The structure of the ATP-bound state of S. cerevisiae phosphofructokinase determined by cryo-electron microscopy

Abstract

Phosphofructokinase (Pfk1, EC 2.7.1.11) plays a key regulatory role in the glycolytic pathway. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inhibited states of the bacterial enzyme, and of the role of the two bacterial effectors. Eukaryotic phosphofructokinases exhibit a far more sophisticated regulatory mechanism, they are more complex structures regulated by a large number of effectors (around 20). Saccharomyces cerevisiae Pfk1 is an 835 kDa hetero-octamer which shows cooperative binding for fructose-6-phosphate (F6P) and non-cooperative binding for ATP. The 3D structure of the F6P-bound state was obtained by cryo-electron microscopy to 1.1 nm resolution. This electron microscopy structure, in combination with molecular replacement using the bacterial enzyme has helped provide initial phases to solve the X-ray structure of the F6P-bound state 12S yeast truncated-tetramer. Biochemical and small-angle X-ray scattering (SAXS) studies had indicated that Pfk1 underwent a large conformational change upon Mg-ATP binding. We have calculated a reconstruction using reference-based 3D projection alignment methods from 0 degrees images acquired from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP. The ATP-bound structure is more extended or open, and the calculated radius of gyration of 7.33 nm (7.0 nm for F6P) is in good agreement with the SAXS data. There is a substantial decrease in the rotational angle between the top and bottom tetramers. Interestingly, all these changes have arisen from a reorientation of the alpha- and beta-subunits in the dimers. The interface region between the alpha- and beta-subunits is now approximately half the size of the one in the F6P-bound structure. This is the first time that the 3D structure of a eukaryotic Pfk1 has been visualized in its T-state (inhibited-state).

Figure 1

a) Area of a micrograph from a vitreous ice preparation. Circles show Pfk1 molecules. Scale bar 100 nm. b) Average periodogram calculated from the whole micrograph showing data to 0.8–0.9 nm resolution. The edge of the periodogram corresponds to 1/0.48nm⁻¹. The arrow points to the last visible ring in the periodogram (Radermacher, et al., 2001; Ruiz, et al., 2003).

Figure 2

a) Surface representation of the 3D reconstruction calculated from 14500 0°-projections in random orientations encompassing 110% of the molecule mass. The top-tetramer is shown in yellow and the bottom-tetramer in blue. The L-shaped α- and β-subunits are shown. The small-subdomains of both α- and β-subunits are labeled with (#), the large subdomains with (*) and two the catalytic-sites per αβ-dimer with (†). Scale bar 10 nm. b) same as (a) front-view or 0°-rotation, c) side-view 36°-rotation, d) side-view 72°-rotation, e) side-view 144°-rotation, f) bottom-view, 90°-rotation, g) top-view, 90°-rotation.

Figure 3

Fourier shell correlation curve (solid line) and five times noise correlation curve (dashed line). The resolution is 1.3 nm with the 0.3 criterion (Rosenthal and Henderson, 2003).

Figure 4

Surface representations of the structure of Pfk1 in the presence of F6P (a) and ATP (b). The two views of each state are related by the noted rotation around the long axis of the molecule, which gives the best fit between the top and bottom tetramers. The large-subdomains of both α- and β-subunits are labeled with (*). Scale bar 10 nm.

Figure 5

Superposition of the structures of Pfk1 in the presence of F6P and in the presence of ATP after alignment of the whole octamer (Chimera representation). The two views (a and b) are related by a 90° rotation around the long axis of the molecule. F6P-bound state (solid yellow), ATP-bound state (transparent blue). The region where the two surfaces coincide is shown in white. The large-subdomains of both α- and β-subunits are labeled with (*). Scale bar 10 nm.

Figure 6

Superposition of the two tetramers of the structures of Pfk1 in the presence of F6P (a) and in the presence of ATP (b) after tetramer alignment (Chimera representation). The two views of each row are related by a 90° rotation around the long axis of the molecule (left panel (side view) and right panel (front view)). Each tetramer is represented in a different color (solid yellow --top tetramer and transparent blue --bottom tetramer). The region where the two surfaces coincide is shown in white. The large-subdomains of both α- and β-subunits are labeled with (*). Scale bar 10 nm.

Figure 7

Superposition of the structures of Pfk1 in the presence of F6P and in the presence of ATP after alignment to the top tetramer of the F6P-bound state (a and b) and to the bottom tetramer of the F6P-bound state (c and d). The top tetramer of the ATP-bound state is shown in a and c while the bottom tetramer of the ATP-bound state is shown in b and d. The two views of each row are related by a 90° rotation around the long axis of the molecule. F6P-bound state (solid yellow), ATP-bound state (transparent blue). The region where the two surfaces coincide is shown in white. The large-subdomains of both α- and β-subunits are labeled with (*). Scale bar 10 nm