TY - JOUR T1 - Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants. JF - J Biol Chem Y1 - 2022 A1 - Mendon, Nivya A1 - Ganie, Rayees A A1 - Kesarwani, Shubham A1 - Dileep, Drisya A1 - Sasi, Sarika A1 - Lama, Prakash A1 - Chandra, Anchal A1 - Sirajuddin, Minhajuddin AB -

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.

VL - 299 IS - 1 ER - TY - JOUR T1 - Biophysical properties of the isolated spike protein binding helix of human ACE2. JF - Biophys J Y1 - 2021 A1 - Das, Anirban A1 - Vishvakarma, Vicky A1 - Dey, Arpan A1 - Dey, Simli A1 - Gupta, Ankur A1 - Das, Mitradip A1 - Vishwakarma, Krishna Kant A1 - Roy, Debsankar Saha A1 - Yadav, Swati A1 - Kesarwani, Shubham A1 - Venkatramani, Ravindra A1 - Maiti, Sudipta KW - Angiotensin-Converting Enzyme 2 KW - COVID-19 KW - Humans KW - Peptidyl-Dipeptidase A KW - Protein Binding KW - SARS-CoV-2 KW - Spike Glycoprotein, Coronavirus AB -

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel β-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

VL - 120 IS - 14 ER - TY - JOUR T1 - Genetically encoded live-cell sensor for tyrosinated microtubules. JF - J Cell Biol Y1 - 2020 A1 - Kesarwani, Shubham A1 - Lama, Prakash A1 - Chandra, Anchal A1 - Reddy, P Purushotam A1 - Jijumon, A S A1 - Bodakuntla, Satish A1 - Rao, Balaji M A1 - Janke, Carsten A1 - Das, Ranabir A1 - Sirajuddin, Minhajuddin AB -

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.

VL - 219 IS - 10 ER - TY - JOUR T1 - Structural insights into actin filament recognition by commonly used cellular actin markers. JF - EMBO J Y1 - 2020 A1 - Kumari, Archana A1 - Kesarwani, Shubham A1 - Javoor, Manjunath G A1 - Vinothkumar, Kutti R A1 - Sirajuddin, Minhajuddin AB -

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.

VL - 39 IS - 14 ER -