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

2019 ◽  
Vol 116 (32) ◽  
pp. 15947-15956 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Specific Transport ◽  
Slc6 Family ◽  
Transport Of Ions ◽  
The Stability ◽  
Dynamics Simulations

Neurotransmitter:sodium symporters (NSSs) 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. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

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 Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Membrane Trafficking ◽  
Cushing’S Disease ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Cushing's Disease ◽  
Binding Motif ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractUbiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations.

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 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 Targeting G-quadruplex Forming Sequences with Cas9

2020 ◽  
Author(s):  
Hamza Balci ◽  
Viktorija Globyte ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Structural Characteristics ◽  
Structural Heterogeneity ◽  
Loop Formation ◽  
Conformational States ◽  
Single Molecule Fret ◽  
G Quadruplex ◽  
The Stability ◽  
The Impact

ABSTRACTClustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into non-canonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of CRISPR-Cas9 complex would be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR-Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the non-target strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson-Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.

scholarly journals Molecular simulations reveal the impact of RAMP1 on ligand binding and dynamics of CGRPR heterodimer

2021 ◽  
Author(s):  
Busecan Aksoydan ◽  
Serdar Durdagi
Keyword(s):  
Ligand Binding ◽  
Pain Perception ◽  
Md Simulations ◽  
Binding Pocket ◽  
Binding Modes ◽  
Cgrp Receptor ◽  
Tm Domain ◽  
The Stability ◽  
Global And Local ◽  
The Impact

The release of the neuropeptide of calcitonin gene-related peptide (CGRP) plays a key role in the mechanisms of migraine pathology and pain perception as it causes vasodilatation, neurogenic inflammation, mast cell degranulation, sensory signal activation and peripheral sensitivity. Although the findings on the effectiveness of CGRP-targeted therapies in migraine provide new information about the pathophysiology of migraine, questions remain on how the CGRP mechanisms fit into the overall migraine theory. The cryo-EM structure of Gs-protein complexed human CGRP receptor (CGRPR) with bound endogenous CGRP neuropeptide paved the way of understanding the insights into the CGRP receptor function. With several molecular modeling approaches, molecular dynamics (MD) simulations and post-MD analyzes, we aimed to investigate the importance of RAMP1 in the stability of calcitonin receptor-like receptor (CLR). Moreover, we compared the binding modes of the CGRP neuropeptide and CGRPR antagonists (i.e., telcagepant and rimegepant) within the presence or absence of RAMP1. We also investigated the global and local effects of bound molecules on CGRPR as well as their effects on the CLR-RAMP1 interaction interfaces. Results showed that although these molecules stay stable at the ectodomain binding site, they can also bind to the orthosteric ligand binding pocket and form the crucial interactions occurred in the CGRP agonism, which may be interpreted as non-specificity of the ligands, however, most of these interactions at orthosteric site are not sustainable or weak. Particularly, RAMP1 may also be important for the stability of TM domain of CLR hereby stabilizing the orthosteric binding pocket.

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 The molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Membrane Trafficking ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Binding Motif ◽  
Deubiquitinating Enzyme ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins, and mutations of the 14-3-3 binding motif in USP8 are related to Cushing′s disease. However, the molecular basis of USP8 enzyme activity regulation remains unclear. Here, we identified amino acids 645–684 of USP8 as an autoinhibitory region, which our pull-down and single-molecule FRET assay results suggested interacts with the catalytic USP domain. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 was found to inhibit USP8 enzyme activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing′s disease caused by USP8 mutations.

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