Introduction

ATP-synthase is the last protein found at the end of the electron transport chain coupling proton pumping to energy production. However, it is classified as a hydrolase protein due to a naturally conserved function of reverting its main activity, the assembly of ATP. In order to stop the hydrolysis reaction, α-proteobacterium is able to produce a protein called ζ-inhibitor. Binding of ζ-inhibitor to the ATP synthase will enable us to create a high resolution structure via X-ray crystallography shown in Figure 1.1.

1. Structure determination

F-ATP synthase is a membrane bound protein pump found in eubacteria and it is widely used to study the structure and functions of ATP synthases. The first one is simpler than its analogue form present in mitochondria but it still contains the same core subunits. F-ATP synthase consists of 2 main entities, a water-soluble domain, F1 and, Fo which is a membrane embedded domain acting as a proton channel transporting proton across the membrane.

Figure 1.1: Structure of the F-ATP synthase from P.denitrificans with ζ-inhibitor(brown) bound to the αβ-catalytic surface of the enzyme. A and C are show different angles of the overall structure, whereas B is the top view showing the δ-subunit (pink), three α-subunits (magenta) and, three β-subunits (blue), of F1 domain. 

The F1 subunit is a catalytic domain consisting of three α-subunits, three β-subunits, and a single γ and ε subunits. The F1 domain is connected to the Fo domain via central and peripheral stalks. The central stalk is part of the F1 complex comprising γ and ε subunits whereas peripheral chains, b and b', are not distinguished by the current structure. However several parts of the peripheral stalk are resolved: the main two are extended α-helices, chain W and V, attached to two α-subunits of the F1 domain via the δ-subunit.

2. Interactions between the F1 domain and the peripheral stalk 

Figure 2.1: Interactions between the F1 domain and the peripheral stalk. A is the top view of the structure showing the α-helix and β-sheet structure of α, β and δ subunits(shown in magenta, blue and pink respectively). B is showing the interactions between the N-termini of αTP with H2,H3,H4 helices, and  N-termini of αE with H1 and H5 helices. The interactions between the αDP subunit and the structural domains of the peripheral stalk is shown in C. 

The δ subunit is composed of 6 α-helices, H1, H2, H3, H4, H5 and H6 interacting with the three α-subunits of the F1 domain, αDP, αE and αTP. The interactions between the N-terminal regions of the αE subunit with the H1 and H5, and between the N terminal regions of the αTP subunit and H2,H3 and H4 are shown in Figure 2.2. Although the structure of αDP is still not well resolved, this region appears to be associated with two unassigned chains, Ch1 and Ch2. The current model states a mobile region connecting the α-subunit to the peripheral stalk via W and V chains.

3. Structure of Membrane Domain

While F1 is exposed to the cytoplasm, Fo is mainly hydrophobic expanding from the bacterial inner membrane. Two essential components of the protein complex are present in the membrane region (Figure 3.1): the motor; consisting of the c-ring and, the stator; shown in the current structure as an array of seven α-helices.

Figure 3.1. Membrane arrangement. View of the c-ring (white), four α-helices corresponding to the a-subunit (light-orange) and, three unassigned α-helices; one Ch3 (purple) parallel to the membrane plane and the other two forming a hairpin ChY (salmon) perpendicular.

The c-ring is made up of twelve c-subunits, each one has a hairpin structure therefore, one side contains both polypeptide ends whereas, the other faces the central stalk.
  

Figure 3.2 Topology of the c-ring.  The periplasmic side contains the N-terminal and C -terminal regions forming an inner and outer ring, respectively. The cytoplasmic side contains the loop region and is confronting the central stalk. Hydrophobicity represented with colours.

4. Transmembrane proton translocation

The pathway of proton translocation starts in a gap between the c-ring and the a-subunit due to the carboxylate group of Glu60 residue contained on each c-subunit. After anticlockwise rotation, the proton will reach the interface between both subunits where the fifth α-helix will release the proton from the now neutralised carboxylic acid by means of the Arg182 residue. The helix containing such residue is assigned to the fifth helix (aH5), because only four α-helices from the a-subunit are resolved in contrast to the six model that can be read from the protein sequence. It has been proposed that the aH5 and aH6 helices may be involved in attracting the proton into the c-ring.
  

Figure 4.1. Proton translocation (A and B) . A describes the pathway as it binds the Glu60 residue on c-subunit and after rotation, it is released to the cytoplasm through interaction with Arg182 reside, what can be seen in B.

Rotor in action

References :

Mendoza-Hoffmann, F., Zarco-Zavala, M., Ortega, R. & Garcia-Trejo, J. (2018) Control of rotation of the F1FO-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF1 proteins. Journal of Bioenergetics and Biomembranes. 50 Available from: doi: 10.1007/s10863-018-9773-9.

Morales-Rios, E., Montgomery, M. G., Leslie, A. G. W. & Walker, J. E. (2015) Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution. Proceedings of the National Academy of Sciences of the United States of America. 112 (43), 13231-13236. Available from: https://www.ncbi.nlm.nih.gov/pubmed/26460036 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4629361/. Available from: doi: 10.1073/pnas.1517542112.