scholarly journals Mechanism of duplex unwinding by coronavirus nsp13 helicases

2020 ◽  
Author(s):  
Xiao Hu ◽  
Wei Hao ◽  
Bo Qin ◽  
Zhiqi Tian ◽  
Ziheng Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Hinge Region ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
List Type ◽  
Single Molecule Fret ◽  
The Difference ◽  
Dna Complex ◽  
Loaded Mechanism

AbstractThe current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family.When dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed.An unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding.Comparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

scholarly journals Monoclonal Antibodies Detect a Single Amino Acid Difference Between the Coat Proteins of Soilborne Wheat Mosaic Virus Isolates: Implications for Virus Structure

1997 ◽  
Vol 87 (3) ◽  
pp. 295-301 ◽  
Author(s):  
Jianping Chen ◽  
Lesley Torrance ◽  
Graham H. Cowan ◽  
Stuart A. MacFarlane ◽  
Gerald Stubbs ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Mosaic Virus ◽  
Single Amino Acid ◽  
Computer Assisted ◽  
Amino Acid Difference ◽  
Coat Proteins ◽  
C Terminus ◽  
Readthrough Protein

Four monoclonal antibodies (MAbs) were prepared against an isolate of soilborne wheat mosaic furovirus from Oklahoma (SBWMV Okl-7). Three MAbs had different reactivities in tests on SBWMV isolates from Nebraska (Lab1), France, and Japan. One MAb (SCR 133) also reacted with oat golden stripe furovirus. None of the MAbs cross-reacted with other rod-shaped viruses including beet necrotic yellow vein furovirus, potato mop-top furovirus, and tobacco rattle tobravirus. Sequence analysis of nucleotides between 334 and 1,000 of RNA 2, the region that encodes the coat protein (CP) and the first 44 amino acids of a readthrough protein, of the four SBWMV isolates revealed up to 27 base changes from the published sequence of a Nebraska field isolate of SBWMV. Most changes were translationally silent, but some caused differences of one to three amino acids in residues located near either the N- or C-terminus of the CPs of the different isolates. Two further single amino acid changes were found at the beginning of the readthrough domain of the CP-readthrough protein. Some of these amino acid changes could be discriminated by MAbs SCR 132, SCR 133, and SCR 134. Peptide scanning (Pepscan) analysis indicated that the epitope recognized by SCR 134 is located near the N-terminus of the CP. SCR 132 was deduced to react with a discontinuous CP epitope near the C-terminus, and SCR 133 reacted with a surface-located continuous epitope also near the C-terminus. Predictions of CP structure from computer-assisted three-dimensional model building, by comparison with the X-ray fiber diffraction structure of tobacco mosaic virus, suggested that the three CP amino acids found to differ between isolates of SBWMV were located near the viral surface and were in regions predicted to be antigenic.

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Single Amino Acid Substitutions in the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Determine Viral Entry and Immunogenicity of a Major Neutralizing Domain

2005 ◽  
Vol 79 (18) ◽  
pp. 11638-11646 ◽  
Author(s):  
Christopher E. Yi ◽  
Lei Ba ◽  
Linqi Zhang ◽  
David D. Ho ◽  
Zhiwei Chen
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Antiviral Agents ◽  
Single Amino Acid ◽  
Major Mechanism ◽  
The Difference

ABSTRACT Neutralizing antibodies (NAbs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) spike (S) glycoprotein confer protection to animals experimentally infected with the pathogenic virus. We and others previously demonstrated that a major mechanism for neutralizing SARS-CoV was through blocking the interaction between the S glycoprotein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). In this study, we used in vivo electroporation DNA immunization and a pseudovirus-based assay to functionally evaluate immunogenicity and viral entry. We characterized the neutralization and viral entry determinants within the ACE2-binding domain of the S glycoprotein. The deletion of a positively charged region SΔ(422-463) abolished the capacity of the S glycoprotein to induce NAbs in mice vaccinated by in vivo DNA electroporation. Moreover, the SΔ(422-463) pseudovirus was unable to infect HEK293T-ACE2 cells. To determine the specific residues that contribute to related phenotypes, we replaced eight basic amino acids with alanine. We found that a single amino acid substitution (R441A) in the full-length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated. However, another substitution (R453A) abolished viral entry while retaining the capacity for inducing NAbs. The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical. Our findings provide direct evidence that these basic residues are essential for immunogenicity of the major neutralizing domain and for viral entry. Our data have implications for the rational design of vaccine and antiviral agents as well as for understanding viral tropism.

scholarly journals A Single Amino Acid Difference Distinguishes Resistant and Susceptible Alleles of the Rice Blast Resistance Gene Pi-ta

2000 ◽  
Vol 12 (11) ◽  
pp. 2033-2045 ◽  
Author(s):  
Gregory T. Bryan ◽  
Kun-Sheng Wu ◽  
Leonard Farrall ◽  
Yulin Jia ◽  
Howard P. Hershey ◽  
...  
Keyword(s):  
Amino Acid ◽  
Resistance Gene ◽  
Rice Blast ◽  
Blast Resistance ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Rice Blast Resistance ◽  
Blast Resistance Gene
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