3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel gamma-subunits

Abstract

The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer ( approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species.

Figure 1

A) Self-organizing map of 0° images from a random conical data set at 0° tilt of early P. pastoris Pfk preparations in the presence of F6P. The numbers indicate the class membership. Scale bar, 10 nm. B) Central slice from a tomographic reconstruction of P. pastoris Pfk preparations in the presence of F6P. Scale bar, 100 nm.

Figure 2

A and B, Tilt pair of P. pastoris Pfk preparations in the presence of ATP; 0° micrograph (A) and tilted micrograph (70°) (B). C and D, Extracted images from the 0° micrograph C) and tilted micrograph D). Scale bar: 100 nm.

Figure 3

Self-organizing map of 0° images from a random conical data set at 0° tilt of P. pastoris Pfk preparations in the presence of ATP. The numbers indicate the class membership. The Black boxes show the classes that were used for the initial multireference alignment step.

Figure 4

Class averages from the last classification step. Indicated is the class number (top) and the number of images included in the class (bottom). Scale bar: 10 nm.

Figure 5

Left) Gallery of surface views in 30 ° steps of the 3D reconstruction of P. pastoris Pfk. Scale bar, 10 nm. Right) Fourier shell correlation curve (solid line) and 5 times noise correlation curve (dashed line).

Figure 6

Surface representation of the 3D reconstruction Pfk from stain embedded preparations of P. pastoris (Pp, left column), cryo preparations of S. cerevisiae (Sc, center column) and stained embedded preparations of S. pombe (Sp, right column). A) “front” view, showing the catalytic surface and B) “side” view. (•) indicates the N-terminal domain of the α-subunits (colored in red) and (x) the N-terminal domain of the β-subunits of P. pastoris and ScPfks and the N-terminal domain of the external α-subunit of S. pombe (all colored in blue), the γ-subunits of P. pastoris are colored in yellow. Surfaces have been manually colored aided by the visualization of surfaces at different thresholds. Scale bar. 10 nm.