@article {2375, title = {Biophysical properties of the isolated spike protein binding helix of human ACE2.}, journal = {Biophys J}, volume = {120}, year = {2021}, month = {2021 07 20}, pages = {2785-2792}, abstract = {

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.

}, keywords = {Angiotensin-Converting Enzyme 2, COVID-19, Humans, Peptidyl-Dipeptidase A, Protein Binding, SARS-CoV-2, Spike Glycoprotein, Coronavirus}, issn = {1542-0086}, doi = {10.1016/j.bpj.2021.06.017}, author = {Das, Anirban and Vishvakarma, Vicky and Dey, Arpan and Dey, Simli and Gupta, Ankur and Das, Mitradip and Vishwakarma, Krishna Kant and Roy, Debsankar Saha and Yadav, Swati and Kesarwani, Shubham and Venkatramani, Ravindra and Maiti, Sudipta} } @article {2290, title = {Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.}, journal = {PLoS Pathog}, volume = {17}, year = {2021}, month = {2021 07}, pages = {e1009706}, abstract = {

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

}, keywords = {Ammonium Chloride, Angiotensin-Converting Enzyme 2, Animals, Antiviral Agents, Cell Line, Chlorocebus aethiops, Chloroquine, Clathrin, COVID-19, Drug Synergism, Endocytosis, Endosomes, Humans, Hydrogen-Ion Concentration, Hydroxychloroquine, Macrolides, Niclosamide, Protein Binding, Protein Domains, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vero Cells, Virus Internalization}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1009706}, author = {Prabhakara, Chaitra and Godbole, Rashmi and Sil, Parijat and Jahnavi, Sowmya and Gulzar, Shah-E-Jahan and van Zanten, Thomas S and Sheth, Dhruv and Subhash, Neeraja and Chandra, Anchal and Shivaraj, Akshatha and Panikulam, Patricia and U, Ibrahim and Nuthakki, Vijay Kumar and Puthiyapurayil, Theja Parassini and Ahmed, Riyaz and Najar, Ashaq Hussain and Lingamallu, Sai Manoz and Das, Snigdhadev and Mahajan, Bhagyashri and Vemula, Praveen and Bharate, Sandip B and Singh, Parvinder Pal and Vishwakarma, Ram and Guha, Arjun and Sundaramurthy, Varadharajan and Mayor, Satyajit} } @article {1185, title = {Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum.}, journal = {Acta Crystallogr F Struct Biol Commun}, volume = {73}, year = {2017}, month = {2017 Jun 01}, pages = {356-362}, abstract = {

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5{\textquoteright}-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 {\r A} resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

}, keywords = {Adenosine Triphosphate, Amino Acid Sequence, Bacterial Proteins, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, Fusobacterium nucleatum, Gene Expression, Genetic Vectors, Hexosamines, Models, Molecular, Phosphotransferases (Alcohol Group Acceptor), Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity}, issn = {2053-230X}, doi = {10.1107/S2053230X17007439}, author = {Caing-Carlsson, Rhawnie and Goyal, Parveen and Sharma, Amit and Ghosh, Swagatha and Setty, Thanuja Gangi and North, Rachel A and Friemann, Rosmarie and Ramaswamy, S} } @article {1204, title = {Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 07 18}, pages = {3632-3646}, abstract = {

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5{\textquoteright}-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2{\textquoteright}-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is \~{}1.3 {\r A} from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

}, keywords = {2,2{\textquoteright}-Dipyridyl, Adenosine Diphosphate Ribose, Alcohol Dehydrogenase, Animals, Catalytic Domain, Crystallography, X-Ray, Formamides, Horses, Kinetics, Liver, Models, Molecular, NAD, Phenanthrolines, Protein Binding, Protein Conformation, Water, Zinc}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00446}, author = {Plapp, Bryce V and Savarimuthu, Baskar Raj and Ferraro, Daniel J and Rubach, Jon K and Brown, Eric N and Ramaswamy, S} } @article {616, title = {Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.}, journal = {Arch Biochem Biophys}, volume = {591}, year = {2016}, month = {2016 Feb 1}, pages = {35-42}, abstract = {

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 {\r A} where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 {\r A} to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 {\r A} from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

}, keywords = {Alcohol Dehydrogenase, Binding Sites, Catalysis, Coenzymes, Computer Simulation, Enzyme Activation, Models, Chemical, Models, Molecular, NAD, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Substrate Specificity, Trifluoroethanol}, issn = {1096-0384}, doi = {10.1016/j.abb.2015.12.009}, author = {Plapp, Bryce V and Charlier, Henry A and Ramaswamy, S} } @article {2114, title = {Metal substitutions incarbonic anhydrase: a halide ion probe study.}, journal = {Biochem Biophys Res Commun}, volume = {66}, year = {1975}, month = {1975 Oct 27}, pages = {1281-6}, keywords = {Animals, Binding Sites, Cadmium, Carbonic Anhydrases, Cattle, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Mercury, Protein Binding, Protein Conformation, Zinc}, issn = {0006-291X}, doi = {10.1016/0006-291x(75)90498-2}, author = {Smith, R J and Bryant, R G} }