scholarly journals Distinct binding modes and structural changes induced by cAMP and cGMP in the GAF domain of Anabaena adenylyl cyclase, CyaB2

2014 ◽  
Author(s):  
Kabir H Biswas ◽  
Suguna Badireddy ◽  
Ganesh S Anand ◽  
Sandhya S Visweswariah
Keyword(s):  
Ligand Binding ◽  
Adenylyl Cyclase ◽  
Structural Changes ◽  
Mutational Analysis ◽  
Allosteric Regulation ◽  
Large Family ◽  
Structural Basis ◽  
Binding Modes ◽  
Cyclase Domain ◽  
Camp And Cgmp

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis indicated distinct modes of binding of cAMP and cGMP to the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by the highly sensitive Bioluminescence Resonance Energy Transfer (BRET) experiments. Amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments, however, revealed the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the structural changes induced by cAMP and cGMP binding to the GAF domain. Thus, our results provide an insight into structural mechanisms of ligand binding to GAF domains in general, which can be utilized in developing molecules that modulate the allosteric regulation by GAF domains in pharmacologically relevant proteins.

scholarly journals Distinct binding modes and structural changes induced by cAMP and cGMP in the GAF domain of Anabaena adenylyl cyclase, CyaB2

2014 ◽  
Author(s):  
Kabir H Biswas ◽  
Suguna Badireddy ◽  
Ganesh S Anand ◽  
Sandhya S Visweswariah
Keyword(s):  
Ligand Binding ◽  
Adenylyl Cyclase ◽  
Structural Changes ◽  
Mutational Analysis ◽  
Allosteric Regulation ◽  
Large Family ◽  
Structural Basis ◽  
Binding Modes ◽  
Cyclase Domain ◽  
Camp And Cgmp

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis indicated distinct modes of binding of cAMP and cGMP to the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by the highly sensitive Bioluminescence Resonance Energy Transfer (BRET) experiments. Amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments, however, revealed the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the structural changes induced by cAMP and cGMP binding to the GAF domain. Thus, our results provide an insight into structural mechanisms of ligand binding to GAF domains in general, which can be utilized in developing molecules that modulate the allosteric regulation by GAF domains in pharmacologically relevant proteins.

scholarly journals Structural basis of ligand binding modes at the human formyl peptide receptor 2

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tong Chen ◽  
Muya Xiong ◽  
Xin Zong ◽  
Yunjun Ge ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Formyl Peptide Receptor ◽  
Structural Basis ◽  
Binding Modes ◽  
Peptide Receptor ◽  
Formyl Peptide

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Abstract Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

scholarly journals Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

Nature ◽  
2018 ◽  
Vol 556 (7702) ◽  
pp. 520-524 ◽  
Author(s):  
Zhenlin Yang ◽  
Shuo Han ◽  
Max Keller ◽  
Anette Kaiser ◽  
Brian J. Bender ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Neuropeptide Y ◽  
Structural Basis ◽  
Binding Modes ◽  
Y1 Receptor

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

2020 ◽  
Author(s):  
Samuel C. Gill ◽  
David Mobley
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Sites ◽  
Degrees Of Freedom ◽  
Dynamics Simulation ◽  
Hiv Integrase ◽  
Binding Modes ◽  
System A ◽  
Multiple Binding ◽  
Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

2017 ◽  
Author(s):  
Samuel Gill ◽  
Nathan M. Lim ◽  
Patrick Grinaway ◽  
Ariën S. Rustenburg ◽  
Josh Fass ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Binding Modes ◽  
Enhanced Sampling ◽  
Recent Success ◽  
Multiple Binding ◽  
Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

2018 ◽  
Author(s):  
Samuel Gill ◽  
Nathan M. Lim ◽  
Patrick Grinaway ◽  
Ariën S. Rustenburg ◽  
Josh Fass ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Binding Modes ◽  
Enhanced Sampling ◽  
Recent Success ◽  
Multiple Binding ◽  
Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

scholarly journals Multi-Targeting Bioactive Compounds Extracted from Essential Oils as Kinase Inhibitors

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2174 ◽  
Author(s):  
Annalisa Maruca ◽  
Delia Lanzillotta ◽  
Roberta Rocca ◽  
Antonio Lupia ◽  
Giosuè Costa ◽  
...  
Keyword(s):  
Essential Oils ◽  
Binding Affinity ◽  
Structural Changes ◽  
Kinase Inhibitors ◽  
Synergistic Effects ◽  
Data Bank ◽  
Binding Modes ◽  
Starting Point ◽  
Target Action ◽  
Potential Resource

Essential oils (EOs) are popular in aromatherapy, a branch of alternative medicine that claims their curative effects. Moreover, several studies reported EOs as potential anti-cancer agents by inducing apoptosis in different cancer cell models. In this study, we have considered EOs as a potential resource of new kinase inhibitors with a polypharmacological profile. On the other hand, computational methods offer the possibility to predict the theoretical activity profile of ligands, discovering dangerous off-targets and/or synergistic effects due to the potential multi-target action. With this aim, we performed a Structure-Based Virtual Screening (SBVS) against X-ray models of several protein kinases selected from the Protein Data Bank (PDB) by using a chemoinformatics database of EOs. By evaluating theoretical binding affinity, 13 molecules were detected among EOs as new potential kinase inhibitors with a multi-target profile. The two compounds with higher percentages in the EOs were studied more in depth by means Induced Fit Docking (IFD) protocol, in order to better predict their binding modes taking into account also structural changes in the receptor. Finally, given its good binding affinity towards five different kinases, cinnamyl cinnamate was biologically tested on different cell lines with the aim to verify the antiproliferative activity. Thus, this work represents a starting point for the optimization of the most promising EOs structure as kinase inhibitors with multi-target features.

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