10 research outputs found
Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics
Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics
The
backbone dihedral parameters of the Amber RNA force field were
improved by fitting using multiple linear regression to potential
energies determined by quantum chemistry calculations. Five backbone
and four glycosidic dihedral parameters were fit simultaneously to
reproduce the potential energies determined by a high-level density
functional theory calculation (B97D3 functional with the AUG-CC-PVTZ
basis set). Umbrella sampling was used to determine conformational
free energies along the dihedral angles, and these better agree with
the population of conformations observed in the protein data bank
for the new parameters than for the conventional parameters. Molecular
dynamics simulations performed on a set of hairpin loops, duplexes
and tetramers with the new parameter set show improved modeling for
the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal
loop of noncanonical pairs, as compared to the conventional parameters.
For the tetramers, the new parameters largely avoid the incorrect
intercalated structures that dominate the conformational samples from
the conventional parameters. For the internal loop, the major conformation
solved by NMR is stable with the new parameters, but not with the
conventional parameters. The new force field performs similarly to
the conventional parameters for the UUCG and GCAA hairpin loops and
the [U(UA)<sub>6</sub>A]<sub>2</sub> duplex
Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics
The
backbone dihedral parameters of the Amber RNA force field were
improved by fitting using multiple linear regression to potential
energies determined by quantum chemistry calculations. Five backbone
and four glycosidic dihedral parameters were fit simultaneously to
reproduce the potential energies determined by a high-level density
functional theory calculation (B97D3 functional with the AUG-CC-PVTZ
basis set). Umbrella sampling was used to determine conformational
free energies along the dihedral angles, and these better agree with
the population of conformations observed in the protein data bank
for the new parameters than for the conventional parameters. Molecular
dynamics simulations performed on a set of hairpin loops, duplexes
and tetramers with the new parameter set show improved modeling for
the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal
loop of noncanonical pairs, as compared to the conventional parameters.
For the tetramers, the new parameters largely avoid the incorrect
intercalated structures that dominate the conformational samples from
the conventional parameters. For the internal loop, the major conformation
solved by NMR is stable with the new parameters, but not with the
conventional parameters. The new force field performs similarly to
the conventional parameters for the UUCG and GCAA hairpin loops and
the [U(UA)<sub>6</sub>A]<sub>2</sub> duplex
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Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.
PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs
Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.
PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs
SARS-CoV-2-specific immune responses in boosted vaccine recipients with breakthrough infections during the Omicron variant surge
BACKGROUND. Breakthrough SARS-CoV-2 infections in vaccinated individuals have been previously associated with suboptimal humoral immunity. However, less is known about breakthrough infections with the Omicron variant. METHODS. We analyzed SARS-CoV-2???specific antibody and cellular responses in healthy vaccine recipients who experienced breakthrough infections a median of 50 days after receiving a booster mRNA vaccine with an ACE2 binding inhibition assay and an ELISpot assay, respectively. RESULTS. We found that high levels of antibodies inhibited vaccine strain spike protein binding to ACE2 but that lower levels inhibited Omicron variant spike protein binding to ACE2 in 4 boosted vaccine recipients prior to infection. The levels of antibodies that inhibited vaccine strain and Omicron spike protein binding after breakthrough in 18 boosted vaccine recipients were similar to levels seen in COVID-19???negative boosted vaccine recipients. In contrast, boosted vaccine recipients had significantly stronger T cell responses to both vaccine strain and Omicron variant spike proteins at the time of breakthrough. CONCLUSION. Our data suggest that breakthrough infections with the Omicron variant can occur despite robust immune responses to the vaccine strain spike protein. FUNDING. This work was supported by the Johns Hopkins COVID-19 Vaccine-related Research Fund and by funds from the National Institute of Allergy and Infectious Disease intramural program as well as awards from the National Cancer Institute (U54CA260491) and the National Institutes of Allergy and Infectious Disease (K08AI156021 and U01AI138897)