RESUMEN
The membrane Fo factor of ATP synthase is highly sensitive to mutations in the proton half-channel leading to the functional blocking of the entire protein. To identify functionally important amino acids for the proton transport, we performed molecular dynamic simulations on the selected mutants of the membrane part of the bacterial FoF1-ATP synthase embedded in a native lipid bilayer: there were nine different mutations of a-subunit residues (aE219, aH245, aN214, aQ252) in the inlet half-channel. The structure proved to be stable to these mutations, although some of them (aH245Y and aQ252L) resulted in minor conformational changes. aH245 and aN214 were crucial for proton transport as they directly facilitated H+ transfer. The substitutions with nonpolar amino acids disrupted the transfer chain and water molecules or neighboring polar side chains could not replace them effectively. aE219 and aQ252 appeared not to be determinative for proton translocation, since an alternative pathway involving a chain of water molecules could compensate the ability of H+ transmembrane movement when they were substituted. Thus, mutations of conserved polar residues significantly affected hydration levels, leading to drastic changes in the occupancy and capacity of the structural water molecule clusters (W1-W3), up to their complete disappearance and consequently to the proton transfer chain disruption.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas , Simulación de Dinámica Molecular , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Mutación , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Bacterianas/química , ATPasas de Translocación de Protón Bacterianas/genéticaRESUMEN
The membrane lipid composition plays an important role in the regulation of membrane protein activity. To probe its influence on proton half-channels' structure in FoF1-ATP synthase, we performed molecular dynamics simulations with the bacterial protein complex (PDB ID: 6VWK) embedded in three types of membranes: a model POPC, a lipid bilayer containing 25% (in vivo), and 75% (bacterial stress) of cardiolipin (CL). The structure proved to be stable regardless of the lipid composition. The presence of CL increased the hydration of half-channels. The merging of two water cavities at the inlet half-channel entrance and a long continuous chain of water molecules directly to cAsp61 from the periplasm were observed. Minor conformational changes in half-channels with the addition of CL caused extremely rare direct transitions between aGlu219-aAsp119, aGlu219-aHis245, and aGln252-cAsp61. Deeper penetration of water molecules (W1-W3) also increased the proton transport continuity. Stable spatial positions of significant amino acid (AA) residue aAsn214 were found under all simulation conditions indicate a prevailing influence of AA-AA or AA-W interactions on the side-chain dynamics. These results allowed us to put forward a model of the proton movement in ATP synthases under conditions close to in vivo and to evaluate the importance of membrane composition in simulations.
RESUMEN
ATP synthase catalyses the formation of ATP from ADP and P(i) and is powered by the diffusion of protons throughout membranes down the proton electrochemical gradient. The protein consists of a water-soluble F(1) and a transmembrane F(0) proton transporter part. It was previously shown that the ring of membrane subunits rotates past a fixed subunit during catalytic cycle of the enzyme. However, many parameters of this movement are still unknown. In the present study the mutual protein movement in the membrane part of F(0)F(1)-ATP syntase has been analysed within the framework of rigid body mechanics. On the base of available experimental data it was shown that electrostatic interaction of two charged amino acids residues is able to supply quite enough energy for the rotation. The initial torque, which caused the rotation, was estimated as 3.7 pN nm and for this pattern the angular movement of c subunits complex could not physically have a period less than 10(-9)s. If membrane viscosity and elastic resistance were taken into account then the time of a whole turnover could rise up to 6.3 x 10(-3)s. It is remarkable that rotation will take place only under condition when the elasticity (Young's) module of the central stalk (gamma subunit and other minor subunits) is less than 5.0 x 10(7)N/m(2). Thus, for generally accepted structural parameters of ATP synthase, two-charge electrostatic interaction model does not permit rotation of the rotor if elastic properties of the central stalk are tougher than mentioned above. In order to explain the rotation under that condition one should either suppose a shorter distance between subunit a and c subunits complex or assume interaction of more than two charged amino acids residues.