This webpage was produced as an assignment for an undergraduate course at Davidson College.
3D Structure of ATP Synthase
CPK Color Scheme
This chime image was obtained from the Protein Data Bank. It is the F1 unit of ATP Synthase from bovine mitochondria. This tutorial will lead you through the structure of ATP Synthase and, hopefully, provide some information on the relationship between structure and function in this important protein. I will focus mainly on the binding properties and proton pumping functions. For more information on the structure-function relationship, please refer to my "my favorite protein" and "my favorite ortholog" pages (links from my home page).
Let's start by displaying the molecule as seven distinct chains. Click here.
Chains A, B, and C are a-subunits, colored dark-blue, blue, blue-green; chains D, E, F are b-subunits, colored green, green-yellow, yellow. The g-subunit is red.
Nucleotides are shown as spacefilling models. Chain D (green) is bDP, and has a bound ADP; Chain E (green-yellow), which has no bound nucleotide, is subunit bE; Chain F (yellow)
is bTP, and has a bound AMP-PNP. The a-subunits all have bound AMP-PNP (as an ATP analogue). hylogenetic clustering of the integral membrane constituents of F-type ATPases generally corresponds to the phylogenies of the organisms of origin,
and consequently the systems in different organisms are probably orthologues. The a subunit of F0 (one copy per complex) spans the membrane five
or six times. The b subunits (2 copies per complex; heterodimeric in plant chloroplasts and blue green bacteria) span the membrane once; and the c
ubunits (called DCCD-binding lipoproteins; 12 copies per complex) span the membrane two times. Some F-type ATPases such as the
a+-translocating ATPase of Acetobacterium woodii probably contains 3 dissimilar but homologous c-subunit proteolipids of 8 and 18 kDa. The a, b and c-subunits of F-type ATPases are homologues to the B, A and c- (or K-) subunits of V-type and A-type ATPases, respectively. Other subunits in these protein complexes are probably homologous to each other, but this fact can not always be demonstrated by statistical analyses of the
sequencs. Thus, for the A-type ATPase of Methanosarcina mazei, theV-type ATPase of yeast, and the F-type ATPase of E. coli, respectively, the
following subunit equivalences have been suggested: A = Vma1 (A) = b; B = Vma2 (B) = a; C = Vma6 (d) = no E. coli F-type ATPase equivalent; Vma8
(D) = g; Vma4 (E) = d; F = Vma7 (F) = e; I = Vphl/stvl = a+b ?, and K = Vma3 (c) = c. Additionally, the yeast v-type ATPase has 3 dissimilar c-subunits:
Vma3(c), Vmal1(c) and Vma6(c), and three subunits, Vma13(H), Vma5(c) and Vma10(G) which are not found in either the A- or F-type ATPases. All of
the yeast vacurlar ATPase subunits have an equivalent subunit in the V-type ATPases of clathrin-coated vesicles of higher eukaryotes (Walker 1990).
This displays the secondary structure of ATP Synthase. Chains A, B, and C are a-subunits, colored dark-blue, blue, blue-green; chains D, E, F are b-subunits, colored green, green-yellow,
yellow. Please be pacient while the image loads.
Now let's look at where hydrogen atoms are pumped through the protein. This "tube" through ATP Synthase provides a tunnel through the membrane. The protons are pumped from the mitochondrial
matrix, through the inner membrane and into the intermembrane space. I refer to this space as a "tunnel" and not a "channel" because it differs greatly from typical proton channels. The most
important distinction is that when being in conducting state, a membrane channel does not require conformational changes for proton translocation, while FO portion of ATP synthase does. The
transfer rate is also too slow for a channel: at voltage of 100 mV textbooks give a rate of about 106 ions per second for an ion channel, more than 100-fold higher than the maximal corresponding
values reported for FO portion. So the latter is a typical example of a proton transporter (the ability to operate as a pump is further confirming it - no channel can do that). However, the term
"proton channels" is often used for certain regions in the membrane proteins that are involved in proton translocation (e.g. proton channels in the cytochrome oxidase, or proton entrance channel
in bacteriorhodopsin). As they never cross the entire membrane, they are sometimes called "proton half-channels". The proton-translocating region of ATP synthase is formed by subunit a and c-subunit
oligomer. There are two certain aminoacid residues that are critically important for proton translocation. The first is an acidic residue (mostly Glu, in some organisms Asp) in the middle of the second
transmembrane alpha-helix of subunit c. The second is an Arg at the last but one transmembrane helix of subunit a. Almost all mutations in those two residues result in a complete loss of activity.
Several other important hydrophillic aminoacid residues are located on subunit a, but their substitution leads only to a partial loss of activity. The currently favored hypothesis of proton transport
through ATP synthase is based on the stochastic rotary mechanism. It is presumed, that the conserved acidic residue on the c-subunit can be deprotonated (i.e. negatively charged) only when facing the
protein-protein interface between a and c subunits, because it is energetically unfavorable to expose a charge into hydrophobic lipid bilayer. Proton enters through one half-channel, binds to the
unprotonated, negatively charged carboxyl group of the c-subunit conserved Glu (or Asp). The latter becomes electrically neutral and can now enter the hydrophobic lipid phase. As soon as it
does, another c-subunit with protonated Glu (Asp) comes from the lipid phase into protein-protein interface area from the other side and releases it's proton through the other half-channel.
Carrying now a negative charge, it cannot go back, but can go one position forward and accept another proton from the first half-channel. (Walker, et al 1990)
Let's take a closer look...
This view shows the configuration of the b-subunits around the g-subunit.
Hydrophobic residues are white, neutral polar residues are green, basic residues are blue, acidic residues are red. The interactions between the different b-subunits and g-subunit are quite different
in the different configurations. In each case, a hydrophobic loop of b, containing residues 273-280, interacts with a specific hydrophobic patch on g, but this differs in the different
configurations. Interaction for subunit b-DP is at the most C-terminal span, and is completely hydrophobic; for subunit b-TP, the interaction is ~5 Å further towards the N-terminus, and
is "discouraged" by a polar patch (Ser-267, Gly-268) on g at this level, so that the interaction is weak; while for b-E the interaction is another ~4 Å towards the N-terminus, and is
the predominantly hydrophobic interaction is strengthened by a H-bond to Thr-259 of g. In this configuration, additional contacts are found in the "catch-loop", composed of residues
311-320, which only in the b-Econfiguration, form at least 3 extra H-bonds with the g-subunit. This loop does not interact with g in the other configurations. (Ko, et al 2000)
ATP is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase (shown in this tutorial) functions as a
rotary molecular motor. In vitro its y-subunit rotates against the surrounding 33 subunits, hydrolysing ATP in three separate catalytic sites on the a and b-subunits. It is widely believed that reverse
rotation of the y-subunit, driven by proton flow through the associated Fo portion of ATP synthase, leads to ATP synthesis in biological systems. Here you can see the y-subunit and how it rotates to the
right around the y-axis. (Walker, et al 1990)
Click here, then click and hold on the protein and select "rotation".
All eukaryotic F-type ATPases pump 3-4 H+ out of mitochondria, or into thylakoids of chloroplasts, per ATP hydrolyzed. Bacterial F-type ATPases pump
3-4 H+ and/or Na+ (depending on the system) out of the cell per ATP hydrolyzed. These enzymes also operate in the opposite direction, synthesizing
ATP when protons flow through the "ATP synthase" down the proton electrochemical gradient (the "proton motive force" or pmf). V-type ATPases may
pump 2-3 H+ per ATP hydrolyzed (Walker 1990)
To see the protein binding its ligand, Click here.
The b-subunits in cartoon, showing the different conformations with the nucleotide binding site occupied. ANP-PNP are white, ADP is blue.
In their nobel prize winning work on ATP Synthase structure, Boyer and Walker (Royal Swedish Academy, 1997) found that instead of the synthesis of ADP to ATP requiring
energy, it was the binding of ADP to and the phosphate to the enzyme and the release of ATP that actually required the energy. Enzymes typically bind and release substrates spontaneously
(Walker et al, 1990), for this, the overall reaction will require energy. This is how ATP Synthase differs from many enzymes. Here, the asymetrical structure comes into play. Each of the
three beta subunits has different couplings to the gamma, delta, and epsilon subunits, yet they function in the exact same way. Boyer discovered that gamma, delta, and epsilon each
opperate in a cylinder formation that is comprised of alpha and beta subunits (Royal Swedish Academy, 1997). The rotation of these cylinders is hypothesized to be caused by the hydrogen
ion flow across the membrane (Walker et al, 1990). The rotation of alpha and beta subunits causes structural changes in the enzyme which lead to functional changes. With each rotation,
the binding ability of the F1 part of the enzyme changes. The interaction of the gamma subunit with the alphas and betas forces "their active surfaces to assume different three-dimmensional
structures" (Walker et al, 1990).
ATP-binding site in b-subunit. The backbone is colored blue, atoms within 6.0 Å of the AMP-PNP (or ATP) are colored by CPK atom colors; the AMP-PNP is colored yellow. Waters are green.