ABSTRACT
The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. The structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to design tau sequences that stabilize its structural ensemble. Our findings reveal that substitutions near the conserved 'PGGG' beta-turn motif can modulate local conformation, more stably engaging in interactions with the 306VQIVYK311 amyloid motif to decrease aggregation in vitro and in cells. Designed tau sequences maintain microtubule binding and explain why 3R isoforms of tau exhibit reduced pathogenesis over 4R isoforms. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, offering insights for therapeutic strategies aimed at reducing protein misfolding in neurodegenerative diseases.
ABSTRACT
Neurodegenerative tauopathies are caused by the transition of tau protein from a monomer to a toxic aggregate. They include Alzheimer disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick disease (PiD). We have previously proposed that tau monomer exists in two conformational ensembles: an inert form (Mi), which does not self-assemble, and seed-competent form (Ms), which self-assembles and templates ordered assembly growth. We proposed that cis/trans isomerization of tau at P301, the site of dominant disease-associated S/L missense mutations, might underlie the transition of wild-type tau to a seed-competent state. Consequently, we created monoclonal antibodies using non-natural antigens consisting of fluorinated proline (P∗) at the analogous P270 in repeat 1 (R1), biased toward the trans-configuration at either the R1/R2 (TENLKHQP∗GGGKVQIINKK) or the R1/R3 (TENLKHQP∗GGGKVQIVYK) interfaces. Two antibodies, MD2.2 and MD3.1, efficiently immunoprecipitated soluble seeds from AD and PSP but not CBD or PiD brain samples. The antibodies efficiently stained brain samples of AD, PSP, and PiD, but not CBD. They did not immunoprecipitate or immunostain tau from the control brain. Creation of potent anti-seed antibodies based on the trans-proline epitope implicates local unfolding around P301 in pathogenesis. MD2.2 and MD3.1 may also be useful for therapy and diagnosis.
Subject(s)
Tauopathies , Humans , Alzheimer Disease/metabolism , Antibodies, Monoclonal/metabolism , Brain/metabolism , Epitopes/metabolism , Pick Disease of the Brain/metabolism , Pick Disease of the Brain/pathology , Proline/metabolism , tau Proteins/metabolism , Tauopathies/metabolismABSTRACT
Amyloid deposition of the microtubule-associated protein tau is associated with neurodegenerative diseases. In frontotemporal dementia with abnormal tau (FTD-tau), missense mutations in tau enhance its aggregation propensity. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation, integrating data from in vitro, in silico and cellular experiments. We find that S320F stabilizes a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif; identify a suppressor mutation that destabilizes S320F-based hydrophobic clustering reversing the phenotype in vitro and in cells; and computationally engineer spontaneously aggregating tau sequences through optimizing nonpolar clusters surrounding the S320 position. We uncover a mechanism for regulating tau aggregation which balances local nonpolar contacts with long-range interactions that sequester amyloid motifs. Understanding this process may permit control of tau aggregation into structural polymorphs to aid the design of reagents targeting disease-specific tau conformations.
Subject(s)
Frontotemporal Dementia , Humans , Frontotemporal Dementia/genetics , Mutation , tau Proteins/metabolism , Mutation, Missense , Amyloid/genetics , Amyloidogenic Proteins/geneticsABSTRACT
The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. The structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to design tau sequences that stabilize its structural ensemble. Our findings reveal that substitutions near the conserved 'PGGG' beta-turn motif can modulate local conformation, more stably engaging in interactions with the 306 VQIVYK 311 amyloid motif to decrease aggregation in vitro and in cells. Designed tau sequences maintain microtubule binding and explain why 3R isoforms of tau exhibit reduced pathogenesis over 4R isoforms. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, offering insights for therapeutic strategies aimed at reducing protein misfolding in neurodegenerative diseases.
ABSTRACT
The vaccination drive against COVID-19 worldwide was quite successful. However, the second wave of infections was even more disastrous. There was a rapid increase in reinfections and human deaths due to the appearance of new SARS-CoV-2 variants. The viral genome mutations in the variants were acquired while passing through different human hosts that could escape antibodies in convalescent or vaccinated individuals. The treatment was based on oxygen supplements and supportive protocols due to the lack of a specific drug. In this study, we identified three lead inhibitors of arylated coumarin derivatives 4,6,8-tri(naphthalen-2-yl)-2H-chromen-2-one (NF1), 8-(4-hydroxyphenyl)-4,6-di(naphthalen-2-yl)-2H-chromen-2-one (NF12) and 8-(4-hydroxyphenyl)-3,6-di(naphthalen-2-yl)-2H-chromen-2-one (NF-13) that showed higher binding affinity towards the junction of SARS-CoV-2 spike glycoprotein (S-protein) and human angiotensin-converting enzyme 2 (ACE2) receptor. Using molecular docking analysis, we identified the putative binding sites of these potent inhibitors. Notably, molecular dynamics (MD) simulation and MM-PBSA studies confirmed that these inhibitors have the potential ability to bind Spike-protein/ACE2 protein complex with minimal energy. Further, the two major concerns are an adaptive mutation of spike proteins- N501Y and D614G which displayed strong affinity towards NF-13 in docking analysis. Additionally, in vitro and in vivo studies are required to confirm the above findings and develop the inhibitors as potential drugs against SARS-CoV-2.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 Drug Treatment , Coumarins/pharmacology , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Oxygen , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Domains , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
TDP-43 proteinopathies is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The N-terminal domain of TDP-43 (NTD) is important to both TDP-43 physiology and TDP-43 proteinopathy. However, its folding and dimerization process is still poorly characterized. In the present study, we have investigated the folding/unfolding of NTD employing all-atom molecular dynamics (MD) simulations in 8 M dimethylsulfoxide (DMSO) at high temperatures. The MD results showed that the unfolding of the NTD at high temperature evolves through the formation of a number of conformational states differing in their stability and free energy. The presence of structurally heterogeneous population of intermediate ensembles was further characterized by the different extents of solvent exposure of Trp80 during unfolding. We suggest that these non-natives unfolded intermediate ensembles may facilitate NTD oligomerization and subsequently TDP-43 oligomerization, which might lead to the formation of irreversible pathological aggregates, characteristics of disease pathogenesis.
ABSTRACT
Multi-targeting enzyme approaches are considered to be the most significant in suppressing pathogen growth and disease control for MDR and XDR-resistant Mycobacterium tuberculosis. The multiple Mur enzymes involved in peptidoglycan biosynthesis play a key role in a cell's growth. Firstly, homology modeling was employed to construct the 3 D structure of the Mur enzymes. The computational approaches, including molecular docking and molecular dynamics simulations and MM-PBSA methods, were performed to explore the detailed interaction mechanism to evaluate the inhibitory activity against targeted proteins. The computational calculations revealed that the best-docked phytochemical compound (gallomyricitrin) inhibits the selected targets: Mur enzymes by forming stable hydrogen bonds. The analysis of RMSD, RMSF, Rg, PCA, DCCM, cross-correlation network, FEL, H-bond, and vector movement reveal that the docked complex of MurA, MurI, MurG, MurC, and MurE is more stable compared to MurB, MurF, MurD, and MurX docked complexes during MD simulations. Moreover, FEL exposed that gallomyricitrin stabilized to the minimum global energy of Mur Enzymes. The PCA, DCCM, and vector movements and binding free energy results provided further evidence for the stability of gallomyricitrin's interactions inside the binding sites by forming hydrogen bonds. The cross-correlation analysis reveals that Mur enzymes exhibit a positive and negative correlated motion between residues in different protein domains. The computational results contribute in several ways to our understanding of inhibition activity and provide a basic insight into the binding activity of gallomyricitrin as a multi-target drug for tuberculosis. Communicated by Ramaswamy H. Sarma.
Subject(s)
Mycobacterium tuberculosis , Molecular Dynamics Simulation , Molecular Docking Simulation , Protein Binding/physiology , Principal Component Analysis , Bacterial Proteins/chemistryABSTRACT
Nearly 80% of human chronic infections are caused due to bacterial biofilm formation. The increased resistance against the conventional antimicrobial agents makes it difficult to treat the biofilm-related infections. The antibiotics resistance developed by planktonic cells has also become a major threat for human. Therefore, we have attempted here to develop an effective alternative strategy to overcome the issues of antibiotics resistance of bacteria. Upon synthesis, biogenic C-dots were combined with lysozymes which were further encapsulated into chitosan nanocarrier to form C-dots carrier (CDC). The as-synthesized C-dots were found irregular shaped and the average size of C-dots and CDC were 8 ± 2 nm and 450 ± 50 nm, respectively. To ensure secure and targeted delivery of C-dots and lysozyme we have employed chitosan, a biodegradable and natural biopolymer, as a delivery system. The study of time-dependent bacterial growth and flow cytometry analysis demonstrated that CDC can exhibit a synergistic bactericidal activity against the antibiotics resistant recombinant E. coli cells. Further, we have shown that the CDC could be a potent agent for both prevention of biofilm formation and eradication of preformed biofilm. In addition, we have observed that our drug delivery system is hemocompatible in nature making it suitable for in vivo applications. Therefore, we believe that the combination therapy of C-dots and lysozyme may be used as an excellent antibacterial and antibiofilm strategy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Carbon/chemistry , Muramidase/chemistry , Quantum Dots/chemistry , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Bacteria/growth & development , Bacteria/ultrastructure , Carbon/pharmacology , Chitosan/chemistry , Drug Carriers/chemistry , Drug Resistance, Bacterial/drug effects , Green Chemistry Technology , Hemolysis/drug effects , Humans , Muramidase/pharmacologyABSTRACT
The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of ß-lactoglobulin (ß-LgA) in aqueous mixture of 8 M urea and 8 M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of ß-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of â¼3.0 kcal mol-1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around â¼30-35 ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (â¼50-150 ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of â¼6.0 kcal mol-1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of ß-LgA in urea and DMSO.Abbreviationsß-LgAß-lactoglobulinDMSOdimethyl sulfoxideFELfree-energy landscapeGdmClguanidinium chlorideIintermediate stateMGmolten globule statePMEparticle mesh EwaldQfraction of native contactsRMSDroot mean square deviationRMSFroot mean square fluctuationRgradius of gyrationSASAsolvent Accessible Surface AreascSASAthe side chain SASATrptryptophanCommunicated by Ramaswamy H. Sarma.
Subject(s)
Dimethyl Sulfoxide , Lactoglobulins , Protein Conformation , Protein Denaturation , Protein Folding , UreaABSTRACT
Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0-4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located ß-sheets. The reduction of 60-65% tertiary contacts in 7.0-8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.
Subject(s)
Carbonic Anhydrase IX/chemistry , Models, Molecular , Protein Unfolding , Urea/chemistry , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Protein Conformation , Protein Stability , Protein Unfolding/drug effects , Solvents , Urea/pharmacologyABSTRACT
Misfolding and aggregation of Cu, Zn Superoxide Dismutase (SOD1) is often found in amyotrophic lateral sclerosis (ALS) patients. The central apo SOD1 barrel was involved in protein maturation and pathological aggregation in ALS. In this work, we employed atomistic molecular dynamics (MD) simulations to study the conformational dynamics of SOD1barrel monomer in different concentrations of trifluoroethanol (TFE). We find concentration dependence unusual structural and dynamical features, characterized by the local unfolding of SOD1barrel. This partially unfolded structure is characterized by the exposure of hydrophobic core, is highly dynamic in nature, and is the precursor of aggregation seen in SOD1barrel. Our computational studies supports the hypothesis of the formation of aggregation 'building blocks' by means of local unfolding of apo monomer as the mechanism of SOD1 fibrillar aggregation. The non-monotonic TFE concentration dependence of protein conformational changes was explored through simulation studies. Our results suggest that altered protein conformation and dynamics within its structure may underlie the aggregation of SOD1 in ALS.