Protein-Ligand Interactions In F1F0-ATP Synthase Enzyme Complex And ATP Synthesis

Interaction between F1F0-ATPase and ATP

Question:

Discuss about the Protein Ligand Interaction.

Introduction

Oxidative phosphorylation is the process that involves the formation of ATP and constitutes the fundamental and most important means of energy production for the cells of animal, plant and bacterial origins (Jonckheere, Smeitink & Rodenburg, 2012). Apart from oxidative phosphorylation, photophosphorylation is also involved in the synthesis of ATP and both these processes are dependent on the F₁F₀-ATPase enzyme complex for catalyzing the ATP synthesis (Nath & Villadsen, 2015). The F₁F₀-ATPase enzyme is also called the ATP synthase enzyme. The source of energy that helps to carry out ATP synthesis is an electrochemical proton gradient. This proton gradient is produced by the electron transfer complexes that are present in the mitochondria, chloroplast or the bacterial membranes (Jonckheere, Smeitink & Rodenburg, 2012).

This essay describes the protein ligand interactions with respect to the F₁F₀-ATP synthase enzyme complex (protein) and ATP(ligand).

Interaction between F₁F₀-ATPase and ATP

In prokaryotes, the F₁F₀-ATPase enzyme complex consists of 8 different sub-units while in mammals the number of sub-units are 16-18. The molecular weight of the enzyme typically ranges between 550-650kDa. It is located in the bacterial plasma membrane, while in plants it is located in the thylakoid membrane of the chloroplasts and mitochondrial inner membranes and in animals it is located in the inner membranes of the mitochondria. The enzyme consists of 2 parts designated F₁ and F₀. The F₁consists of 5 sub-units, designated α₃, β₃, γ, δ and ε. The F₀ part on the other hand consists of 3 sub-units designated a, b and c. The stoichiometry of the a, b and c sub-units of the F₀ is ab₂c_10-14(Hahn et al., 2016). The 2 parts of the enzyme complex, which includes F₁ and F₀ are connected to each other by 2 stalks, a central one and the other a peripheral one. The central stalk consists of the γ and ε sub-units, while the peripheral stalk comprises the δ and b sub-units. Interactions between F₁ and F₀are said to be coupled and disruption of the interactions results in the loss of energy transduction and the system assumes an uncoupled state. Each of the 3 β sub-units present in the F₁consists of catalytic sites for ATP synthesis. The molecular interactions involved in the synthesis of ATP by the F₁F₀-ATPase was proposed by Paul Boyer and it was called the rotational catalysis mechanism or the binding change or alternating site hypothesis (Capaldi & Aggeler, 2002). According to this mechanism or hypothesis, the 3 active sites of the F₁ part of the enzyme complex participates in the catalysis of ATP synthesis. One of the β sub-units binds ADP and Pi, thereby assuming the β-ADP conformation. The sub-unit then assumes a β-ATP conformation by tightly binding to ATP and bringing about an equilibration of both ATP and ADP/Pi on the surface of the enzyme.

Proton transport and the mechanism of ATP synthesis

On assuming the β-empty conformation, the sub-unit loses its affinity for ATP and as a result the newly synthesized ATP then is released from the enzyme complex. The next round of catalysis begins when the β sub-unit again binds to ADP and Pi and assumes the β-ADP conformation. During the catalysis reactions, the 3 sub-units interacts and assumes the conformations in such a way that one is in the β-ATP conformations, while another is in the β-ADP conformation and the third is in the β-empty conformation. The conformational changes are brought about by the passage of protons through the F₀ portion of the F₁F₀-ATPase. The streaming of the protons through the pore of F₀, results in the rotation of the cylinder part of F₀ composed of the c sub-units and the γ sub-units along its long axis. The rotation is about 120 degrees and results in the contact of the γ sub-unit with each and every catalytic sub-unit, thereby forcing them to assume the β-empty conformations (Nelson, Cox & Lehninger, 2012). These conformations of the β sub-units are also called the open, partly open and closed conformations, where the open conformation binds ATP or ADP/Pi. Binding of ATP and the closure of the open site, results in a conformational change of the remaining catalytic sub-units that results in the partly open sub-unit to attain the open conformation and the closed sub-unit to attain the partly open conformation (Capaldi & Aggeler, 2002). Proton transport is dependent on the a and c sub-units of the F₀ portion of the F₁F₀-ATPase. And the amino acid residues aspartate at position 61 of the c sub-units and Arginine at position 210 of the a sub-units are critical for carrying out the function of the proton transport. The rotation of the c sub-units takes place relative to that of the a sub-unit. The rotor stalk composed of the γ and ε sub-units are firmly attached to the c sub-units at the base, while interacting with the α and β sub-units at the top. The stator stalk consists of the b₂ and δ sub-units, where the b₂ interacts with the a sub-units and the δ interacts with the α sub-units. Moreover, the arginine residue at position 376 of the α sub-unit is involved in the generation of the transition state structure of the catalytic sub-units during steady state catalysis. The positive charge of αArg376 along with those of βLys155 and βArg182, results in the formation of a Pi binding pocket. Binding of Pi prevents the binding of ATP. Upon formation of the partly open β-ADP conformation, the movement of the C- terminal region of the enzyme along with the nucleotide binding domain results in the re-positioning of the catalytic side chains, designated βGlu181 and βArg182. Thus, this step involves priming of the catalytic site for subsequent catalytic steps. The next step involves transition from ADP/ Pi to ATP. This is brought about by the rotation of the γ sub-unit that results in the movement of the α sub-unit towards the β and the subsequent movement of the αArg376 and βArg182 towards ADP. Co-ordination of a Mg²⁺ along with the residues αArg376, βLys155 and βArg182 brings about the synthesis of ATP. The γ sub-unit then undergoes further rotation resulting in the withdrawal of the αArg376 from the catalytic site, thereby causing the release of the ATP (Senior, Nadanaciva & Weber, 2002; Martin et al., 2015).

Classical inhibitor of ATP synthase, DCCD

                                           Figure 1: The arrangements of the sub-units of the ATP synthase enzyme complex.

                                                                     (Source: Capaldi & Aggeler, 2002)

Figure 2: Proton motive force and ATP synthesis mechanism.

(Source: Senior, Nadanaciva & Weber, 2002)

Figure 3: The various conformations of the catalytic site of the enzyme.

(Source: Source: Capaldi & Aggeler, 2002)

The oxidative phosphorylation is the metabolic pathway that is involved in the oxidation of nutrients in order to release energy in the form of ATP. Oxidative phosphorylation involves the occurrence of redox reactions with the help of which electrons are transferred to electron acceptors like oxygen from the electron donors. The flow of electrons is called the energy transport chain. The energy released is used in the transport of protons. Here the ATP synthase enzyme utilizes the energy of the proton gradient to convert ADP to ATP. It is the last enzyme of the oxidative phosphorylation pathway (Bazil, Beard & Vinnakota, 2016). Apart from oxidative phosphorylation, photophosphorylation is another process that utilizes the ATP synthase enzyme to carry out phosphorylation of ADP to ATP during photosynthesis. Th transfer of electrons helps to generate a proton motive force that helps in the activation of the ATP synthase enzyme (Junge & Nelson, 2015).

The DCCD is the classical inhibitor of the ATP synthase. It functions by modification of the proton binding site present in the c sub-unit. It causes steric hindrance of the movement of the c sub-unit relative to the a sub-unit, thereby preventing the rotation of the F₀F₁ complex (Toei & Noji, 2013).

Conclusion

Thus, it can be concluded that the ATP synthase is an integral part of the metabolism and energy production pathways of various organisms and the intricate molecular interactions of ATP with the enzyme complex constitutes the highlight of such an important metabolic reaction.

Reference List

Bazil, J. N., Beard, D. A., & Vinnakota, K. C. (2016). Catalytic coupling of oxidative phosphorylation, ATP demand, and reactive oxygen species generation. Biophysical journal, 110(4), 962-971.

Capaldi, R. A., & Aggeler, R. (2002). Mechanism of the F1F0-type ATP synthase, a biological rotary motor. Trends in biochemical sciences, 27(3), 154-160.

Hahn, A., Parey, K., Bublitz, M., Mills, D. J., Zickermann, V., Vonck, J., ... & Meier, T. (2016). Structure of a complete ATP synthase dimer reveals the molecular basis of inner mitochondrial membrane morphology. Molecular cell, 63(3), 445-456.

Jonckheere, A. I., Smeitink, J. A., & Rodenburg, R. J. (2012). Mitochondrial ATP synthase: architecture, function and pathology. Journal of inherited metabolic disease, 35(2), 211-225.

Junge, W., & Nelson, N. (2015). ATP synthase. Annual review of biochemistry, 84, 631-657.

Martin, J., Hudson, J., Hornung, T., & Frasch, W. D. (2015). Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase. Journal of Biological Chemistry, 290(17), 10717-10728.

Nath, S., & Villadsen, J. (2015). Oxidative phosphorylation revisited. Biotechnology and bioengineering, 112(3), 429-437.

Nelson, D., Cox, M., & Lehninger, A. (2012). Lehninger Principles of biochemistry (6th ed.). New York: W.H.Freeman and Co.

Senior, A. E., Nadanaciva, S., & Weber, J. (2002). The molecular mechanism of ATP synthesis by F1F0-ATP synthase. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1553(3), 188-211.

Toei, M., & Noji, H. (2013). Single-molecule analysis of F0F1-ATP synthase inhibited by N, N-dicyclohexylcarbodiimide. Journal of Biological Chemistry, 288(36), 25717-25726.