Biological Research Centre, Szeged
Centre of Excellence of the European Union

Proton pumping by a membranous molecular motor, the vacuolar proton-ATPase (V-ATPase)

The V-ATPase is a membrane-bound molecular rotary engine, which converts the chemical energy from ATP hydrolysis to the rotation of the rotor domain via a torque between specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and rotor domains. The V-ATPase plays an important role in diseases like osteoporosis and in the metastasis of tumours. Therefore, specific inhibition of certain sub-classes of the V-ATPase family has direct medical and pharmaceutical relevance. To date there is no atomic resolution structure of the V-ATPase known and its mechanism of function is not known either.

In extensive international collaborations we have shown earlier that the c subunit of the Vo domain is a representative of the ductins family of highly homologous and structurally similar membrane proteins. We have determined the assembly of the subunit c ring in native membranes as a hexamer of four-helix bundles and found that highly specific V-ATPase inhibitors perturb the lipid-protein interface of the Vo domain. We localized a unique cysteine and the glutamic acid residue, essential for proton transport, which were found to face lipids.

The primary long-term objective is the better understanding of structure-function relationship and the identification of functionally relevant structural changes in the engine. This project is aimed at the study of the proton pumping and ATP hydrolyzing functions of the Vo and V1 domains, respectively, their connection and interaction; the arrangement and interactions, also with lipids, of the Vo subunits; the rotation of the rotor domain; structural stability and the effect of structural and functional agents, e.g. specific inhibitors, on all these features in intact vacuoles, vacuolar vesicles and in reconstituted V-ATPase-lipid vesicles. We aim to develop the structural models of the intra-membranous a and c subunits based on our structural data, whereas the function will be interpreted in physical models.

Protein insertion, folding and assembly in membranes and on membrane surfaces

Protein folding in general and membrane protein folding in particular are most challenging problems in biophysics today, because membrane lipids and proteins are coupled structurally, dynamically and functionally. The protein-lipid interface takes several different forms, all of which are crucial to biology. For biological membranes the protein-lipid interface may be either polar in the case of surface-bound or absorbed peripheral proteins, or apolar in the case of integral trans-membrane proteins. Focusing on the protein-lipid interface requires studies on the structure, the dynamics and the function of both membrane proteins and lipids. We are also interested in this problem, because the work on membrane proteins requires they be inserted and assembled properly in the bilayer to achieve functional reconstitution. Our objective is to obtain experimental data on factors guiding insertion, folding and assembly of proteins and polypeptides in membranes and on membrane surfaces. These data are used to guide and constrain molecular and physical models. We pay increasing attention to theoretical approaches to membrane protein folding. At present, we focus on three groups of proteins: trans-membrane helix (TMH; V-ATPase subunits and polypeptides), beta-barrel (E. coli outer membrane proteins) and soluble proteins interacting with bio- and model membranes (lysozyme).

Approaches and techniques

The working strategy for the above membrane-protein systems is that structural, dynamic and thermodynamic data on native and reconstituted membranes are measured, during permanent control of the biological function, wherever possible, using a range of biophysical techniques, which are then consistently interpreted in detailed molecular models and related to the biological function. Data are obtained with a variety of techniques and their combinations. These include Fourier-transform infrared (FTIR), site-specific spin-labelling and spin-trapping electron paramagnetic resonance (EPR), polarized attenuated total internal reflection FTIR, UV, visible and fluorescence spectroscopy; high-sensitivity differential scanning calorimetry (DSC); theory and computation (physical models, spectrum simulations and molecular modelling). This approach can be termed as function-controlled spectroscopy-based structural biology.