scholarly journals Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane

2021 ◽  
Vol 22 (4) ◽  
pp. 2138
Author(s):  
Lorna Chebon-Bore ◽  
Taremekedzwa Allan Sanyanga ◽  
Colleen Varaidzo Manyumwa ◽  
Afrah Khairallah ◽  
Özlem Tastan Bishop
Keyword(s):  
Plasmodium Falciparum ◽  
Active Site ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Point Mutations ◽  
Md Simulations ◽  
Resistance Mechanisms ◽  
Phospholipid Bilayer ◽  
Active Site Residues ◽  
Presence And Absence

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.

Molecular docking and molecular dynamics simulations of fumarate hydratase and its mutant H235N complexed with pyromellitic acid and citrate

2017 ◽  
Vol 15 (06) ◽  
pp. 1750026 ◽  
Author(s):  
S. Subasri ◽  
Santosh Kumar Chaudhary ◽  
K. Sekar ◽  
Manish Kesherwani ◽  
D. Velmurugan
Keyword(s):  
Molecular Dynamics ◽  
Active Site ◽  
Model Building ◽  
Hydrophobic Interactions ◽  
Conformational Stability ◽  
Fumarate Hydratase ◽  
Md Simulations ◽  
Major Effect ◽  
Active Site Residues ◽  
Pyromellitic Acid

Fumarase catalyzes the reversible, stereospecific hydration/dehydration of fumarate to L-malate during the Kreb’s cycle. In the crystal structure of the tetrameric fumarase, it was found that some of the active site residues S145, T147, N188 G364 and H235 had water-mediated hydrogen bonding interactions with pyromellitic acid and citrate which help to the protonation state for the conversion of fumarate to malate. When His 235 is mutated with Asn (H235N), water-mediated interactions were lost due to the shifting of active site water molecule by 0.7 Å away. Molecular dynamics (MD) simulations were also carried out by NAMD and analyzed using Assisted Model Building with Energy Refinement (AMBER) program to better understand the conformational stability and other aspects during the binding of pyromellitic acid and citrate with native and mutant FH. The role of hydrogen bonds and hydrophobic interactions was also analyzed. The present study confirms that the H235N mutation has a major effect on the catalytic activity of fumarase which is evident from the biochemical studies.

scholarly journals Prediction of Function Determining and Buried Residues Through Analysis of Saturation Mutagenesis Datasets

2021 ◽  
Vol 8 ◽  
Author(s):  
Munmun Bhasin ◽  
Raghavan Varadarajan
Keyword(s):  
Active Site ◽  
Protein Function ◽  
Structural Information ◽  
Three Dimensional ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Large Numbers ◽  
Sensitivity Data ◽  
Sensitivity Specificity

Mutational scanning can be used to probe effects of large numbers of point mutations on protein function. Positions affected by mutation are primarily at either buried or at exposed residues directly involved in function, hereafter designated as active-site residues. In the absence of prior structural information, it has not been easy to distinguish between these two categories of residues. We curated and analyzed a set of twelve published deep mutational scanning datasets. The analysis revealed differential patterns of mutational sensitivity and substitution preferences at buried and exposed positions. Prediction of buried-sites solely from the mutational sensitivity data was facilitated by incorporating predicted sequence-based accessibility values. For active-site residues we observed mean sensitivity, specificity and accuracy of 61, 90 and 88% respectively. For buried residues the corresponding figures were 59, 90 and 84% while for exposed non active-site residues these were 98, 44 and 82% respectively. We also identified positions which did not follow these general trends and might require further experimental re-validation. This analysis highlights the ability of deep mutational scans to provide important structural and functional insights, even in the absence of three-dimensional structures determined using conventional structure determination techniques, and also discuss some limitations of the methodology.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Molecular docking and pharmacokinetic screening of eucalyptol (1,8 cineole) from eucalyptus essential oil against SARS-CoV-2

2020 ◽  
Vol 12 (3) ◽  
pp. 536-545
Author(s):  
Arun D. SHARMA ◽  
Inderjeet KAUR
Keyword(s):  
Molecular Docking ◽  
Essential Oil ◽  
Active Site ◽  
In Silico ◽  
Hydrophobic Interactions ◽  
Rna Virus ◽  
In Silico Analysis ◽  
Active Site Residues ◽  
Virus Family

SARS-CoV-2 (COVID-19), member of corona virus family, is a positive single stranded RNA virus. Due to lack of drugs it is spreading its tentacles across the world. Being associated with cough, fever, and respiratory distress, this disease caused more than 15% mortality worldwide. Mpro/3CLpro has recently been regarded as a suitable target for drug design due to its vital role in virus replication. The current study focused on the inhibitory activity of eucalyptol (1,8 cineole), an essential oil component from eucalyptus oil, against Mpro/3CLprofrom SARS-CoV-2. Till date there is no work is undertaken on in-silico analysis of this compound against Mpro/3CLproof SARS-CoV-2. Molecular docking studies were conducted by using 1-click dock tool and Patchdock analysis. In-silico absorption, distribution, metabolism, excretion and toxicity (ADMET) profile were also studied. The calculated parameters such as docking score indicated effective binding of eucalyptol to COVID-19 Mpro protein. Active site prediction revealed the involvement of active site residues in ligand binding. Interactions results indicated that, Mpro/3CLpro/eucalyptol complexes forms hydrophobic interactions. ADMET studies provided guidelines and mechanistic scope for identification of potent anti-COVID 19 drug. Therefore, eucalyptol may represent potential herbal treatment to act as COVID-19 Mpro/3CLproinhibitor, a finding which must be validated in vivo.

scholarly journals Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

2020 ◽  
Vol 295 (19) ◽  
pp. 6472-6481
Author(s):  
Kyle J. Mamounis ◽  
Erik T. Yukl ◽  
Victor L. Davidson
Keyword(s):  
Active Site ◽  
Protein Function ◽  
Marine Bacterium ◽  
Substrate Binding ◽  
Active Site Residues ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Hill Coefficients ◽  
The Hill ◽  
Glycine Oxidase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

scholarly journals Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

2018 ◽  
Author(s):  
Ayşegül Özen ◽  
Kuan-Hung Lin ◽  
Keith P Romano ◽  
Davide Tavella ◽  
Alicia Newton ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Md Simulations ◽  
Drug Susceptibility ◽  
Active Site Residues ◽  
Allosteric Coupling ◽  
Direct Acting ◽  
Distal Mutation ◽  
Active Site Mutations

AbstractHepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 Å away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the α-2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

Navigating Research Toward the Re-emerging Nipah Virus- A New Piece to the Puzzle

2019 ◽  
Vol 25 (12) ◽  
pp. 1392-1401
Author(s):  
Pritika Ramharack ◽  
Nikita Devnarain ◽  
Letitia Shunmugam ◽  
Mahmoud E.S. Soliman
Keyword(s):  
Active Site ◽  
Hydrophobic Interactions ◽  
Nipah Virus ◽  
Homology Model ◽  
Future Research ◽  
Active Site Residues ◽  
Comprehensive Overview ◽  
Computational Tools ◽  
Plot Analysis ◽  
Potential Inhibitors

Background: The recent Nipah virus (NiV) outbreak in India has caused a state of chaos, with potential to become the next international pandemic. There is still a great deal to learn about NiV for the development of a potent treatment against it. The NiV non-structural proteins play important roles in the lifecycle of the virus, with the RNA-dependent RNA-polymerase (RdRp) being a vital component in viral replication. In this study, we not only provide a comprehensive overview of all the literature concerning NiV, we also propose a model of the NiV RdRp and screen for potential inhibitors of the viral enzyme. Objectives: In this study, computational tools were utilized in the design of a NiV RdRp homology model. The active site of RdRp was then identified and potential inhibitors of the protein were discovered with the use of pharmacophore-based screening. Methods: In this study, computational tools were utilized in the design of a NiV RdRp homology model. The active site of RdRp was then identified and potential inhibitors of the protein were discovered with the use of pharmacophore-based screening. Results: Ramachandran plot analysis revealed a favourable model. Upon binding of nucleoside analog, 4’- Azidocytidine, active site residues Trp1714 and Ser1713 took part in stabilizing hydrogen bonds, while Thr1716, Ser1478, Ser1476 and Glu1465 contributed to hydrophobic interactions. Pharmacophore based screening yielded 18 hits, of which ZINC00085930 demonstrated the most optimal binding energy (-8.1 kcal/mol), validating its use for further analysis as an inhibitor of NiV. Conclusion: In this study we provide a critical guide, elucidating on the in silico requirements of the drug design and discovery process against NiV. This material lays a foundation for future research into the design and development of drugs that inhibit NiV.

scholarly journals Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

2007 ◽  
Vol 401 (3) ◽  
pp. 635-644 ◽  
Author(s):  
Juthamart Piromjitpong ◽  
Jantana Wongsantichon ◽  
Albert J. Ketterman
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Glutathione Transferases ◽  
Active Site Residues ◽  
Interface Area ◽  
Subunit Interface ◽  
Alternatively Spliced ◽  
Active Site Conformation ◽  
Interface Residues

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.

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