scholarly journals Mechanistic basis for ubiquitin modulation of a protein energy landscape

2021 ◽  
Vol 118 (12) ◽  
pp. e2025126118
Author(s):  
Emma C. Carroll ◽  
Naomi R. Latorraca ◽  
Johanna M. Lindner ◽  
Brendan C. Maguire ◽  
Jeffrey G. Pelton ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
26S Proteasome ◽  
Dynamics Simulation ◽  
Structural Motif ◽  
Local Context ◽  
Exchange Mass Spectrometry ◽  
Substrate Protein ◽  
Protein Energy ◽  
C Terminus ◽  
Mechanistic Basis

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

scholarly journals Mechanistic basis for ubiquitin modulation of a protein energy landscape

2020 ◽  
Author(s):  
Emma C. Carroll ◽  
Naomi R. Latorraca ◽  
Johanna M. Lindner ◽  
Brendan C. Maguire ◽  
Jeff G. Pelton ◽  
...  
Keyword(s):  
Quality Control ◽  
Posttranslational Modification ◽  
Energy Landscape ◽  
26S Proteasome ◽  
Dynamics Simulation ◽  
Structural Motif ◽  
Protein Energy ◽  
C Terminus ◽  
Model Protein ◽  
Mechanistic Basis

AbstractUbiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words)Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a protein’s energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate–ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

scholarly journals Virtual Screening for Potential Inhibitors of Human Hexokinase II for the Development of Anti-Dengue Therapeutics

BioTech ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 1
Author(s):  
Suriyea Tanbin ◽  
Fazia Adyani Ahmad Fuad ◽  
Azzmer Azzar Abdul Hamid
Keyword(s):  
Hydrogen Bonds ◽  
Binding Energies ◽  
Host Cells ◽  
Dynamics Simulation ◽  
Hexokinase Ii ◽  
Tropical Regions ◽  
Lipinski’S Rule ◽  
Viral Propagation ◽  
C Terminus ◽  
Similarity Scores

Dengue fever, which is a disease caused by the dengue virus (DENV), is a major unsolved issue in many tropical and sub-tropical regions of the world. The absence of treatment that effectively prevent further viral propagation inside the human’s body resulted in a high number of deaths globally each year. Thus, novel anti-dengue therapies are required for effective treatment. Human hexokinase II (HKII), which is the first enzyme in the glycolytic pathway, is an important drug target due to its significant impact on viral replication and survival in host cells. In this study, 23.1 million compounds were computationally-screened against HKII using the Ultrafast Shape Recognition with a CREDO Atom Types (USRCAT) algorithm. In total, 300 compounds with the highest similarity scores relative to three reference molecules, known as Alpha-D-glucose (GLC), Beta-D-glucose-6-phosphate (BG6), and 2-deoxyglucose (2DG), were aligned. Of these 300 compounds, 165 were chosen for further structure-based screening, based on their similarity scores, ADME analysis, the Lipinski’s Rule of Five, and virtual toxicity test results. The selected analogues were subsequently docked against each domain of the HKII structure (PDB ID: 2NZT) using AutoDock Vina programme. The three top-ranked compounds for each query were then selected from the docking results based on their binding energy, the number of hydrogen bonds formed, and the specific catalytic residues. The best docking results for each analogue were observed for the C-terminus of Chain B. The top-ranked analogues of GLC, compound 10, compound 26, and compound 58, showed predicted binding energies of −7.2, −7.0, and −6.10 kcal/mol and 7, 5, and 2 hydrogen bonds, respectively. The analogues of BG6, compound 30, compound 36, and compound 38, showed predicted binding energies of −7.8, −7.4, and −7.0 kcal/mol and 11, 9, and 5 hydrogen bonds, while the top three analogues of 2DG, known as compound 1, compound 4, and compound 31, showed predicted binding energies of −6.8, −6.3, and −6.3 kcal/mol and 4, 3, and 1 hydrogen bonds, sequentially. The highest-ranked compounds in the docking analysis were then selected for molecular dynamics simulation, where compound 10, compound 30, and compound 1, which are the analogues of GLC, BG6, and 2DG, have shown strong protein-ligand stability with an RMSD value of ±5.0 A° with a 5 H bond, ±4.0 A° with an 8 H bond, and ±0.5 A° with a 2 H bond, respectively, compared to the reference molecules throughout the 20 ns simulation time. Therefore, by using the computational studies, we proposed novel compounds, which may act as potential drugs against DENV by inhibiting HKII’s activity.

scholarly journals Mechanistic Basis for Ubiquitin Modulation of a Protein Energy Landscape

2021 ◽  
Vol 120 (3) ◽  
pp. 126a
Author(s):  
Emma Carroll ◽  
Naomi R. Latorraca ◽  
Johanna Lindner ◽  
Jeff Pelton ◽  
Brendan Maguire ◽  
...  
Keyword(s):  
Energy Landscape ◽  
Protein Energy ◽  
Mechanistic Basis ◽  
Protein Energy Landscape

scholarly journals Prediction of GluN2B-CT1290-1310/DAPK1 Interaction by Protein–Peptide Docking and Molecular Dynamics Simulation

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 3018 ◽  
Author(s):  
Gao Tu ◽  
Tingting Fu ◽  
Fengyuan Yang ◽  
Lixia Yao ◽  
Weiwei Xue ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Electrostatic Interactions ◽  
Binding Free Energy ◽  
Computational Simulation ◽  
Critical Role ◽  
Dynamics Simulation ◽  
Peptide Docking ◽  
C Terminus ◽  
Free Energy Decomposition

The interaction of death-associated protein kinase 1 (DAPK1) with the 2B subunit (GluN2B) C-terminus of N-methyl-D-aspartate receptor (NMDAR) plays a critical role in the pathophysiology of depression and is considered a potential target for the structure-based discovery of new antidepressants. However, the 3D structures of C-terminus residues 1290–1310 of GluN2B (GluN2B-CT1290-1310) remain elusive and the interaction between GluN2B-CT1290-1310 and DAPK1 is unknown. In this study, the mechanism of interaction between DAPK1 and GluN2B-CT1290-1310 was predicted by computational simulation methods including protein–peptide docking and molecular dynamics (MD) simulation. Based on the equilibrated MD trajectory, the total binding free energy between GluN2B-CT1290-1310 and DAPK1 was computed by the mechanics generalized born surface area (MM/GBSA) approach. The simulation results showed that hydrophobic, van der Waals, and electrostatic interactions are responsible for the binding of GluN2B-CT1290–1310/DAPK1. Moreover, through per-residue free energy decomposition and in silico alanine scanning analysis, hotspot residues between GluN2B-CT1290-1310 and DAPK1 interface were identified. In conclusion, this work predicted the binding mode and quantitatively characterized the protein–peptide interface, which will aid in the discovery of novel drugs targeting the GluN2B-CT1290-1310 and DAPK1 interface.

scholarly journals Mechanism for antigenic peptide selection by endoplasmic reticulum aminopeptidase 1

2019 ◽  
Vol 116 (52) ◽  
pp. 26709-26716 ◽  
Author(s):  
Petros Giastas ◽  
Anastasia Mpakali ◽  
Athanasios Papakyriakou ◽  
Aggelos Lelis ◽  
Paraskevi Kokkala ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Motif ◽  
Internal Cavity ◽  
Sequence Specificity ◽  
Substrate Selection ◽  
Class I Mhc ◽  
Mhc I ◽  
Intracellular Enzyme ◽  
C Terminus ◽  
Endoplasmic Reticulum Aminopeptidase 1

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.

scholarly journals MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

2020 ◽  
Author(s):  
Ryan Ard ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Michael Phan ◽  
Radoslav Zinoviev ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Pdz Domain ◽  
Diacylglycerol Kinase ◽  
Binding Motif ◽  
Membrane Blebbing ◽  
Rhoa Activity ◽  
C Terminus ◽  
Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

scholarly journals Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

2017 ◽  
Vol 474 (3) ◽  
pp. 385-398 ◽  
Author(s):  
Ryan P. Emptage ◽  
Mark A. Lemmon ◽  
Kathryn M. Ferguson
Keyword(s):  
Membrane Surface ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Disease States ◽  
C Terminus ◽  
Regulatory Module ◽  
Correct Location ◽  
Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

scholarly journals Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

2019 ◽  
Vol 116 (15) ◽  
pp. 7333-7342 ◽  
Author(s):  
Xiang Ye ◽  
Jiabei Lin ◽  
Leland Mayne ◽  
James Shorter ◽  
S. Walter Englander
Keyword(s):  
Hydrogen Exchange ◽  
Atp Hydrolysis ◽  
Physiological Solution ◽  
Mass Spectrometry Analysis ◽  
Structural Element ◽  
Spectrometry Analysis ◽  
Exchange Mass Spectrometry ◽  
Substrate Protein ◽  
Central Pore ◽  
The One

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

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