@article {6592, title = {Cryo-EM research in India.}, journal = {Structure}, volume = {32}, year = {2024}, month = {2024 Feb 01}, pages = {113-119}, abstract = {

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

}, keywords = {Cryoelectron Microscopy, India}, issn = {1878-4186}, doi = {10.1016/j.str.2024.01.002}, author = {Shukla, Arun K and Banerjee, Manidipa and Singh, Appu K and Penmatsa, Aravind and Dutta, Somnath and Anand, Ruchi and Sirajuddin, Minhajuddin and Srivastava, Shanti Swaroop} } @article {2423, title = {Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.}, journal = {Nat Cell Biol}, volume = {24}, year = {2022}, month = {2022 Feb}, pages = {253-267}, abstract = {

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

}, issn = {1476-4679}, doi = {10.1038/s41556-021-00825-4}, author = {Jijumon, A S and Bodakuntla, Satish and Genova, Mariya and Bangera, Mamata and Sackett, Violet and Besse, Laetitia and Maksut, Fatlinda and Henriot, Veronique and Magiera, Maria M and Sirajuddin, Minhajuddin and Janke, Carsten} } @article {3640, title = {Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants.}, journal = {J Biol Chem}, volume = {299}, year = {2022}, month = {2022 Nov 22}, pages = {102732}, abstract = {

The emergence of new escape mutants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-angiotensin-converting enzyme 2 interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif of the spike protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, using mCherry fluorescence-based reporter assay, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.

}, issn = {1083-351X}, doi = {10.1016/j.jbc.2022.102732}, author = {Mendon, Nivya and Ganie, Rayees A and Kesarwani, Shubham and Dileep, Drisya and Sasi, Sarika and Lama, Prakash and Chandra, Anchal and Sirajuddin, Minhajuddin} } @article {2148, title = {Genetically encoded live-cell sensor for tyrosinated microtubules.}, journal = {J Cell Biol}, volume = {219}, year = {2020}, month = {2020 Oct 05}, abstract = {

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

}, issn = {1540-8140}, doi = {10.1083/jcb.201912107}, author = {Kesarwani, Shubham and Lama, Prakash and Chandra, Anchal and Reddy, P Purushotam and Jijumon, A S and Bodakuntla, Satish and Rao, Balaji M and Janke, Carsten and Das, Ranabir and Sirajuddin, Minhajuddin} } @article {2106, title = {Structural insights into actin filament recognition by commonly used cellular actin markers.}, journal = {EMBO J}, volume = {39}, year = {2020}, month = {2020 Jul 15}, pages = {e104006}, 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.

}, issn = {1460-2075}, doi = {10.15252/embj.2019104006}, author = {Kumari, Archana and Kesarwani, Shubham and Javoor, Manjunath G and Vinothkumar, Kutti R and Sirajuddin, Minhajuddin} } @article {1187, title = {Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel.}, journal = {J Biol Chem}, year = {2018}, month = {2018 May 18}, abstract = {

The microtubule protein tubulin is a heterodimer comprising α/β subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight β-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic C-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin{\textquoteright}s role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Using recombinant yeast α/β-tubulin constructs with α-CTTs, β-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that either α-CTTs or β-CTTs block VDAC pore and that the efficiency of blockage by individual CTTs spans two orders of magnitude, depending on the CTT isotype. β-Tubulin constructs, notably β3, blocked VDAC most effectively. We quantitatively describe these experimental results using a physical model that accounts only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly depending on the isotype, thus suggesting an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.001569}, author = {Rostovtseva, Tatiana K and Gurnev, Philip A and Hoogerheide, David P and Rovini, Amandine and Sirajuddin, Minhajuddin and Bezrukov, Sergey M} }