Controlling the Heterodimerisation of the Phytosulfokine Receptor 1 (PSKR1) via Island Loop Modulation

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.

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Controlling the Heterodimerisation of the Phytosulfokine Receptor 1 (PSKR1) via Island Loop Modulation

10.26434/chemrxiv.13333952.v2 ◽

2020 ◽

Author(s):

Joao Victor de Souza Cunha ◽

Matthew Kondal ◽

Piotr Zaborniak ◽

Ryland Cairns ◽

Agnieszka K. Bronowska

Keyword(s):

Small Molecules ◽

Binding Site ◽

Cell Communication ◽

Md Simulations ◽

Free Energy Calculations ◽

Agricultural Applications ◽

Lrr Domain ◽

Signalling Cascade ◽

New Crop ◽

Resolution Structure

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.

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Controlling the Heterodimerisation of the Phytosulfokine Receptor 1 (PSKR1) via Island Loop Modulation

10.26434/chemrxiv.13333952.v1 ◽

2020 ◽

Author(s):

Joao Victor de Souza Cunha ◽

Matthew Kondal ◽

Piotr Zaborniak ◽

Ryland Cairns ◽

Agnieszka K. Bronowska

Keyword(s):

Small Molecules ◽

Binding Site ◽

Cell Communication ◽

Md Simulations ◽

Free Energy Calculations ◽

Agricultural Applications ◽

Lrr Domain ◽

Signalling Cascade ◽

New Crop ◽

Resolution Structure

Phytosulfokine (PSK) is a phytohormone responsible for cell-to-cell communication in plants, playing pivotal role in plant development and growth. The binding of PSK to its cognate receptor, PSKR1, is modulated by the formation of a binding site located between leucine-rich repeat (LRR) domain of PSKR1 and the loop located in the receptor’s island domain (ID). The atomic resolution structure of the extracellular PSKR1 bound to PSK has been reported, however, the intrinsic dynamics of PSK binding and the architecture of PSKR1 binding site remain to be understood. In this work, we used atomistic molecular dynamics (MD) simulations and free energy calculations to elucidate how the PSKR1 island domain (ID) loop forms and binds PSK. Moreover, we report a novel “druggable” binding site which could be exploited for the targeted modulation of the PSKR1-PSK binding by small molecules. We expect that our results will open new ways to modulate the PSK signalling cascade via small molecules, which can result in new crop control and agricultural applications.

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Accounting for the Central Role of Interfacial Water in Protein-Ligand Binding Free Energy Calculations

10.26434/chemrxiv.12668816 ◽

2020 ◽

Author(s):

Ido Ben-Shalom ◽

Zhixiong Lin ◽

Brian Radak ◽

Charles Lin ◽

Woody Sherman ◽

...

Keyword(s):

Free Energy ◽

Binding Site ◽

Binding Free Energy ◽

Md Simulations ◽

Simulation Software ◽

Free Energy Calculations ◽

Computer Hardware ◽

Water Molecules ◽

Energy Calculations ◽

Binding Free Energy Calculations

Rigorous binding free energy methods in drug discovery are growing in popularity due to a combination of methodological advances, improvements in computer hardware, and workflow automation. These calculations typically use molecular dynamics (MD) to sample from the Boltzmann distribution of conformational states. However, when part or all the binding site is inaccessible to bulk solvent, the time needed for water molecules to equilibrate between bulk solvent and the binding site can be well beyond what is practical with standard MD. This sampling limitation is problematic in relative binding free energy calculations, which compute the reversible work of converting Ligand 1 to Ligand 2 within the binding site. Thus, if Ligand 1 is smaller and/or more polar than Ligand 2, the perturbation may allow additional water molecules to occupy a region of the binding site. However, this change in hydration may not be captured by standard MD simulations and may therefore lead to errors in the computed free energy. We recently developed a hybrid Monte Carlo/MD (MC/MD) method, which speeds the equilibration of water between bulk solvent and buried cavities, while sampling from the intended distribution of states. Here, we report on the use of this approach in the context of alchemical binding free energy calculations. We find that using MC/MD markedly improves the accuracy of the calculations and also reduces hysteresis between the forward and reverse perturbations, relative to matched calculations using only MD with or without the crystallographic water molecules. The present method is available for use in the AMBER simulation software.<br>

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Accounting for the Central Role of Interfacial Water in Protein-Ligand Binding Free Energy Calculations

10.26434/chemrxiv.12668816.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ido Ben-Shalom ◽

Zhixiong Lin ◽

Brian Radak ◽

Charles Lin ◽

Woody Sherman ◽

...

Keyword(s):

Free Energy ◽

Binding Site ◽

Binding Free Energy ◽

Md Simulations ◽

Simulation Software ◽

Free Energy Calculations ◽

Computer Hardware ◽

Water Molecules ◽

Energy Calculations ◽

Binding Free Energy Calculations

Rigorous binding free energy methods in drug discovery are growing in popularity due to a combination of methodological advances, improvements in computer hardware, and workflow automation. These calculations typically use molecular dynamics (MD) to sample from the Boltzmann distribution of conformational states. However, when part or all the binding site is inaccessible to bulk solvent, the time needed for water molecules to equilibrate between bulk solvent and the binding site can be well beyond what is practical with standard MD. This sampling limitation is problematic in relative binding free energy calculations, which compute the reversible work of converting Ligand 1 to Ligand 2 within the binding site. Thus, if Ligand 1 is smaller and/or more polar than Ligand 2, the perturbation may allow additional water molecules to occupy a region of the binding site. However, this change in hydration may not be captured by standard MD simulations and may therefore lead to errors in the computed free energy. We recently developed a hybrid Monte Carlo/MD (MC/MD) method, which speeds the equilibration of water between bulk solvent and buried cavities, while sampling from the intended distribution of states. Here, we report on the use of this approach in the context of alchemical binding free energy calculations. We find that using MC/MD markedly improves the accuracy of the calculations and also reduces hysteresis between the forward and reverse perturbations, relative to matched calculations using only MD with or without the crystallographic water molecules. The present method is available for use in the AMBER simulation software.<br>

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How does a small molecule bind at a cryptic binding site?

10.1101/2021.03.31.437917 ◽

2021 ◽

Author(s):

Yibing Shan ◽

Venkatesh P. Mysore ◽

Abba E. Leffler ◽

Eric T. Kim ◽

Shiori Sagawa ◽

...

Keyword(s):

Small Molecules ◽

Binding Site ◽

Small Molecule ◽

Protein Interactions ◽

Binding Sites ◽

Rational Design ◽

Structural Information ◽

Interleukin 2 ◽

Md Simulations ◽

Drug Binding

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are "cryptic": When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands' binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.

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

10.1101/2021.06.14.448350 ◽

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.

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Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123933 ◽

1992 ◽

Vol 50 (1) ◽

pp. 706-707

Author(s):

Ji-da Dai ◽

M. Joseph Costello ◽

Lawrence I. Gilbert

Keyword(s):

Gap Junctions ◽

Small Molecules ◽

Manduca Sexta ◽

Freeze Fracture ◽

Cell Communication ◽

Cellular Level ◽

Thin Sections ◽

Prothoracic Glands ◽

Polyhydroxylated Steroids ◽

Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

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Unveiling the Structural Insights into the Selective Inhibition of Protein Kinase D1

Current Pharmaceutical Design ◽

10.2174/1381612825666190527095510 ◽

2019 ◽

Vol 25 (10) ◽

pp. 1059-1074 ◽

Cited By ~ 1

Author(s):

Raju Dash ◽

Md. Arifuzzaman ◽

Sarmistha Mitra ◽

Md. Abdul Hannan ◽

Nurul Absar ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Protein Kinase ◽

Binding Site ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Selective Inhibitors ◽

Conserved Residues ◽

Protein Kinase D1 ◽

Binding Mechanisms

Background: Although protein kinase D1 (PKD1) has been proved to be an efficient target for anticancer drug development, lack of structural details and substrate binding mechanisms are the main obstacles for the development of selective inhibitors with therapeutic benefits. Objective: The present study described the in silico dynamics behaviors of PKD1 in binding with selective and non-selective inhibitors and revealed the critical binding site residues for the selective kinase inhibition. Methods: Here, the three dimensional model of PKD1 was initially constructed by homology modeling along with binding site characterization to explore the non-conserved residues. Subsequently, two known inhibitors were docked to the catalytic site and the detailed ligand binding mechanisms and post binding dyanmics were investigated by molecular dynamics simulation and binding free energy calculations. Results: According to the binding site analysis, PKD1 serves several non-conserved residues in the G-loop, hinge and catalytic subunits. Among them, the residues including Leu662, His663, and Asp665 from hinge region made polar interactions with selective PKD1 inhibitor in docking simulation, which were further validated by the molecular dynamics simulation. Both inhibitors strongly influenced the structural dynamics of PKD1 and their computed binding free energies were in accordance with experimental bioactivity data. Conclusion: The identified non-conserved residues likely to play critical role on molecular reorganization and inhibitor selectivity. Taken together, this study explained the molecular basis of PKD1 specific inhibition, which may help to design new selective inhibitors for better therapies to overcome cancer and PKD1 dysregulated disorders.

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A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

International Journal of Molecular Sciences ◽

10.3390/ijms22031417 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1417

Author(s):

Azeem Danish ◽

Robin Gedschold ◽

Sonja Hinz ◽

Anke C. Schiedel ◽

Dominik Thimm ◽

...

Keyword(s):

Gap Junctions ◽

Small Molecules ◽

High Throughput Screening ◽

Cell Communication ◽

Receptor Activation ◽

Adenosine A2a Receptor ◽

Intracellular Camp ◽

Donor Cells ◽

A2a Receptor ◽

Adenosine A2a

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

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