The Structure of CF

The hydrophilic CF1 - part can be separated from the membrane integrated CF0 -part. First insights into the structure of CF1 came from electron microscopy and image analysis of negatively stained particles that show a pseudo hexagonal arrangement of a3p3 with ySe located asymmetrically in the centre [13, 14]. These projection maps still lacked three-dimensional information. Only later localization of subunit S of the E. coli ATP synthase by immuno electron microscopy revealed its location at the top, outside of the a3p3-complex [65]; whereas, y and £ form the central stalk that is anchored inside the a3p3-hexagon by two long helices of y [45, 71] - In the pseudo-hexagonal view of a3p3 - the centers of mass of the a-subunits extend further from the two central helices of y than those of the P-subunits, which also enables distinguishing between a and P subunits in low-resolution maps.

The three aP-pairs in a3p3y can adopt different conformations. In the mitochondrial complex, the orientation of the central stalk relative to the aP-pairs deter-

Figure 9.2 Superposition of the CFrpart of the three dimensional map of CF0F, derived from electron microscopy (grey) with models of the subunits. The a-subunits are shown in magenta, P in red, y in yellow, e in brown, S in light blue, I and II in dark blue. The homology models of y and e are calculated from the a3p3 y S template (center, 1E79, mitochondrial S is homologous to chloroplast e). The aP-pairs are derived from the chloroplast a3p3(center, 1FXO). The homology model of the N-terminal region of S was based on the structure of S from E. coli (center, 1ABV), the models of subunit I and II are both based on the structure of the dimerization region of the b-subunits from E. coli (center, 1L2P).

Figure 9.2 Superposition of the CFrpart of the three dimensional map of CF0F, derived from electron microscopy (grey) with models of the subunits. The a-subunits are shown in magenta, P in red, y in yellow, e in brown, S in light blue, I and II in dark blue. The homology models of y and e are calculated from the a3p3 y S template (center, 1E79, mitochondrial S is homologous to chloroplast e). The aP-pairs are derived from the chloroplast a3p3(center, 1FXO). The homology model of the N-terminal region of S was based on the structure of S from E. coli (center, 1ABV), the models of subunit I and II are both based on the structure of the dimerization region of the b-subunits from E. coli (center, 1L2P).

mines the conformational state of the catalytic nucleotide binding sites. These sites are located on the P- subunits at the interface to the adjacent a subunits. In the mitochondrial a3p3y8e-complex, one of the catalytic binding sites is empty (E), one has an ADP (D) loosely bound, and the third has a tightly bound non-hydrolysable ATP analogue(AMPPNP) (T).

This is different in the a3p3ye complex of chloroplasts [41, 42], where the crystal-lographic threefold axis superimposes with the pseudo-sixfold axis of the complex, making the aP - pairs indistinguishable and the electron density for y and £ un-interpretable. However, the aP pair is well resolved and shows a similar conformation as the ap-pair of the mitochondrial complex in the D or T conformation, but is incompatible with an aP-pair in the E state. The significance of this difference to the mitochondrial enzyme is still unclear.

In order to obtain a pseudo-atomic model for CF1, the structure of the mitochondrial (aP)3y8£ (1E79, -45]) was used as a template (Figure 9.2 center). The three copies of the aP-pairs of CF1 (1FX0, [41], Figure 9.2 centre) and the modeled y and £ subunits were matched to the template. The newly generated sub-complex was placed into the upper half of the 3D-map of CF0F1 (Figure 9.2, right). The model accounted for most of the density in CF1, except for a region at the top of a3P3 and at the periphery of one of the a-subunits. Into the region at the top, the modeled N-terminal domain of the S-subunit was placed adjacent to an a-subunit as determined by NMR-spectroscopy. This left room for the C-terminal domain of S at the central top of a3P3, in agreement with the localization by immuno- electron microscopy in EF0F1 [65], Models of the dimerization region of subunits I and II were fitted into the unaccounted density at the periphery of the a-subunit to which S is bound. The positioning of subunits I and II in close proximity to the a-subunit is in agreement with cross-linking data between the a- and the b-subunits in E.

coli [ 72] . Figure 9.2 , right, shows a superposition of the 3D -map derived from electron cryo -microscopy (grey) with a3p3 from chloroplasts and the homology models of y £, S and I + II (in color).

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