Chemical IN04 Inhibits the Kinase Domain not the ROC Domain of LRRK1: Results from Homology Modeling and Molecular Docking

Background: Bone loss is the most common reason for broken bones among the elderly. An ideal agent for treatment of bone loss should have both osteoclast inhibitory and osteoblast stimulatory functions. Leucine rich repeat kinase 1 (LRRK1) is a novel target for alternative anti-resorptive drugs to treat osteoporosis and osteoporotic fractures. Recently a chemical IN04, Methyl 3-[(([5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-thio-acetyl)-amino]-benzoate, has been identified as a potential LRRK1 inhibitor. Objective: The aim of this work is to investigate how the chemical IN04 interacts with LRRK1 and inhibits its activity. Methods: A structural model of LRRK1 kinase domain was constructed with SWISS-MODEL. The human protein kinase ROCO4 (PDB ID: 4YZN) was chosen as the template based on sequence homology, structural and phylogenetic analysis. In addition, a homology model of the LRRK1 ROC domain was also prepared based on the LRRK2 ROC domain structure (PDB ID: 2ZEJ). The interactions of IN04 with the active sites in the LRRK1 kinase domain and ROC domain were investigated by SwissDock. Results: IN04 was docked into the active site of the LRRK1 kinase domain with similar interactions as ATP comparable to the ligand bound to homologous kinases. Many rational binding modes of IN04 to LRRK1 kinase domain were investigated and the most likely binding pose containing multiple hydrogen bonds and a salt bridge was discovered. However, IN04 cannot fit into the GDP-binding site of the ROC domain. Conclusion: Chemical IN04 inhibits LRRK1 by binding to the active site of the kinase domain but not the ROC domain.

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Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding

Biochemical Journal ◽

10.1042/bj20130350 ◽

2013 ◽

Vol 452 (3) ◽

pp. 423-432 ◽

Cited By ~ 7

Author(s):

Radika Soysa ◽

Hanka Venselaar ◽

Jacqueline Poston ◽

Buddy Ullman ◽

Marie-Pierre Hasne

Keyword(s):

Trypanosoma Cruzi ◽

Structural Model ◽

Three Dimensional ◽

Salt Bridge ◽

Homology Model ◽

Binding Pocket ◽

Dimensional Structure ◽

Helical Structures ◽

Structural Determinants ◽

Key Residues

The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247–Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

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Potential Ebola drug targets – filling the gap: a critical step forward towards the design and discovery of potential drugs

Biologia ◽

10.1515/biolog-2017-0012 ◽

2017 ◽

Vol 72 (1) ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Marissa Balmith ◽

Mahmoud E.S. Soliman

Keyword(s):

Binding Site ◽

Active Site ◽

Active Sites ◽

Binding Free Energy ◽

Three Dimensional ◽

Computational Prediction ◽

Homology Model ◽

Free Energy Calculations ◽

Biologically Active ◽

World Health

AbstractAmong the classified neglected infectious diseases, the Ebola virus (EboV) remains a challenging epidemic. This deadly virus has been reported as a category A bioweapon organism by the World Health Organization due to the serious threat it poses. To date, Ebola drug discovery proves challenging. Proteins need to be targeted at the relevant biologically active site for drug or inhibitor binding to be effective. Due to insufficient experimental data to confirm the biologically active binding site for novel protein targets, researchers often rely on computational prediction methods to identify binding sites. Many computational studies have attempted to identify the biological active site for EboV proteins, however, the methods employed are not sufficiently validated. This has prompted us to provide a comprehensive molecular understanding of the various targets of the EboV, including three-dimensional structures, active site identification and further validation. Herein we report the account of a three-dimensional homology model of the unresolved EboV RNA-dependent RNA polymerase (RdRp), as well as a comprehensive analysis of the binding site residues of all proteins of the EboV. Docking-aided active site determination was carried out to identify possible active sites on the homology model of RdRp. Binding free energy calculations revealed subtle differences in the binding at each site. These results can also provide some potential clues for further design of novel inhibitors to treat this killer virus and is a critical cornerstone of research into the EboV.

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Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

10.26434/chemrxiv.12084468.v1 ◽

2020 ◽

Author(s):

Sourav Pal ◽

Dr. Arindam Talukdar

Keyword(s):

Active Site ◽

Drug Targets ◽

Active Sites ◽

Structural Alignment ◽

Homology Model ◽

Active Site Residues ◽

Protein Targets ◽

Rational Development ◽

Novel Coronavirus ◽

Approved Drugs

<p>The recent pandemic due to the novel coronavirus SARS-CoV-2 (COVID-19) is causing significant mortality worldwide. However, there is a lack of specific drugs which can either prevent or treat the patient suffering from COVID-19. To understand the SARS-CoV-2 receptor recognition causing infectivity and pathogenesis, we have compiled a list of 20 probable drug targets on host and virus based on viral life cycle along with their PDB IDs for the rational development of future antivirals. We have prepared nine homology model for vital proteins for which no crystal structure is reported, which includes protein from host, viral membrane proteins and essential non-structural proteins (NSPs) of virus. The generated models were validated followed by Ramachandran plot along with their sequence and structural alignment. The active site residues of all the protein models are calculated by utilizing COACH meta-server and also cross verified with the CASTp webservers. All the active sites of the homology build proteins were evaluated after superimposition of the closely related X-ray crystallized structure bound with the co-crystal ligands. These information present in the manuscript can be used for the discovery effort towards new antivirals as well as repurposing FDA approved drugs against SARS-CoV-2.</p><br>

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Inhibitory Proteins Block Substrate Access to the Active Site of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB

10.1101/2021.07.09.451828 ◽

2021 ◽

Author(s):

Sandra Olenic ◽

Lim Heo ◽

Michael Feig ◽

Lee Kroos

Keyword(s):

Bacillus Subtilis ◽

Active Site ◽

Active Sites ◽

Structural Model ◽

Terminal Region ◽

Cross Linking ◽

Intramembrane Proteases ◽

Inhibitory Proteins ◽

Partial Homology ◽

Active Site Region

Intramembrane proteases of diverse signaling pathways use membrane-embedded active sites to cleave membrane-associated substrates. Interactions of intramembrane metalloproteases with modulators are poorly understood. Inhibition of Bacillus subtilis intramembrane metalloprotease SpoIVFB requires BofA and SpoIVFA, which transiently prevent cleavage of Pro-σK during endosporulation. Three conserved BofA residues (N48, N61, T64) in or near predicted transmembrane segment (TMS) 2 were found to be required for SpoIVFB inhibition. Disulfide cross-linking indicated that BofA TMS2 occupies the SpoIVFB active site region. BofA and SpoIVFA neither prevented SpoIVFB from interacting with Pro-σK in co-purification assays nor interfered with cross-linking between the C-terminal regions of Pro-σK and SpoIVFB. However, BofA and SpoIVFA did interfere with cross-linking between the N-terminal Proregion of Pro-σK and the SpoIVFB active site region and interdomain linker. A BofA variant lacking predicted TMS1, in combination with SpoIVFA, was less effective at interfering with some of the cross-links and slightly less effective at inhibiting cleavage of Pro-σK by SpoIVFB. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and Pro-σK, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that N48 in BofA TMS2 interacts with T64 (and possibly N61) of BofA to stabilize a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB. Thus, the two inhibitory proteins block access of the Pro-σK N-terminal region to the SpoIVFB active site region. Our findings may inform efforts to develop selective inhibitors of intramembrane metalloproteases.

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Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715015497 ◽

2015 ◽

Vol 71 (10) ◽

pp. 2032-2039 ◽

Cited By ~ 1

Author(s):

Roland Pfoh ◽

Emil F. Pai ◽

Vivian Saridakis

Keyword(s):

Active Site ◽

Active Sites ◽

Methanobacterium Thermoautotrophicum ◽

Site Directed Mutagenesis ◽

Binding Modes ◽

Wild Type ◽

Active Site Residues ◽

Nicotinamide Mononucleotide ◽

Nicotinamide Mononucleotide Adenylyltransferase ◽

Phosphate Groups

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD+and NaAD+. The crystal structure of NMNAT fromMethanobacterium thermoautotrophicumcomplexed with NAD+and SO42−revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed inEscherichia coliall of these NMNAT mutants trapped a molecule of NADP+in their active sites. This NADP+was bound in a conformation that was quite different from that displayed by NAD+in the native enzyme complex. When NADP+was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP+in the active site of NMNAT, indicating plasticity of the active site.

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Comments on the recent crystal structure of TsaBDE complex of bacterial t6A biosynthesis system and its significance for understanding TC-AMP processing

10.1101/563171 ◽

2019 ◽

Author(s):

Boguslaw Stec

Keyword(s):

Crystal Structure ◽

Active Sites ◽

Structural Model ◽

Thermotoga Maritima ◽

Biosynthesis Pathway ◽

Binding Modes ◽

Anticodon Loop ◽

Structural Details ◽

Unstable Intermediate ◽

Trna Binding

ABSTRACTThe N(6)-threonylcarbamoyl adenosine (t6A) modification at position 37 of a tRNA of the anticodon loop is universal and central to the translational fidelity of all known organisms. The ternary complex of TsaBDE is the central and essential workstation for t6A biosynthesis in bacteria. The recently published crystal structure of Thermotoga maritima (T.maritima) TsaBDE complex (Missoury et al., 2018) has ~15% incorrectly-placed, misplaced/mistraced, or missing residues. These structural errors have precipitated incorrect conclusions about the disordering of the active site and inferred action of the TsaE element. In this report, we rectify the published structural model of the T.maritima TsaBDE complex. In stark contrast, a corrected structural model of TsaBDE shows that both active sites of the TsaD element are fully occupied with threonylcarbamoyladenosine (TC-AMP), an unstable intermediate chemical moiety of the t6A biosynthesis pathway. This observation has profound implications for understanding the funneling of intermediates in the t6A pathway and also in helping to elucidate tRNA binding modes. Based on the structural details described in here we propose a unifying principle for binding the tRNA to the TsaD subunit of the complex which is universally required in all known t6A modification pathways.

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Protein and substrate flexibility contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

10.1101/2021.02.18.431797 ◽

2021 ◽

Author(s):

Madhuri Gade ◽

Li Lynn Tan ◽

Mahakaran Sandhu ◽

Jospeh S. Brock ◽

Andie Delaney ◽

...

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Elevated Level ◽

Active Sites ◽

Conformational Dynamics ◽

Cancer Cell Line ◽

Evolutionary Divergence ◽

Binding Modes ◽

Catalytic Promiscuity ◽

Stable Conformation

AbstractProtein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to increased mobility of an active site loop. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the combined effects of both enzyme and substrate dynamics.

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Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

10.26434/chemrxiv.12084468 ◽

2020 ◽

Author(s):

Sourav Pal ◽

Dr. Arindam Talukdar

Keyword(s):

Active Site ◽

Drug Targets ◽

Active Sites ◽

Structural Alignment ◽

Homology Model ◽

Active Site Residues ◽

Protein Targets ◽

Rational Development ◽

Novel Coronavirus ◽

Approved Drugs

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Calcium and vitamin D nutrition and bone disease of the elderly

Public Health Nutrition ◽

10.1079/phn2001140 ◽

2001 ◽

Vol 4 (2b) ◽

pp. 547-559 ◽

Cited By ~ 148

Author(s):

C Gennari

Keyword(s):

Vitamin D ◽

Bone Loss ◽

Bone Mass ◽

Vitamin D Deficiency ◽

Bone Health ◽

Calcium Intake ◽

Osteoporotic Fractures ◽

The Elderly ◽

Vitamin D Insufficiency ◽

High Incidence

AbstractOsteoporosis, a systemic skeletal disease characterized by a low bone mass, is a major public health problem in EC member states because of the high incidence of fragility fractures, especially hip and vertebral fracture. In EC member states the high incidence of osteoporotic fractures leads to considerable mortality, morbidity, reduced mobility and decreased quality of life. In 1995 the number of hip fractures in 15 countries of EC has been 382.000 and the estimated total care cost of about 9 billion of ECUs. Given the magnitude of the problem public health measures are important for preventive intervention.Skeletal bone mass is determined by a combination of endogenous (genetic, hormonal) and exogenous (nutritional, physical activity) factors. Nutrition plays an important role in bone health. The two nutrients essential for bone health are calcium and vitamin D. Reduced supplies of calcium are associated with a reduced bone mass and osteoporosis, whereas a chronic and severe vitamin D deficiency leads to osteomalacia, a metabolic bone disease characterized by a decreased mineralization of bone. Vitamin D insufficiency, the preclinical phase of vitamin D deficiency, is most commonly found in the elderly. The major causes of vitamin D deficiency and insufficiency are decreased renal hydroxylation of vitamin D, poor nutrition, scarce exposition to sunlight and a decline in the synthesis of vitamin D in the skin.The daily average calcium intake in Europe has been evaluated in the SENECA study concerning the diet of elderly people from 19 towns of 10 European countries. In about one third of subjects the dietary calcium intake results were very low, between 300 and 600 mg/day in women, and 350 and 700 mg/day in men. Calcium supplements reduce the rate of bone loss in osteoporotic patients. Some recent studies have reported a significant positive effect of calcium treatment not only on bone mass but also on fracture incidence. The SENECA study, has also shown that vitamin D insufficiency is frequent in elderly populations in Europe. There are a number of studies on the effects of vitamin D supplementation on bone loss in the elderly, showing that supplementations with daily doses of 400–800 IU of vitamin D, given alone or in combination with calcium, are able to reverse vitamin D insufficiency, to prevent bone loss and to improve bone density in the elderly.In recent years, there has been much uncertainty about the intake of calcium for various ages and physiological states. In 1998, the expert committee of the European Community in the Report on Osteoporosis-Action on prevention, has given the recommended daily dietary allowances (RDA) for calcium at all stage of life. For the elderly population, above age 65 the RDA is 700–800 mg/day. The main source of calcium in the diet are dairy products (milk, yoghurts and cheese) fish (sardines with bones), few vegetables and fruits. The optimal way to achieve adequate calcium intake is through the diet. However, when dietary sources are scarce or not well tolerated, calcium supplementation may be used. Calcium is generally well tolerated and reports of significant side-effects are rare.Adequate sunlight exposure may prevent and cure vitamin D insufficiency. However, the sunlight exposure or the ultraviolet irradiation are limited by concern about skin cancer and skin disease. The most rational approach to reducing vitamin D insufficiency is supplementation. In Europe, the RDA is 400–800 IU (10–20 μg) daily for people aged 65 years or over. This dose is safe and free of side effects.In conclusion, in Europe a low calcium intake and a suboptimal vitamin D status are very common in the elderly. Evidence supports routine supplementation for these people at risk of osteoporosis, by providing a daily intake of 700–800 mg of calcium and 400–800 IU of vitamin D. This is an effective, safe and cheap means of preventing osteoporotic fractures.

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On the Adsorption of Aspartate Derivatives to Calcite Surfaces in Aqueous Environment

10.26434/chemrxiv.11558430.v1 ◽

2020 ◽

Author(s):

Robert Stepic ◽

Lara Jurković ◽

Ksenia Klementyeva ◽

Marko Ukrainczyk ◽

Matija Gredičak ◽

...

Keyword(s):

Active Sites ◽

Realistic Model ◽

Binding Energies ◽

Inhibition Mechanism ◽

Aqueous Environment ◽

Binding Modes ◽

Carboxylate Groups ◽

Dissolved Ions ◽

Living Organisms ◽

Dynamics Simulations

In many living organisms, biomolecules interact favorably with various surfaces of calcium carbonate. In this work, we have considered the interactions of aspartate (Asp) derivatives, as models of complex biomolecules, with calcite. Using kinetic growth experiments, we have investigated the inhibition of calcite growth by Asp, Asp2 and Asp3.This entailed the determination of a step-pinning growth regime as well as the evaluation of the adsorption constants and binding free energies for the three species to calcite crystals. These latter values are compared to free energy profiles obtained from fully atomistic molecular dynamics simulations. When using a flat (104) calcite surface in the models, the measured trend of binding energies is poorly reproduced. However, a more realistic model comprised of a surface with an island containing edges and corners, yields binding energies that compare very well with experiments. Surprisingly, we find that most binding modes involve the positively charged, ammonium group. Moreover, while attachment of the negatively charged carboxylate groups is also frequently observed, it is always balanced by the aqueous solvation of an equal or greater number of carboxylates. These effects are observed on all calcite features including edges and corners, the latter being associated with dominant affinities to Asp derivatives. As these features are also precisely the active sites for crystal growth, the experimental and theoretical results point strongly to a growth inhibition mechanism whereby these sites become blocked, preventing further attachment of dissolved ions and halting further growth.

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