Fig. 9: Arg237 of Kae1 is positioned in the active site of Bud32 and is essential for regulating its ATPase activity.
From: Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32
A Cryo-EM density in mesh representation and fitted atomic model in cartoon representation of Kae1 and Bud32 in the structure of KEOPS bound to tRNA in its native-like conformation. Arg237 of Kae1 is ~3.6 Å from the active site residue Asp451 in Bud32. The position of ATP was modeled from the crystal structure of phosphorylase kinase (PDB ID: 2PHK). B Weblogo sequences showing the conservation of Arg237 in Kae1. C Cartoon representation showing the crystal structure of phosphorylase kinase bound to substrate peptide (labeled Ser-acceptor) and the ATP analog AMP-PnP (PDB 2PHK) superimposed on the Bud32 subunit of KEOPS bound to tRNA in a native-like conformation. Box shows detailed view of the active site region of Bud32 and its interaction with Kae1. Arg237 in Kae1 is ~3.1 Å from the phospho-acceptor site of the peptide substrate. D GST pull-down analysis of GST-Cgi121 binding to Kae1 WT and R237A mutant in the presence or absence of Bud32. Samples were analyzed by Coomassie-stained SDS PAGE (n = 2 technical replicates). E Competitive displacement of an Alexa-647 labeled CCA-tail probe (647-CCA) from KEOPS WT or the R237A mutant by titration of tRNALys. Displacement of the 647-CCA probe was monitored by fluorescence polarization (FP). Respective mean IC50 values for the displacement are shown (n = 3 technical replicates, error bars represent ± SD). F ATPase activity analysis of KEOPS reconstituted with Kae1 WT or the Arg237Ala mutant in the presence and absence of tRNALys. Activity was monitored using the ADP Glo assay. Displayed results center represent the mean luminescence for each reaction condition, error bars represent ±SD (n = 3 technical replicates). G t6A modification activity analysis of KEOPS reconstituted with Kae1 WT or the Arg237Ala mutant. tRNALys was used as substrate. Representative HPLC profiles of nucleoside composition for each reaction are shown at left. Quantification shows center of mean t6A content normalized to the content of uridine and error bars represent ±SD (n = 3 technical replicates). Source data are provided as a Source Data file.
