The EcCLC antiporter embedded in lipidic liquid crystalline films - molecular dynamics simulations and electrochemical methods.

Lipidic-liquid crystalline nanostructures (lipidic cubic phases), which are biomimetic and stable in an excess of water, were used as a convenient environment to investigate the transport properties of the membrane antiporter E. coli CLC-1 (EcCLC). The chloride ion transfer by EcCLC was studied by all-atom molecular dynamics simulations combined with electrochemical methods at pH 7 and pH 5. The cubic phase film was used as the membrane between the chloride donor and receiving compartments and it was placed on the glassy carbon electrode and immersed in the chloride solution. Structural characterization of lipidic mesoscopic systems with and without the incorporation of EcCLC was performed using small-angle X-ray scattering. The EcCLC transported chloride ions more efficiently at more acidic pH, and the resistance of the film decreased at lower pH. 4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) employed as an inhibitor of the protein was shown to decrease the transport efficiency upon hydrolysis to DADS at both pH 7 and pH 5. The molecular dynamics simulations, performed for the first time in lipidic cubic phases for EcCLC, allowed studying the collective movements of chloride ions which can help in elucidating the mechanism of transporting the ions by the EcCLC antiporter. The protein modified lipidic cubic phase film is a convenient and simple system for screening potential inhibitors of integral membrane proteins, as demonstrated by the example of the EcCLC antiporter. The use of lipidic cubic phases may also be important for the further development of new electrochemical sensors for membrane proteins and enzyme electrodes.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis.

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Specificities of Protein Homology Modeling for Allosteric Drug Design.

The allosteric binding sites are usually located in the flexible areas of proteins, which are hardly visible in the crystal structures. However, there are notable exceptions like allosteric sites in receptors in class B and C of GPCRs, which are located within a well-defined bundle of transmembrane helices. Class B and C evolved from class A and even after swapping of orthosteric and allosteric sites the central binding site persisted and it can be used for easy design of allosteric drugs. However, studying the ligand binding to the allosteric sites in the most populated class A of GPCRs is still a challenge, since they are located mostly in unresolved parts of the receptor's structure, and especially N-terminus. This chapter provides an example of cannabinoid CB1 receptor N-terminal homology modeling, ligand-guided modeling of the allosteric site in GABA receptor, as well as C-linker modeling in the potassium ion channels where the allosteric phospholipid ligand PIP2 is bound.