Crystal structures of the Gon7/Pcc1 and Bud32/Cgi121 complexes provide a model for the complete yeast KEOPS complex.
Bottom Line: We further show that yeast KEOPS, unlike its archaeal counterpart, exists as a heteropentamer in which Gon7, Pcc1, Kae1, Bud32 and Cgi121 also adopt a linear arrangement.We constructed a model of yeast KEOPS that provides structural insight into the role of Gon7.The model also revealed the presence of a highly positively charged crater surrounding the entrance of Kae1 that likely binds tRNA.
Affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, CNRS, Université de Paris Sud XI, Bâtiment 430, 91405 Orsay, France.Show MeSH
Mentions: We made use of the ensemble of available structures of KEOPS subcomplexes to construct the complete archaeal complex showing that Pcc1, Kae1, Bud32 and Cgi121 are arranged in a linear manner. The Kae1 subunit is situated at the center of the complex, while Pcc1 binds to its N-terminal and Bud32 to its C-terminal lobe. Cgi121 binds to Bud32 at the opposite site of Kae1 (Supplementary Figure S1). We speculated that Gon7 would bind to one of the extremities of the yeast KEOPS complex, associating either with Pcc1 or with Cgi121. Deletion of the Gon7 gene is deleterious for cell life, but not the deletion of Cgi121, suggesting that Gon7 might interact with Pcc1 rather than with Cgi121. We therefore first co-expressed Gon7 and His-tagged Pcc1 from a polycistronic plasmid and observed that both proteins co-purified and form a stable complex (Supplementary Figure S2C). We obtained good quality preparations of the Gon7/Pcc1 complex that yielded diffracting crystals. The X-ray crystal structure of the Pcc1/Gon7 complex was determined using Se-methionine labeling. There are three copies of the Gon7/Pcc1 heterodimer in the asymmetric unit. Gon7 is partially ordered since electron density could only be observed for the peptide regions comprised between residues 3 to 21, 63 to 113. The missing regions are about the same for the three copies of Gon7 in the asymmetric unit, suggesting that the lack of electron density is due to disorder. Gon7 is made of an anti-parallel two-stranded β-sheet (residues 3–21) and a long α-helix (residues 63–95) that are packed to form the structured core of the protein (Figure 1A). Two copies of Gon7 in the asymmetric unit have a supplementary α-helix (residues 98–113) that is pointing away from the structured core, contacting the helical region of a neighboring Gon7/Pcc1 heterodimer in the crystal (not shown). The structure of Pcc1 (88 residues) is well defined between residues 8 (or 9, depending on the copy) and 91 (three histidines from the 6His tag have defined electron density). Pcc1 forms a three-stranded anti-parallel β-sheet with two helices packed on one face. The structure of yeast Pcc1 is very similar to that of P. furiosus Pcc1 (17), with a Z-score of 7.7 and an RMSD of 1.09 Å for 65 aligned Cα atoms. Dimer formation between Gon7 and Pcc1 involves packing of two helices and formation of an anti-parallel β-sheet. The first β-strand of Gon7 aligns with the first β-strand of Pcc1, creating a continuous anti-parallel five-stranded β-sheet (Figure 1A, left panel). Complex formation buries about 2400 Å2 for a total of 11 700 Å2 accessible surface area. The most important contribution to the dimer interface results from the packing of the α1-helix of Gon7 against the α2-helix of Pcc1 (Figure 1A, right panel). Fifteen hydrogen bonds and five salt bridges stabilize the interface. The well-conserved residues from the Gon7 α1 helix directly interact with Pcc1: hydrophobic residues form the core of the interface while polar and charged residues form specific hydrogen bonds at the periphery (for instance the totally conserved Arg72 of Gon7 H-bonds to Glu83 of Pcc1). As shown in Figure 1C, the sequence of the β1 strand of Gon7 is not well conserved but the majority of its interactions with Pcc1 consist of hydrogen bonds between main chain atoms. The main non-structured region of Gon7 is situated between β2 and α1 and is not close to the heterodimer interface. Despite its disorder, this region contains a few very well-conserved sequence stretches and is globally better conserved than the interacting β1 strand. This suggests that this region might be functionally important and might be involved in interaction with other KEOPS partners or substrate tRNA. The very acidic C-terminal peptide of Gon7 is also disordered.
Affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, CNRS, Université de Paris Sud XI, Bâtiment 430, 91405 Orsay, France.