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)

RESUMEN

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

Proton translocation through the Fo fraction of FoF1-ATP synthase is one of the crucial processes in the catalytic cycle of the enzyme. However, the exact trace of protons movement has not been finally established yet because the location and structure of the half-channels are still the subject of investigation. We described the possible network of polar amino acids residues and water molecules that can favor the preferential proton pathway using molecular dynamics simulation of the membrane part of the E. coli ATP synthase embedded in the lipid bilayer and water environment. The inlet half-channel was a complex structure with two entrances in the form of aqueous lacunae and a highly conservative proton transfer chain near Asp61 of c-subunit including amino acids residues and three structural water molecules (W1-W3), while the outlet half-channel was just a water cavity through which a proton can easily move into the cytoplasm. Moreover, the side chains of Asn214 and Gln252 of a-subunit had the stable spatial positions (SP1-SP3). аAsn214 in position SP3 and аGln252 in SP1, SP2 were oriented towards cAsp61 and could presumably protonate it via W1. Herewith aAsn214 in SP1, SP2 was oriented to aHis245. Thus, the proton transfer chain is always unclosed, and switching between positions SP1/SP2 and SP3 of aAsn214 determines the time of proton transport and the movement in this region is the rate-limiting step. In addition, we found another rare position SP3, in which aGln252 is oriented to aAsn116 and aSer144, located outside of the "main H+ route" and being a dead end. The new findings would help to evaluate the whole process of the proton translocation through FoF1-ATP synthase.

Asunto(s)

RESUMEN

The FoF1-ATP synthase is one of the key enzymes in supplying energy production in almost all living systems. In this paper, we provide a theoretical description of its catalytic cycle using combined mathematical methods. These methods include Langevin dynamics for the rotation of the central protein core and the Monte-Carlo method to model nucleotide and proton binding. This model is the first in which ATP synthesis and hydrolysis can occur depending on the nucleotide concentration and system conditions. The main advantage of the presented model is the possibility of obtaining results for both single-molecular protein-machines and large ensembles of proteins. The calculated rates are close to the experimentally measured rates for a single enzyme. The model has been formalised as a computer simulation that allows researchers to evaluate ATP production in different types of living cells.

Asunto(s)

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.

Asunto(s)