scholarly journals A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

2020 ◽  
Author(s):  
Veronika Thallmair ◽  
Lea Schultz ◽  
Siewert J. Marrink ◽  
Dominik Oliver ◽  
Sebastian Thallmair
Keyword(s):  
Binding Site ◽  
Md Simulations ◽  
Binding Pocket ◽  
Binding Mode ◽  
Phosphatidyl Serine ◽  
Coarse Grained ◽  
Membrane Association ◽  
Membrane Interface ◽  
Binding Properties ◽  
Protein Membrane

ABSTRACTPhosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic.Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico.Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

scholarly journals Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

2017 ◽  
Vol 73 (4) ◽  
pp. 294-315 ◽  
Author(s):  
Kimberly A. Stanek ◽  
Jennifer Patterson-West ◽  
Peter S. Randolph ◽  
Cameron Mura
Keyword(s):  
Rna Binding ◽  
Binding Pocket ◽  
Binding Mode ◽  
Evolutionary Conservation ◽  
Aquifex Aeolicus ◽  
Binding Properties ◽  
Bacterial Phyla ◽  
Mrna Targets ◽  
Small Regulatory Rnas ◽  
And Function

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

Discovery of novel sphingosine kinase 1 inhibitorsvia structure-based hierarchical virtual screening

MedChemComm ◽  
2015 ◽  
Vol 6 (3) ◽  
pp. 413-417 ◽  
Author(s):  
Xiaojian Wang ◽  
Chenbin Sun ◽  
Liang Fang ◽  
Dali Yin
Keyword(s):  
Virtual Screening ◽  
Sphingosine Kinase ◽  
Md Simulations ◽  
Binding Pocket ◽  
Binding Mode ◽  
Positive Control ◽  
Biological Evaluation ◽  
U937 Cells ◽  
Sphingosine Kinase 1 ◽  
Inhibitory Activities

Hierarchical structure-based virtual screening against the sphingosine kinase 1(SphK1) binding pocket was performed. 25 compounds were selected for biological evaluation. Compound 25 exhibited comparable SphK1 and SphK2 inhibitory activities and anti-proliferative effects on U937 cells to the positive control N,N-dimethylsphingosine (DMS) 1. Further molecule dynamic (MD) simulations revealed the binding mode between SphK1 and 25.

scholarly journals Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

2016 ◽  
Author(s):  
Kimberly A Stanek ◽  
Jennifer P West ◽  
Peter S Randolph ◽  
Cameron Mura
Keyword(s):  
Rna Binding ◽  
Binding Pocket ◽  
Binding Mode ◽  
Evolutionary Conservation ◽  
Thermophilic Bacterium ◽  
Aquifex Aeolicus ◽  
Binding Properties ◽  
Bacterial Phyla ◽  
Mrna Targets ◽  
Small Regulatory Rnas

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-Å resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.AbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings, with at least two distinct surfaces that bind RNA. Recently, another binding site—dubbed the ‘lateral rim’—has been implicated in sRNA•mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 Å resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

scholarly journals OR24-01 Mutational Study of the GPR119 Receptor Binding Site

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Matthew D Rosales ◽  
Frank Dean ◽  
Evangelia Kotsikorou
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Specific Activity ◽  
Md Simulations ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Receptor Model ◽  
Wild Type

Abstract The GPR119 receptor, a class A G-protein coupled receptor located in the pancreatic β cells, induces insulin production when activated. Due to its specific activity, the pharmaceutical industry has identified GPR119 as a target for the treatment for type 2 diabetes. The lack of a GRP119 crystal structure has hindered the study of the receptor so our laboratory developed GPR119 active and inactive homology models. Docking studies with the inactive receptor model indicated that two leucine residues facing the binding pocket, L5.43(169) and L6.52(242), may be involved in ligand activation. Additionally, a serine at the extracellular end of the pocket, S1.32(4), may help orient of the ligand in the binding pocket via hydrogen bonding. To gain further insight into the role of these residues and the receptor activation mechanism, molecular dynamics (MD) simulations and in vitro cAMP assays of the wild type and mutant receptors were employed. The software NAMD employing the CHARMM force field was used to carry out MD simulations of the active receptor model bound with the agonist AR231453 embedded in a hydrated lipid bilayer. Preliminary results indicate that L6.52(242), located on transmembrane helix (TMH) 6, does not face directly into the binding site and does not interact with the ligand, while L5.43(169), located on TMH5, does face into the binding site, potentially interacting directly with the ligand. Also, S1.32(4), because of its extracellular location, is solvated instead of interacting with the ligand. The in vitro studies overall support the MD simulations. The mutations L6.52(242)M and L6.52(242)A appear to have minimal to no effect on agonist-induced cAMP production, compared to the wild type. In contrast, the L5.43(169)M and L5.43(169)A mutations decrease the potency of activation by AR231453, indicating that L5.43(169) changes the shape of the binding pocket, affecting ligand binding and activation. Finally, the cAMP assays show that the S1.32(4)A mutant also shows decreased activity compared to the wild type, implying that the ligand may be losing a hydrogen bonding interaction when S1.32(4) is mutated to alanine.

scholarly journals Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Meng-Hsuan Lin ◽  
Chao-Cheng Cho ◽  
Yi-Chih Chiu ◽  
Chia-Yu Chien ◽  
Yi-Ping Huang ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Binding Mode ◽  
Biological Functions ◽  
Binding Properties ◽  
Binding Ability ◽  
Nad Metabolism ◽  
Ligand Selectivity ◽  
Physiological Temperature ◽  
Binding Behavior ◽  
Macro Domain

AbstractThe macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

scholarly journals Exploring a new ligand binding site of G protein-coupled receptors

2018 ◽  
Vol 9 (31) ◽  
pp. 6480-6489 ◽  
Author(s):  
H. C. Stephen Chan ◽  
Jingjing Wang ◽  
Krzysztof Palczewski ◽  
Slawomir Filipek ◽  
Horst Vogel ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Md Simulations ◽  
Binding Pocket ◽  
G Protein Coupled Receptors ◽  
Ligand Binding Site ◽  
Endogenous Ligand ◽  
G Protein Coupled

A new binding pocket of the endogenous ligand has been discovered by MD simulations.

scholarly journals Prediction of essential binding domains for the endocannabinoid N-arachidonoylethanolamine (AEA) in the brain cannabinoid CB1 receptor

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0229879
Author(s):  
Joong-Youn Shim
Keyword(s):  
Cannabis Sativa ◽  
Md Simulations ◽  
Binding Pocket ◽  
Binding Mode ◽  
Cb1 Receptor ◽  
Cannabinoid Cb1 Receptor ◽  
X Ray ◽  
Binding Domains ◽  
Long Time ◽  
The Brain

Δ9-tetrahydrocannabinol (Δ9-THC), the main active ingredient of Cannabis sativa (marijuana), interacts with the human brain cannabinoid (CB1) receptor and mimics pharmacological effects of endocannabinoids (eCBs) like N-arachidonylethanolamide (AEA). Due to its flexible nature of AEA structure with more than 15 rotatable bonds, establishing its binding mode to the CB1 receptor is elusive. The aim of the present study was to explore possible binding conformations of AEA within the binding pocket of the CB1 receptor confirmed in the recently available X-ray crystal structures of the CB1 receptor and predict essential AEA binding domains. We performed long time molecular dynamics (MD) simulations of plausible AEA docking poses until its receptor binding interactions became optimally established. Our simulation results revealed that AEA favors to bind to the hydrophobic channel (HC) of the CB1 receptor, suggesting that HC holds essential significance in AEA binding to the CB1 receptor. Our results also suggest that the Helix 2 (H2)/H3 region of the CB1 receptor is an AEA binding subsite privileged over the H7 region.

scholarly journals Enhancing Sampling of Water Rearrangements on Ligand Binding: A Comparison of Techniques

2021 ◽  
Author(s):  
Yunhui Ge ◽  
David C. Wych ◽  
Marley L. Samways ◽  
Michael E. Wall ◽  
Jonathan W. Essex ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Binding Site ◽  
Md Simulation ◽  
Direct Analysis ◽  
Md Simulations ◽  
Binding Mode ◽  
Bulk Water ◽  
Free Energy Calculations ◽  
Water Sampling ◽  
Ligand Interactions

Water often plays a key role in protein structure, molecular recognition, and mediating protein-ligand interactions. Thus, free energy calculations must adequately sample water motions, which often proves challenging in typical MD simulation timescales. Thus, the accuracy of methods relying on MD simulations ends up limited by slow water sampling. Particularly, as a ligand is removed or modified, bulk water may not have time to fill or rearrange in the binding site. In this work, we focus on several molecular dynamics (MD) simulation-based methods attempting to help address water motions and occupancies: BLUES, using nonequilibrium candidate Monte Carlo (NCMC); grand, using grand canonical Monte Carlo (GCMC); and normal MD. We assess the accuracy and efficiency of these methods in sampling water motions. We selected a range of systems with varying numbers of waters in the binding site, as well as those where water occupancy is coupled to the identity or binding mode of the ligand. We analyzed water motions and occupancies using both clustering of trajectories and direct analysis of electron density maps. Our results suggest both BLUES and grand enhance water sampling relative to normal MD and grand is more robust than BLUES, but also that water sampling remains a major challenge for all of the methods tested. The lessons we learned for these methods and systems are discussed.

A New Reactive Ketenaminal: Synthesis, Coupling Reaction, Tautomeric Study, Docking and Antimicrobial Evaluation of the Products

2020 ◽  
Vol 16 (6) ◽  
pp. 761-773
Author(s):  
Huda K. Mahmoud ◽  
Hanadi A. Katouah ◽  
Marwa F. Harras ◽  
Thoraya A. Farghaly
Keyword(s):  
Binding Site ◽  
Antimicrobial Agents ◽  
Coupling Reaction ◽  
Docking Study ◽  
Binding Mode ◽  
Binding Energies ◽  
Simple Method ◽  
Antimicrobial Evaluation ◽  
New Compounds ◽  
Active Methylene

Background: One of the most successful reagents used in the synthesis of the reactive enaminone is DMF-DMA, but it is very expensive with harmful effects on the human health and reacts with special compounds to generate the enaminone such as active methylene centers. Aim: In this article, we synthesized a new ketenaminal by simple method with inexpensive reagents (through desulfurization in diphenylether). Methods: Thus, a novel reactive ketenaminal (enaminone) was synthesized from the desulfurization of 2-((2-(4-chlorophenyl)-2-oxoethyl)thio)-5,7-bis(4-methoxyphenyl)pyrido[2,3-d]pyrimidin- 4(3H)-one with diphenylether. The starting keteneaminal was coupled with diazotized anilines via the known coupling conditions to give a new series of 2-(4-chlorophenyl)-1-(2-(arylhydrazono)-2- oxoethyl)-5,7-bis(4-methoxy-phenyl)pyrido[2,3-d]pyrimidin-4(1H)-ones. Results: The structures of the new compounds were elucidated based on their IR, 1H-NMR, 13CNMR, and Mass spectra. Moreover, the potency of these compounds as antimicrobial agents has been evaluated. The results showed that some of the products have high activity nearly equal to that of the used standard antibiotic. Additionally, the docking study was done to get the binding mode of the synthesized compounds with the binding site of the DHFR enzyme. The results of molecular docking of the synthesized arylhydrazono compounds are able to fit in DHFR binding site with binding energies ranging from -4.989 to -8.178 Kcal/mol. Conclusion: Our goal was achieved in this context by the synthesis of new ketenaminal from inexpensive reagents, which was utilized in the preparation of bioactive arylhydrazone derivatives.

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