemical studies show that the three-dimensional environment of the ribosome enables the interaction of select loop regions in KsgA with the ribosome helices paramount to maintain selectivity.

}, issn = {1554-8937}, doi = {10.1021/acschembio.1c00732}, author = {Singh, Juhi and Raina, Rahul and Vinothkumar, Kutti R and Anand, Ruchi} } @article {108057, title = {Interaction of human erythrocyte catalase with air-water interface in cryoEM.}, journal = {Microscopy (Oxf)}, volume = {71}, year = {2022}, month = {02/2022}, pages = {i51-i59}, abstract = {

One of the key goals in single-particle cryo-microscopy is to obtain a uniform distribution of particle orientations, so that the three-dimensional structure has isotropic resolution in Fourier space. A common problem arises from the interaction of protein molecules with the air-water interface that exists on both surfaces of the thin film of liquid that is formed prior to plunge-freezing into liquid ethane. Some proteins and other macromolecular complexes are disrupted by interaction with the air-water interface. Other proteins or macromolecules either become concentrated through their interaction with the interface or are excluded because they bind strongly to some other part of the grid or the filter paper used in blotting. In this paper, the interaction of human erythrocyte catalase with the air-water interface is investigated and minimized by the addition of certain detergents. Detergents can form an amphipathic monolayer at the air-water interface that creates a barrier and leaves the molecules free to adopt a variety of orientations, thus facilitating the 3D structure determination. These results suggest that further characterization and development of detergents for cryo-microscopy plunge-freezing would be useful.

}, doi = {10.1093/jmicro/dfab037 }, author = {Chen, S. and Li, J. and Vinothkumar, Kutti R and Henderson, R.} } @article {99104, title = {Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.}, journal = {Cell Rep}, volume = {36}, year = {2021}, month = {2021 Aug 31}, pages = {109648}, abstract = {

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109648}, author = {Nasrallah, Chady and Cannone, Giuseppe and Briot, Julie and Rottier, Karine and Berizzi, Alice E and Huang, Chia-Ying and Quast, Robert B and Hoh, Francois and Ban{\`e}res, Jean-Louis and Malhaire, Fanny and Berto, Ludovic and Dumazer, Ana{\"e}lle and Font-Ingles, Joan and G{\'o}mez-Santacana, Xavier and Catena, Juanlo and Kniazeff, Julie and Goudet, Cyril and Llebaria, Amadeu and Pin, Jean-Philippe and Vinothkumar, Kutti R and Lebon, Guillaume} } @article {100624, title = {Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in tracheoblasts.}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Oct 08}, abstract = {

Progenitors of the thoracic tracheal system of adult (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an HO generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/ Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of HO in minutes. The findings presented reveal that HO activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

}, issn = {2050-084X}, doi = {10.7554/eLife.68636}, author = {Kizhedathu, Amrutha and Chhajed, Piyush and Yeramala, Lahari and Sain Basu, Deblina and Mukherjee, Tina and Vinothkumar, Kutti R and Guha, Arjun} } @article {93665, title = {Expanding capabilities and infrastructure for cryo-EM.}, journal = {Nat Methods}, volume = {18}, year = {2021}, month = {2021 May}, pages = {437-438}, issn = {1548-7105}, doi = {10.1038/s41592-021-01139-x}, author = {Vinothkumar, Kutti R} } @article {79721, title = {A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.}, journal = {Angew Chem Int Ed Engl}, year = {2020}, month = {2020 May 25}, abstract = {

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

}, issn = {1521-3773}, doi = {10.1002/anie.202005332}, author = {Arya, Chetan Kumar and Yadav, Swati and Fine, Jonathan and Casanal, Ana and Chopra, Gaurav and Ramanathan, Gurunath and Vinothkumar, Kutti R and Subramanian, Ramaswamy} } @article {80091, title = {Comparison of CryoEM and X-ray structures of dimethylformamidase.}, journal = {Prog Biophys Mol Biol}, year = {2020}, month = {2020 Jul 28}, abstract = {

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 {\textdegree}C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

}, issn = {1873-1732}, doi = {10.1016/j.pbiomolbio.2020.06.008}, author = {Vinothkumar, Kutti R and Arya, Chetan and Ramanathan, Gurunath and Subramanian, Ramaswamy} } @article {79728, title = {Structural insights into actin filament recognition by commonly used cellular actin markers}, journal = {EMBO J}, volume = {39}, year = {2020}, month = {07/2020}, abstract = {

Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

}, doi = {10.15252/embj.2019104006 }, author = {Kumari, Archana and Kesarwani, Shubham and Javoor, Manjunath G and Vinothkumar, Kutti R and Sirajuddin, Minhajuddin} } @article {64899, title = {Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Sep 11}, pages = {4127}, abstract = {

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-11931-1}, author = {Sathyanarayanan, Nitish and Cannone, Giuseppe and Gakhar, Lokesh and Katagihallimath, Nainesh and Sowdhamini, Ramanathan and Ramaswamy, Subramanian and Vinothkumar, Kutti R} } @article {62762, title = {Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Jul 11}, pages = {3058}, abstract = {

The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 {\r A} structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to αβ of F-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5{\textquoteright}-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-11084-1}, author = {Nirwan, Neha and Itoh, Yuzuru and Singh, Pratima and Bandyopadhyay, Sutirtha and Vinothkumar, Kutti R and Amunts, Alexey and Saikrishnan, Kayarat} }