T‐705‐modified ssRNA in complex with Lassa virus nucleoprotein exhibits nucleotide splaying and increased water influx into the RNA‐binding pocket

Background: Lassa virus (LV) infection is a endemic disease from West Africa posing threat to the entire world. A thorough understanding of the mechanistic workings of the genome products of LV may be key to developing drugs candidates for the treatment of LV infection. Methods: Molecular dynamics simulation has been used to provide insight into the mechanistic basis for total loss of ssRNA interaction in nucleoprotein (NP) K309A, partial loss in S247A, and no loss in S237A by following the hydrogen bond interaction, water influx into the ssRNA pocket and glycosidic torsion angle (?) of the ssRNA. Results: The results revealed that K309A mutation is associated with complete loss of salt-bridge interaction between lysine ?-amino and U4-O2P phosphodiester linkage but not in S237A where S247-OG atom played a redundant role. S247A is also associated with partial loss of hydrogen bond between OG atom of S247 and C5-O2P phosphodiester bond as T178-OG1 group seem to have a seemingly redundant interaction with C5-O2P. While S247A only is also associated with alteration of ? rotation in U6/C7, both K309A and S247 but not S237A is associated with increased water influx into the ssRNA binding pocket. Conclusion: K309A mutation may result in non-viable Lassa viron as loss of ssRNA interaction may negatively affect genome biochemistry, semi-viable Lassa viron in S247A mutation may be due to loss of 3D arrangement of ssRNA due to splayed out nucleotides.

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Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011922 ◽

2019 ◽

Vol 295 (6) ◽

pp. 1551-1564 ◽

Cited By ~ 5

Author(s):

Kelly E. Du Pont ◽

Russell B. Davidson ◽

Martin McCullagh ◽

Brian J. Geiss

Keyword(s):

Molecular Dynamics ◽

Structural Changes ◽

Rna Binding ◽

Atp Hydrolysis ◽

Nonstructural Protein ◽

Binding Pocket ◽

Energy Transduction ◽

Helicase Domain ◽

Genome Replication ◽

Turnover Rates

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

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Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317000031 ◽

2017 ◽

Vol 73 (4) ◽

pp. 294-315 ◽

Cited By ~ 12

Author(s):

Kimberly A. Stanek ◽

Jennifer Patterson-West ◽

Peter S. Randolph ◽

Cameron Mura

Keyword(s):

Rna Binding ◽

Binding Pocket ◽

Binding Mode ◽

Evolutionary Conservation ◽

Aquifex Aeolicus ◽

Binding Properties ◽

Bacterial Phyla ◽

Mrna Targets ◽

Small Regulatory Rnas ◽

And Function

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

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Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

10.1101/078733 ◽

2016 ◽

Author(s):

Kimberly A Stanek ◽

Jennifer P West ◽

Peter S Randolph ◽

Cameron Mura

Keyword(s):

Rna Binding ◽

Binding Pocket ◽

Binding Mode ◽

Evolutionary Conservation ◽

Thermophilic Bacterium ◽

Aquifex Aeolicus ◽

Binding Properties ◽

Bacterial Phyla ◽

Mrna Targets ◽

Small Regulatory Rnas

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-Å resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.AbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings, with at least two distinct surfaces that bind RNA. Recently, another binding site—dubbed the ‘lateral rim’—has been implicated in sRNA•mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 Å resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

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1H-NMR characterization of l-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis

Biochemical Journal ◽

10.1042/bj3150895 ◽

1996 ◽

Vol 315 (3) ◽

pp. 895-900 ◽

Cited By ~ 5

Author(s):

Vasudevan RAMESH ◽

Tom BROWN

Keyword(s):

Bacillus Subtilis ◽

Complex Formation ◽

1H Nmr ◽

Rna Binding ◽

Chemical Exchange ◽

Chemical Shifts ◽

Large Change ◽

Binding Pocket ◽

Nmr Study ◽

Nmr Characterization

A 1H-NMR study of the binding of L-tryptophan to the trp RNA-binding attenuation protein of Bacillus subtilis (TRAP), an ondecamer (91.6 kDa), has been implemented. The assignment of the aromatic indole ring proton resonances of the bound tryptophan ligand has been successfully carried out by two-dimensional chemical exchange experiments. The observation of only a single set of chemical shifts of the bound ligand demonstrates that the tryptophan binding site is identical in all the 11 subunits of the protein. Further, the large change in ligand chemical shifts suggests that the conformation of tryptophan ligand undergoes a significant rearrangement after complex formation with TRAP. This is further substantiated by the extensive ligand-induced chemical shift changes observed to the protein resonances and identification of several strong ligand–protein intermolecular nuclear Overhauser effects. A correlation of these preliminary NMR data with the X-ray crystal structure of the TRAP–tryptophan complex also suggests, tentatively, that the observed changes to the NMR spectra of the protein might correspond to changes associated with residues surrounding the tryptophan binding pocket owing to complex formation.

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Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)69073-8 ◽

1988 ◽

Vol 263 (7) ◽

pp. 3307-3313

Author(s):

B M Merrill ◽

K L Stone ◽

F Cobianchi ◽

S H Wilson ◽

K R Williams

Keyword(s):

Nucleic Acid ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Pocket ◽

Nucleic Acid Binding ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Form Part ◽

Nuclear Ribonucleoprotein

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Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin–antitoxin system

IUCrJ ◽

10.1107/s2052252521000452 ◽

2021 ◽

Vol 8 (3) ◽

Author(s):

Chenglong Jin ◽

Sung-Min Kang ◽

Do-Hee Kim ◽

Bong-Jin Lee

Keyword(s):

Klebsiella Pneumoniae ◽

Rna Binding ◽

Complex Structure ◽

Binding Pocket ◽

Ribonuclease Activity ◽

Bacterial Toxin ◽

Binding Modes ◽

Catalytic Core ◽

Physiological Processes ◽

Distinct Features

Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.

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Residues R192 and K225 in RNA-binding pocket of tobacco vein banding mosaic virus CP control virus cell-to-cell movement and replication

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-20-0265-r ◽

2021 ◽

Author(s):

Zhi-Yong Yan ◽

Xiao-Jie Xu ◽

Le Fang ◽

De-Jie Cheng ◽

Yan-Ping Tian ◽

...

Keyword(s):

Mosaic Virus ◽

Rna Binding ◽

Cell Movement ◽

Binding Pocket ◽

Binding Activity ◽

Watermelon Mosaic Virus ◽

Wild Type ◽

Three Dimensional Model ◽

Mobility Shift ◽

Virus Particles

Potyviruses move to neighboring cells in the form of virus particles or coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic potyvirus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions of 192 and 225 (R192 and K225) either with alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP-RNA binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.

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