scholarly journals Local-to-global signal transduction at the core of the Mn2+ sensing riboswitch

2019 ◽  
Author(s):  
Krishna C Suddala ◽  
Ian R Price ◽  
Michal Janeček ◽  
Petra Kührová ◽  
Shiba Dandpat ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Metal Ions ◽  
Single Molecule ◽  
Md Simulations ◽  
Xanthomonas Oryzae ◽  
Manganese Ion ◽  
The Core ◽  
Ion Sensing ◽  
Single Molecule Fret ◽  
Global Signal

The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 μs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

scholarly journals Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Krishna C. Suddala ◽  
Ian R. Price ◽  
Shiba S. Dandpat ◽  
Michal Janeček ◽  
Petra Kührová ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Metal Ions ◽  
Single Molecule ◽  
Md Simulations ◽  
Xanthomonas Oryzae ◽  
Rna Dynamics ◽  
The Core ◽  
Single Molecule Fret ◽  
Global Signal ◽  
Structural Perturbations

Abstract The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

scholarly journals Transport mechanism of a glutamate transporter homologue GltPh

2016 ◽  
Vol 44 (3) ◽  
pp. 898-904 ◽  
Author(s):  
Yurui Ji ◽  
Vincent L.G. Postis ◽  
Yingying Wang ◽  
Mark Bartlam ◽  
Adrian Goldman
Keyword(s):  
Single Molecule ◽  
Transport Mechanism ◽  
Glutamate Transporter ◽  
Md Simulations ◽  
Research Progress ◽  
Pyrococcus Horikoshii ◽  
Single Molecule Fret ◽  
Neurotransmitter Glutamate ◽  
Central Nervous Systems ◽  
Recent Research Progress

Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter.

scholarly journals The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

2019 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Md Simulations ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Single Molecule Fret ◽  
Allosteric Mechanism ◽  
Specific Transport ◽  
Slc6 Family ◽  
The Stability

AbstractNeurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

scholarly journals Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

2021 ◽  
Author(s):  
Steffen Wolf ◽  
Benedikt Sohmen ◽  
Björn Hellenkamp ◽  
Johann Thurn ◽  
Gerhard Stock ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Md Simulations ◽  
Large Protein ◽  
Single Molecule Fret ◽  
Fluorescence Methods ◽  
Dynamics Simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.

scholarly journals Prokaryotic Argonaute From Archaeoglobus Fulgidus Interacts With DNA as a Homodimer

2020 ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Acid Interaction ◽  
Action Mechanism ◽  
Piwi Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Domains Of Life ◽  
And Function

Abstract Argonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

scholarly journals Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

2019 ◽  
Author(s):  
Antonio N. Calabrese ◽  
Bob Schiffrin ◽  
Matthew Watson ◽  
Theodoros K. Karamanos ◽  
Martin Walko ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outer Membrane Protein ◽  
Conformational Dynamics ◽  
Core Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Dynamics Simulations

AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

scholarly journals Dynamics of Histone H3 Tails in Mononucleosomes Studied by Single-Molecule FRET and MD Simulations

2018 ◽  
Vol 114 (3) ◽  
pp. 256a-257a
Author(s):  
Kathrin Lehmann ◽  
Ruihan Zhang ◽  
Suren Felekyan ◽  
Ralf Kühnemuth ◽  
Katalin Toth
Keyword(s):  
Single Molecule ◽  
Histone H3 ◽  
Md Simulations ◽  
Single Molecule Fret

scholarly journals Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Acid Interaction ◽  
Action Mechanism ◽  
Piwi Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Domains Of Life ◽  
And Function

AbstractArgonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

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