F1_ATPase

F1 F0-ATPase is a protein found in chloroplasts, eubacteria, and mitochondria. It is a membrane bound enzyme complex that combines adenosine triphosphate (ATP) synthesis and hydrolysis with the transport of protons, generated by oxidative phosphorylation or photosynthesis, across a membrane (1). It harnesses the energy from a proton gradient by a proton channel to drive the synthesis of ATP. ATP is a nucleoside that transports chemical energy within cells for metabolism; thus, the F1 F0-ATPase is an important protein that scientists study (2). However, no drug complexes for this protein exist. F1 F0-ATPase contains two domains: a globular F1 catalytic domain and a proton-translocating F0 domain that is membrane bound. A central stalk and a stator connect the F1 and F0 domains (3). F1-ATPase from the Bos taurus (PDB ID: 1e79) is located in the mitochondria. This F1-ATPase contains the globular domain and the central stalk. However, it does not contain the F0 domain (this paper will explore the F1-ATPase but will refer to the F0 region to aid in explanation of the F1 region) (4). F1-ATPase has a molecular weight of 371528.59 Da and an isoelectric point at a pH of 5.75 (5). The protein has 9 subunits that are contained in two joining domains: the globular domain that consists of the catalytic alpha and beta subunits and the central stalk domain that penetrates into the globular domain. The central stalk contains the gamma, delta, and epsilon units (4).

The method of discovering the crystal structure of the bovine F1-ATPase involved inhibiting it with dicyclohexylcarbodiimide (DCCD) and then using a spectator dialysis membrane to identify the 3D structure. X-ray diffraction was used to view the crystals. A majority of the structure of the protein was revealed after this process. The catalytic globular region of the F1-ATPase is comprised of three alpha subunits (residues 19–510) and three beta subunits (residues 9–474 of two β subunits and 9–475 of the third β subunit) (4). The gamma, delta and epsilon subunits are shown forming the foot or rotary element of the protein as well as the other subunits that make up the globular protein. However, some residues of the protein are not revealed. These regions include the N-terminal regions of the alpha/beta subunits, the C-terminal regions of the beta subunits, two loop regions of the five and four residues in the gamma subunit, residues 1–14 and the C-terminal residue (146) of the delta subunit, and three residues (48–50) at the C-terminus of the epsilon subunit (4).

The catalytic subunits are arranged alternatively around an asymmetrical antiparallel alpha helical coiled coil of the gamma subunit of the central stalk. The alpha and beta subunits have almost identical structures. Nucleotides bind to both subunits but the beta subunits contain the catalytic nucleotide-binding site (2). Only two of the three beta subunits participate in catalysis since one beta subunit does not contain a catalytic site. The alpha subunits do not participate in catalysis. The two catalytic active beta subunits can have three different conformations: open, loose, and tight. Each corresponds to the affinity for substrates. The open site has a low affinity for substrates, the loose can bind substrates reversibly, and the tight has a high affinity for substrates. In the tight conformation, ATP is produced spontaneously from adenosine diphosphate (ADP) and inorganic phosphate (Pi). This concept of changing the affinity supports the “binding change mechanism” of catalysis that explains that there are different affinities for each catalytic site. The process of conformational change begins with the rotation of the asymmetric central stalk during ATP hydrolysis. This rotation occurs in 120° steps and changes the caralytic site affinities during each step. In the ATPase, the rotation of the central stalk is the important coupling element in the protein (4).

The central stalk domain of the F1-ATPase consists of the gamma (residues 1–61, 67–96 and 101–272), delta (residues 15–145), and epsilon subunits (residues 1–47). This central stalk penetrates into the F1 sububit's catalytic area where the three α subunits and three β subunits are arranged (4). As previously stated, the rotation of the central stalk is the important coupling agent in the protein since it assists in the transfer of energy from the F0 domain to the F1 domain. This energy transfer causes both of the domains to rotate (3). This occurs because the F0 domain uses the power from the proton gradient to force the F1 domain to produce ATP. As the F0 domain rotates, it turns the central stalk, causing the F1 domain to also rotate. This process is reversible (1).

F1-ATPase contains six ligands. Three of them: dicyclohexylurea, glycerol, and a sulfate ion, were used during the crystallization of the protein and have no biological function with the protein in cells. The three ligands that play a major role in the biological function are a magnesium metal ion, ATP, and ADP. The magnesium ion is bound to the ADP and ATP, which are bound to the alpha and beta subunits of the catalytic globular F1 domain. Residues in the βDP subunit (the others are called βTP and βE) that contribute to the binding site of ATP or ADP include Val-164 and Met-167 from helix B and Val-420 and Phe-424 from the C-terminal domain. The rotation of the central stalk begins in the γ subunit. Three carboxyl groups (γAsp-194, γAsp-195 and γAsp-97) in the visible lower portion of the base increase downwards, signifying that, in ATP synthase, they might cooperate with the basic amino acids in the loop regions on the c-ring portion of the subunit, a part of the F0 domain (4). It is currently thought that a trans-membrane proton gradient drives rotation of the c-subunit ring of F0, which is then coupled to movement of the central stalk. The rotation of the central stalk causes conformational changes in the catalytic sites located in the F1 domain leading to the synthesis of ATP (3).

F1-ATPase contains a variety of secondary structures. The only beta sheets found in the protein are in the delta subunit and in the gamma subunit. The gamma subunit contains a five-stranded beta sheet. Alpha helices exist on the central stalk subunits and the alpha/beta complex. The other secondary structures in the protein are in the form of random coils and they exist in every subunit connecting the different helices and sheets. One example of secondary structure interactions occurs in the delta subunit. The bovine δ subunit has two domains: an N-terminal β-sandwich (residues 15–98) and a C-terminal α-helical hairpin (residues 105–145). The β-sandwich consists of 10 β-strands with a hydrophobic interior. A 310-helix (residues 99–104) is in the loop connecting the β-sandwich to the two C-terminal helices. One example of hydrogen bonding in the delta subunit occurs with the Arg-23 and Lys-27 amino acid residues. These are located in the beta sandwich and they assist in the stabilization of the secondary structure. There are other ionic and hydrogen bonding in F1-ATPase’s central stalk domain. One example is with the residue Trp-4, which is located at the interface of γ and δ subunits. Another residue, Tyr-11, forms part of the hydrophobic pocket for Trp-4, which allows the Trp-4 to hydrogen bond to the δ subunit (4). Another example of ionic interactions involving the central stalk includes the interaction with the alpha/beta subunits of the F1 domain, which ultimately leads to the rotation of the F1 domain and the synthesis of ATP. An Arg residue at position 75 interacts with βDPGlu-395 and αEAsp-409 (the three alpha subunits are called αE, αTP, and αDP) in the region where the γ subunit emerges from the alpha/beta domain. This is known as the DELSEED region. Residue γArg-75 is in a loop, linking strand 1 (residues 68–71) to helix b (residues 77–96) of the gamma subunit (4). The γ subunit rotation in F1-ATPase is known to proceed in 120° steps, signifying that three steady conformational intermediates are present in the rotary catalytic cycle as discussed before. Polar interactions such as those involving γArg-75 may contribute to the stabilization of these intermediate conformations (3).

The F1-ATPase is involved in the electron transport chain in the mitochondria that produces ATP for the cell. This indicates that the F1-ATPase is something that all organisms and cells need to engage in cellular activity since ATP is a necessity for this metabolic activity (4). The optimal ATPase is in the heart of the Bos taurus, based on its biochemistry and structure. This is because beef heart has a high concentration of mitochondria in cardiac muscle (1). Another organism that is useful for the exploration of the F1-ATPase and which has the same function as the bovine F1-ATPase is the Rattus rattus (black rat; PDB ID: 1MAB). The ATPase in the liver of the Rattus rattus was investigated (6). This F1-ATPase has a molecular weight of 136531.4 Da and an isoelectric point at a pH of 5.85 (5). According to the PSI-Blast of the bovine F1-ATPase, the E-value of the black rat’s F1-ATPase is 0.0. The PSI blast is used to find similarity in the primary structure of a given protein query. An E-value is assigned for similarity. The lower the E-value, the more similar the proteins are. An E-value below 0.05 indicates high similarity (7). The value is the lowest possible for the black rat; thus, it is identical to the bovine protein in terms of the primary structure. The Dali server is another system used to compare protein structure. It compares the 3D structure of the protein query. The 3D structure can reveal functional clues about the protein. This server compares the 3D structures based on a Z-score. The higher the Z-score, the more the 3D structures are equivalent. A score above two indicates similar folds. The Z score for the black rat F1-ATPase compared to the protein of the bovine is equal to 55.9 (8). This indicates that it is similar but there are some deviations. One major difference between the two 3D structures is a conformational alteration that is seen in the catalytic globular region that consists of the alpha and beta complex. The bovine ATPase is a two-nucleotide structure that has only nucleotides at 2/3 of the beta subunits. The empty beta subunit has a substantially different conformation from the other two. The rat ATPase has nucleotides at 3/3 of the beta subunits. Instead of having two of the three beta subunits participate in catalysis since one beta subunit does not contain a nucleotide, the black rat’s F1-ATPase has a nucleotide at all three of the beta subunits (6). Besides this difference, the rest of the 3D structure of the black rat’s F1-ATPase is similar to the F1-ATPase in the cow. For example, ATP and ADP are the ligands that participate in ATP synthesis. There are metal ions present in the protein that were used during the crystallization process. The black rat has a phosphate ion in addition to the magnesium ion (6). This is one other minor difference between the rat and bovine structure: the magnesium ion in addition to the phosphate ion bind to ADP and ATP in the rat ATPase. One other similarity between the two proteins is that they both have a gamma subunit in the middle of the alpha and beta complex, which attaches to the central stalk of the protein (6). Another F1-ATPase that is under much study is in the Saccharomyces cerevisiae (PDB ID: 2HLD) (9). The yeast F1-ATPase, when compared with the bovine F1-ATPase, has a Z-score of 62.7 and an E-value of 0.0 (7,8). This means that the 3D structure is different from the bovine F1-ATPase. There are many similarities between the two proteins. First, the residues on the active sites of the bovine and yeast beta complexes are identical. Second, the yeast ATPase also has the gamma subunit attached to the alpha and beta subunit (9). Although the three protein structures compared are slightly different in their 3D structures, the function of the protein remains the same.