Interaction of small organic molecules with the sodium pump

Marika Janovská

Doctoral thesis

Abstract:

INTRODUCTION The balance between concentrations of Na and K ions across cell membranes is a fundamental property which is utilized in numerous physiological processes. The establishment of the concentration gradient is carried out by Na+/K+-ATPase (also called sodium pump), which was first identified in the late 1950s in Aarhus by the Danish scientist J. C. Skou. Na+/K+-ATPase is heterodimeric membrane protein consisting of three subunits (?, ?, and ?). It belongs to the family of P-type ATPases, which use energy for ion translocation through the cell membrane from ATP hydrolysis and the pumping process involves a phosphorylated intermediate. For the proper function of Na+/K+-ATPase, Mg2+ cations must be present in cytoplasm as an essential cofactor (Skou 1960). Because of importance of Na+/K+-ATPase in many physiological processes, inhibition of its activity can cause serious illnesses in humans and can be the main reason for the toxicological effects of numerous drugs. Recently, high-resolution crystal structure of Na+/K+-ATPase was described (Morth et al. 2007, Shinoda et al. 2009). Large cytoplasmic loop of Na+/K+-ATPase located between fourth and fifth transmembrane helices of its ?-subunit (C45) contains both phosphorylation and ATP-binding sites. It was shown that it retains its tertiary structure even when separated from the rest of the protein, and can thus still bind ATP (Kubala et al. 2003), and can be expressed in bacterial cells with histidine tag (Gatto et al. 1998). Alkaloids are one of the largest groups of plant secondary metabolites, and they are interesting for their biological activities. Sanguinarine (SG), the benzo[c]phenanthridine alkaloid, exhibits multiple biological effects, in particular antimicrobial and antiinflammatory activities, for which it is used in dental hygiene products and feed additives. On the other hand, SG is linked to toxic effects on humans such as the formation of leukoplakia and the syndrome termed epidemic dropsy. From the chemical point of view, in a solution, there is an acido-basic equilibrium between the cationic- (SG+) and pseudobase (SGOH) forms of SG, and in the gastrointestinal tract, SG is converted to biologically inactive dihydrosanguinarine (DHSG). All the forms exhibit fluorescence. Na+/K+-ATPase has been proposed as a possible target of these alkaloids. METHODS We combined the steady-state and time-resolved fluorescence techniques to distinguish fluorescence properties of the individual SG forms and to study their changes expected upon the interactions with proteins. We used methods of intristic tryptophan fluorescence quenching methods and molecular dynamic simulations to study conformational changes of isolated large cytoplasmic loop of Na+/K+-ATPase (C45) induced by cytoplasmic ligand binding (i. e., Mg2+ and/or ATP). C45 loop was expressed in E. coli. Heterologous expression of recombinant proteins in E. coli is routine method in our lab. RESULTS AND DISCUSSION In the study, where the SG was incubated with Na+/K+-ATPase, evaluation of the SG fluorescence characteristics provided valuable information about the structural details of the SG/enzyme interactions. We were able to identify, which form of SG (SG+ or SGOH) and/or DHSG are able to interact with Na+/K+-ATPase. Moreover, we were able to identify SG+-binding site on Na+/K+-ATPase. Our experiments with isolated large cytoplasmic loop of Na+/K+-ATPase revealed that binding of ATP in the absence of Mg2+ induced the open C45 conformation, while presence of Mg2+ stabilized the closed conformation. Moreover, we introduced a novel method for monitoring changes in electrostatic surface potential (ESP) caused by natural cytoplasmic ligands binding. Our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule

Keywords

Na+/K+-ATPase, sanguinarine, fluorescence, interactions, isolated large cytoplasmic loop of Na+/K+-ATPase, tryptophan fluorescence quenching, ligand binding

Language used: English

Date on which the thesis was submitted / produced: 10. 8. 2010

Thesis defence

• Date of defence: 10. 8. 2010
• Supervisor: Martin Kubala

Univerzita Palackého

Přírodovědecká fakulta

Doctoral programme / field:
Fyzika / Biofyzika