scholarly journals NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics

2021 ◽  
Vol 12 ◽  
Author(s):  
Priyesh Mohanty ◽  
Kiran Sankar Chatterjee ◽  
Ranabir Das
Keyword(s):  
Conformational Changes ◽  
Salt Bridge ◽  
Md Simulations ◽  
Enteric Bacteria ◽  
Covalent Attachment ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
Non Covalent Interactions ◽  
Ring E3 ◽  
Covalent Interactions

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter’s inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD’s catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αβ subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

scholarly journals Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

2018 ◽  
Author(s):  
Meng-Yin Li ◽  
Yi-Lun Ying ◽  
Xi-Xin Fu ◽  
Jie Yu ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Ionic Current ◽  
Md Simulations ◽  
Energy Spectra ◽  
Membrane Channels ◽  
Non Covalent Interactions ◽  
Protein Channels ◽  
Covalent Interactions ◽  
Molecule Interaction

Millions of years of evolution have produced membrane protein channels capable of efficiently moving ions across the cell membrane. The underlying fundamental mechanisms that facilitate these actions greatly contribute to the weak non-covalent interactions. However, uncovering these dynamic interactions and its synergic network effects still remains challenging in both experimental techniques and molecule dynamics (MD) simulations. Here, we present a rational strategy that combines MD simulations and frequency-energy spectroscopy to identify and quantify the role of non-covalent interactions in carrier transport through membrane protein channels, as encoded in traditional single channel recording or ionic current. We employed wild-type aerolysin transporting of methylcytosine and cytosine as a model to explore the dynamic ionic signatures with non-stationary and non-linear frequency analysis. Our data illuminate that methylcytosine experiences strong non-covalent interactions with the aerolysin nanopore at Region 1 around R220 than cytosine, which produces characteristic frequency-energy spectra. Furthermore, we experimentally validate the obtained hypothesis from frequency-energy spectra by designing single-site mutation of K238G which creates significantly enhanced non-covalent interactions for the recognition of methylcytosine. The frequency-energy spectrum of ions flowing inside membrane channels constitutes a single-molecule interaction spectrum, which bridges the gap between traditional ionic current recording and the MD simulations, facilitating the qualitative and quantitive description of the non-covalent interactions inside membrane channels.

scholarly journals The relation between intrinsic protein conformational changes and ligand binding

2020 ◽  
Author(s):  
Marijn de Boer
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Conformational Equilibrium ◽  
Biological Function ◽  
Free Protein ◽  
Classical Statistical Mechanics ◽  
Protein Conformational Changes ◽  
Ligand Free ◽  
Non Covalent Interactions ◽  
Covalent Interactions

1ABSTRACTStructural changes in proteins allow them to exist in several conformations. Non-covalent interactions with ligands drive the structural changes, thereby allowing the protein to perform its biological function. Recent findings suggest that many proteins are always in an equilibrium of different conformations and that each of these conformations can be formed by both the ligand-free and ligand-bound protein. By using classical statistical mechanics, we derived the equilibrium probabilities of forming a conformation with and without ligand. We found, under certain conditions, that increasing the probability of forming a conformation by the ligand-free protein also increases the probability of forming the same conformation when the protein has a ligand bound. Further, we found that changes in the conformational equilibrium of the ligand-free protein can increase or decrease the affinity for the ligand.

scholarly journals Columnar Aggregates of Azobenzene Stars: Exploring Intermolecular Interactions, Structure, and Stability in Atomistic Simulations

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7598
Author(s):  
Markus Koch ◽  
Marina Saphiannikova ◽  
Olga Guskova
Keyword(s):  
Hydrogen Bonds ◽  
Md Simulations ◽  
Binding Energies ◽  
Atomistic Simulations ◽  
Π Interactions ◽  
Different Types ◽  
Non Covalent Interactions ◽  
Experimental Works ◽  
Supramolecular Aggregates ◽  
Covalent Interactions

We present a simulation study of supramolecular aggregates formed by three-arm azobenzene (Azo) stars with a benzene-1,3,5-tricarboxamide (BTA) core in water. Previous experimental works by other research groups demonstrate that such Azo stars assemble into needle-like structures with light-responsive properties. Disregarding the response to light, we intend to characterize the equilibrium state of this system on the molecular scale. In particular, we aim to develop a thorough understanding of the binding mechanism between the molecules and analyze the structural properties of columnar stacks of Azo stars. Our study employs fully atomistic molecular dynamics (MD) simulations to model pre-assembled aggregates with various sizes and arrangements in water. In our detailed approach, we decompose the binding energies of the aggregates into the contributions due to the different types of non-covalent interactions and the contributions of the functional groups in the Azo stars. Initially, we investigate the origin and strength of the non-covalent interactions within a stacked dimer. Based on these findings, three arrangements of longer columnar stacks are prepared and equilibrated. We confirm that the binding energies of the stacks are mainly composed of π–π interactions between the conjugated parts of the molecules and hydrogen bonds formed between the stacked BTA cores. Our study quantifies the strength of these interactions and shows that the π–π interactions, especially between the Azo moieties, dominate the binding energies. We clarify that hydrogen bonds, which are predominant in BTA stacks, have only secondary energetic contributions in stacks of Azo stars but remain necessary stabilizers. Both types of interactions, π–π stacking and H-bonds, are required to maintain the columnar arrangement of the aggregates.

scholarly journals Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation

2019 ◽  
Author(s):  
Emma Branigan ◽  
J. Carlos Penedo ◽  
Ronald T. Hay
Keyword(s):  
Single Molecule ◽  
Active Form ◽  
Resonance Energy Transfer ◽  
E3 Ligase ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Closed Conformation ◽  
Ring E3 Ligase ◽  
Ring E3

AbstractUbiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate for catalysis by locking it into a “closed” conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.

scholarly journals Deamidation disrupts native and transient contacts to weaken the interaction between UBC13 and RING-finger E3 ligases

2019 ◽  
Author(s):  
Priyesh Mohanty ◽  
Rashmi ◽  
Batul Ismail Habibullah ◽  
Arun G S ◽  
Ranabir Das
Keyword(s):  
Shigella Flexneri ◽  
Ring Finger ◽  
Salt Bridge ◽  
Enteric Bacteria ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Ring Complex ◽  
Ubiquitin Conjugating Enzyme ◽  
Transient Interactions

AbstractThe deamidase OspI from enteric bacteriaShigella flexnerideamidates a glutamine residue in the host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate (Q100E). Consequently, its polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream NF-κB inflammatory response is inactivated. The precise role of deamidation in inactivating the UBC13/TRAF6 complex is unknown. We report that deamidation inhibits the interaction between UBC13 and TRAF6 RING-domain (TRAF6RING) by perturbing both the native and transient interactions. Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a critical intermolecular salt-bridge at the native UBC13/TRAF6RINGinterface. Moreover, the salt-bridge competition prevents transient interactions necessary to form a typical UBC13/RING complex. Repulsion between E100 and the negatively charged surface of RING also prevents transient interactions in the UBC13/RING complex. Our findings highlight a mechanism where a post-translational modification perturbs the conformation and stability of transient complexes to inhibit protein-protein association.

scholarly journals Conformational Transition Pathways in Major Facilitator Superfamily Transporters

2019 ◽  
Author(s):  
Dylan Ogden ◽  
Kalyan Immadisetty ◽  
Mahmoud Moradi
Keyword(s):  
Conformational Changes ◽  
Glucose Transporter ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Glucose Transporter 1 ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

scholarly journals Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

2018 ◽  
Author(s):  
Meng-Yin Li ◽  
Yi-Lun Ying ◽  
Xi-Xin Fu ◽  
Jie Yu ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Ionic Current ◽  
Md Simulations ◽  
Energy Spectra ◽  
Membrane Channels ◽  
Non Covalent Interactions ◽  
Protein Channels ◽  
Covalent Interactions ◽  
Molecule Interaction

Millions of years of evolution have produced membrane protein channels capable of efficiently moving ions across the cell membrane. The underlying fundamental mechanisms that facilitate these actions greatly contribute to the weak non-covalent interactions. However, uncovering these dynamic interactions and its synergic network effects still remains challenging in both experimental techniques and molecule dynamics (MD) simulations. Here, we present a rational strategy that combines MD simulations and frequency-energy spectroscopy to identify and quantify the role of non-covalent interactions in carrier transport through membrane protein channels, as encoded in traditional single channel recording or ionic current. We employed wild-type aerolysin transporting of methylcytosine and cytosine as a model to explore the dynamic ionic signatures with non-stationary and non-linear frequency analysis. Our data illuminate that methylcytosine experiences strong non-covalent interactions with the aerolysin nanopore at Region 1 around R220 than cytosine, which produces characteristic frequency-energy spectra. Furthermore, we experimentally validate the obtained hypothesis from frequency-energy spectra by designing single-site mutation of K238G which creates significantly enhanced non-covalent interactions for the recognition of methylcytosine. The frequency-energy spectrum of ions flowing inside membrane channels constitutes a single-molecule interaction spectrum, which bridges the gap between traditional ionic current recording and the MD simulations, facilitating the qualitative and quantitive description of the non-covalent interactions inside membrane channels.

Conformational changes of DNA induced by a trans-azobenzene derivative via non-covalent interactions

2018 ◽  
Vol 20 (35) ◽  
pp. 22645-22651 ◽  
Author(s):  
Hong Zhang ◽  
Haohao Fu ◽  
Xueguang Shao ◽  
Christophe Chipot ◽  
Antonio Monari ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Non Covalent Interactions ◽  
Covalent Interactions ◽  
Trans Azobenzene

The presence of only one interacting azobenzene is not sufficient to lead to a global conformational change from B- to A-DNA.

scholarly journals RIP-MD: a tool to study residue interaction networks in protein molecular dynamics

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5998 ◽  
Author(s):  
Sebastián Contreras-Riquelme ◽  
Jose-Antonio Garate ◽  
Tomas Perez-Acle ◽  
Alberto J.M. Martin
Keyword(s):  
Molecular Dynamics ◽  
Protein Structures ◽  
Md Simulations ◽  
Relevant Information ◽  
Interaction Networks ◽  
Salt Bridges ◽  
Residue Interaction ◽  
Non Covalent Interactions ◽  
Protein Molecular Dynamics ◽  
Covalent Interactions

Protein structure is not static; residues undergo conformational rearrangements and, in doing so, create, stabilize or break non-covalent interactions. Molecular dynamics (MD) is a technique used to simulate these movements with atomic resolution. However, given the data-intensive nature of the technique, gathering relevant information from MD simulations is a complex and time consuming process requiring several computational tools to perform these analyses. Among different approaches, the study of residue interaction networks (RINs) has proven to facilitate the study of protein structures. In a RIN, nodes represent amino-acid residues and the connections between them depict non-covalent interactions. Here, we describe residue interaction networks in protein molecular dynamics (RIP-MD), a visual molecular dynamics (VMD) plugin to facilitate the study of RINs using trajectories obtained from MD simulations of proteins. Our software generates RINs from MD trajectory files. The non-covalent interactions defined by RIP-MD include H-bonds, salt bridges, VdWs, cation-π, π–π, Arginine–Arginine, and Coulomb interactions. In addition, RIP-MD also computes interactions based on distances between Cαs and disulfide bridges. The results of the analysis are shown in an user friendly interface. Moreover, the user can take advantage of the VMD visualization capacities, whereby through some effortless steps, it is possible to select and visualize interactions described for a single, several or all residues in a MD trajectory. Network and descriptive table files are also generated, allowing their further study in other specialized platforms. Our method was written in python in a parallelized fashion. This characteristic allows the analysis of large systems impossible to handle otherwise. RIP-MD is available at http://www.dlab.cl/ripmd.

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