20 research outputs found
The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches
Ligand
specificity is an essential requirement for all riboswitches.
Some variant riboswitches utilize a common structural motif, yet through
subtle sequence differences, they are able to selectively respond
to different small molecule ligands and regulate downstream gene expression.
These variants discriminate between structurally and chemically similar
ligands. Crystal structures provide insight into how specificity is
achieved. However, ligand specificity cannot always be explained solely
by nucleotides in direct contact with the ligand. The cyclic dinucleotide
variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP
riboswitches, that were distinguished based on the identity of a single
nucleotide in contact with the ligand. Here we report a variant riboswitch
with a mutation at a second ligand-contacting position that is promiscuous
for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference
for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT,
was used to quantitatively assess thousands of sites in the first-
and second-shells of ligand contact for impacts on ligand specificity
and promiscuity. In addition to nucleotides in direct ligand contact,
nucleotides more distal from the binding site, within the J1/2 linker
and the terminator helix, were identified that impact ligand specificity.
These findings provide an example of how nucleotides outside the ligand
binding pocket influence the riboswitch specificity. Moreover, these
distal nucleotides could be used to predict promiscuous sequences
The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches
Ligand
specificity is an essential requirement for all riboswitches.
Some variant riboswitches utilize a common structural motif, yet through
subtle sequence differences, they are able to selectively respond
to different small molecule ligands and regulate downstream gene expression.
These variants discriminate between structurally and chemically similar
ligands. Crystal structures provide insight into how specificity is
achieved. However, ligand specificity cannot always be explained solely
by nucleotides in direct contact with the ligand. The cyclic dinucleotide
variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP
riboswitches, that were distinguished based on the identity of a single
nucleotide in contact with the ligand. Here we report a variant riboswitch
with a mutation at a second ligand-contacting position that is promiscuous
for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference
for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT,
was used to quantitatively assess thousands of sites in the first-
and second-shells of ligand contact for impacts on ligand specificity
and promiscuity. In addition to nucleotides in direct ligand contact,
nucleotides more distal from the binding site, within the J1/2 linker
and the terminator helix, were identified that impact ligand specificity.
These findings provide an example of how nucleotides outside the ligand
binding pocket influence the riboswitch specificity. Moreover, these
distal nucleotides could be used to predict promiscuous sequences
The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches
Ligand
specificity is an essential requirement for all riboswitches.
Some variant riboswitches utilize a common structural motif, yet through
subtle sequence differences, they are able to selectively respond
to different small molecule ligands and regulate downstream gene expression.
These variants discriminate between structurally and chemically similar
ligands. Crystal structures provide insight into how specificity is
achieved. However, ligand specificity cannot always be explained solely
by nucleotides in direct contact with the ligand. The cyclic dinucleotide
variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP
riboswitches, that were distinguished based on the identity of a single
nucleotide in contact with the ligand. Here we report a variant riboswitch
with a mutation at a second ligand-contacting position that is promiscuous
for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference
for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT,
was used to quantitatively assess thousands of sites in the first-
and second-shells of ligand contact for impacts on ligand specificity
and promiscuity. In addition to nucleotides in direct ligand contact,
nucleotides more distal from the binding site, within the J1/2 linker
and the terminator helix, were identified that impact ligand specificity.
These findings provide an example of how nucleotides outside the ligand
binding pocket influence the riboswitch specificity. Moreover, these
distal nucleotides could be used to predict promiscuous sequences
Transition State Charge Stabilization and Acid–Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE
The bacterial toxin RelE is a ribosome-dependent
endoribonuclease.
It is part of a type II toxin–antitoxin system that contributes
to antibiotic resistance and biofilm formation. During amino acid
starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting
protein translation. RelE is structurally similar to microbial RNases
that employ general acid–base catalysis to facilitate RNA cleavage.
The RelE active site is atypical for acid–base catalysis, in
that it is enriched with positively charged residues and lacks the
prototypical histidine-glutamate catalytic pair, making the mechanism
of mRNA cleavage unclear. In this study, we use a single-turnover
kinetic analysis to measure the effect of pH and phosphorothioate
substitution on the rate constant for cleavage of mRNA by wild-type
RelE and seven active-site mutants. Mutation and thio effects indicate
a major role for stabilization of increased negative change in the
transition state by arginine 61. The wild-type RelE cleavage rate
constant is pH-independent, but the reaction catalyzed by many of
the mutants is strongly dependent on pH, suggestive of general acid–base
catalysis. pH–rate curves indicate that wild-type RelE operates
with the p<i>K</i><sub>a</sub> of at least one catalytic
residue significantly downshifted by the local environment. Mutation
of any single active-site residue is sufficient to disrupt this microenvironment
and revert the shifted p<i>K</i><sub>a</sub> back above
neutrality. pH–rate curves are consistent with K54 functioning
as a general base and R81 as a general acid. The capacity of RelE
to effect a large p<i>K</i><sub>a</sub> shift and facilitate
a common catalytic mechanism by uncommon means furthers our understanding
of other atypical enzymatic active sites
Bacterial Toxin RelE: A Highly Efficient Ribonuclease with Exquisite Substrate Specificity Using Atypical Catalytic Residues
The toxin RelE is a ribosome-dependent
endoribonuclease implicated
in diverse cellular processes, including persistence. During amino
acid starvation, RelE inhibits translation by cleaving ribosomal A-site
mRNA. Although RelE is structurally similar to other microbial endoribonucleases,
the active-site amino acid composition differs substantially and lacks
obvious candidates for general acid–base functionality. Highly
conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround
the mRNA scissile phosphate, and specific 16S rRNA contacts further
contribute to substrate positioning. We used a single-turnover kinetic
assay to evaluate the catalytic importance of individual residues
in the RelE active site. Within the context of the ribosome, RelE
rapidly cleaves A-site mRNA at a rate similar to those of traditional
ribonucleases. Single-turnover rate constants decreased between 10<sup>2</sup>- and 10<sup>6</sup>-fold for the RelE active-site mutants
of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis
via transition-state charge stabilization and leaving-group protonation,
in addition to achieving in-line substrate positioning in cooperation
with the ribosome. This kinetic analysis complements structural information
to provide a foundation for understanding the molecular mechanism
of this atypical endoribonuclease
Structural and Biochemical Characterization of Linear Dinucleotide Analogues Bound to the c-di-GMP-I Aptamer
The cyclic dinucleotide c-di-GMP regulates lifestyle
transitions in many bacteria, such as the change from a free motile
state to a biofilm-forming community. Riboswitches that bind this
second messenger are important downstream targets in this bacterial
signaling pathway. The breakdown of c-di-GMP in the cell is accomplished
enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding
riboswitches must be able to discriminate between their cognate cyclic
ligand and linear dinucleotides in order to be selective biological
switches. It has been reported that the c-di-GMP-I riboswitch binds
c-di-GMP 5 orders of magnitude better than the linear pGpG, but the
cause of this large energetic difference in binding is unknown. Here
we report binding data and crystal structures of several linear c-di-GMP
analogues in complex with the c-di-GMP-I riboswitch. These data reveal
the parameters for phosphate recognition and the structural basis
of linear dinucleotide binding to the riboswitch. Additionally, the
pH dependence of binding shows that exclusion of pGpG is not due to
the additional negative charge on the ligand. These data reveal principles
that, along with published work, will contribute to the design of
c-di-GMP analogues with properties desirable for use as chemical tools
and potential therapeutics
Nuclease-Resistant c‑di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors
The
ability of bacteria to sense environmental cues and adapt is
essential for their survival. The use of second-messenger signaling
molecules to translate these cues into a physiological response is
a common mechanism employed by bacteria. The second messenger 3′–5′-cyclic
diadenosine monophosphate (c-di-AMP) has been linked to a diverse
set of biological processes involved in maintaining cell viability
and homeostasis, as well as pathogenicity. A complex network of both
protein and RNA receptors inside the cell activates specific pathways
and mediates phenotypic outputs in response to c-di-AMP. Structural
analysis of these RNA and protein receptors has revealed the different
recognition elements employed by these effectors to bind the same
small molecule. Herein, using a series of c-di-AMP analogues, we probed
the interactions made with a riboswitch and a phosphodiesterase protein
to identify the features important for c-di-AMP binding and recognition.
We found that the <i>ydaO</i> riboswitch binds c-di-AMP
in two discrete sites with near identical affinity and a Hill coefficient
of 1.6. The <i>ydaO</i> riboswitch distinguishes between
c-di-AMP and structurally related second messengers by discriminating
against an amine at the C2 position more than a carbonyl at the C6
position. We also identified phosphate-modified analogues that bind
both the <i>ydaO</i> RNA and GdpP protein with high affinity,
whereas symmetrically modified ribose analogues exhibited a substantial
decrease in ydaO affinity but retained high affinity for GdpP. These
ligand modifications resulted in increased resistance to enzyme-catalyzed
hydrolysis by the GdpP enzyme. Together, these data suggest that these
c-di-AMP analogues could be useful as chemical tools to specifically
target subsections of second-messenger signaling pathways
Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore
<div><p>The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F<sup>-</sup>), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a ‘double-barreled’ channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant <i>Arabidopsis thaliana</i> and an animal <i>Amphimedon queenslandica</i>, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast <i>Saccharomyces cerevisiae</i>. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are ‘single-barreled’ transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.</p></div
Growth rescue of <i>fex1Δfex2Δ</i> yeast expressing previously uncharacterized FEX-like genes.
<p>(A) The strains were grown on YPD and YPD containing 5 mM NaF to determine rescue phenotype. Plant <i>FEX-At</i> is a putative FEX gene from <i>A</i>. <i>thaliana</i> cloned into the p426GPD vector. Animal <i>FEX-Aq</i> is a putative FEX gene from <i>A</i>. <i>queenslandica</i> cloned into the p426GPD vector. Yeast rescue plasmid (pRS416-<i>FEX1</i>) was used as a positive control and empty vector (p426GPD) was used as a negative control. (B) Quantification of the growth tolerance to NaF of yeast strains described in A.</p
Model of Fex1p from <i>S</i>. <i>cerevisiae</i>.
<p>Comparison of the residues in two pores for Fex1p-<i>Sc</i> (red residues are in N-terminal domain and blue are in C-terminal domain) and Fluc-<i>Bp</i> (white). Residues in Pore II and not Pore I of FEX are similar to bacterial Fluc.</p