scholarly journals Fluoride resistance and transport by riboswitch-controlled CLC antiporters

2012 ◽  
Vol 109 (38) ◽  
pp. 15289-15294 ◽  
Author(s):  
Randy B. Stockbridge ◽  
Hyun-Ho Lim ◽  
Renee Otten ◽  
Carole Williams ◽  
Tania Shane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Membrane Transport ◽  
Lipid Bilayer ◽  
Anion Binding ◽  
Planar Lipid Bilayer ◽  
Sequence Alignments ◽  
Conserved Residues ◽  
Anion Selectivity ◽  
Osmotic Response

A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F-. We establish here that a set of randomly selected representatives from this “CLCF” clade protect Escherichia coli from F- toxicity, and that the purified proteins catalyze transport of F- in liposomes. Sequence alignments and membrane transport experiments using 19F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLCF proteins apart from all other known CLCs. First, CLCFs lack conserved residues that form the anion binding site in canonical CLCs. Second, CLCFs exhibit high anion selectivity for F- over Cl-. Third, at a residue thought to distinguish CLC channels and transporters, CLCFs bear a channel-like valine rather than a transporter-like glutamate, and yet are F-/H+ antiporters. Finally, F-/H+ exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1.

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

2019 ◽  
Vol 25 (10) ◽  
pp. 1059-1074 ◽  
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.

scholarly journals Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle

2020 ◽  
Vol 22 (1) ◽  
pp. 366
Author(s):  
Mao Arai ◽  
Tomohiro Miura ◽  
Yuriko Ito ◽  
Takatoshi Kinoshita ◽  
Masahiro Higuchi
Keyword(s):  
Binding Site ◽  
Lipid Bilayer ◽  
Structural Changes ◽  
Bilayer Membrane ◽  
Lipid Bilayer Membrane ◽  
Target Protein ◽  
Self Organization ◽  
Specific Response ◽  
Target Proteins ◽  
Recognition Ability

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

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