Abstract
Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and P i -dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.
Highlights
Flaveria trinervia was determined by molecular replacement at 2.9 Å resolution using the maize structure
An overall root mean square deviation (RMSD) of 4.8 Å was calculated from a structural alignment of the individual monomers of the phosphate dikinase (PPDK) dimer in 5JVJ, indicating a substantial difference in their conformation
The presence of two structurally distinct conformations of the PPDK monomer (NBD open versus closed) within a single crystal structure may suggest that these structural asymmetries reflect functional asymmetries in substrate binding and/or catalytic turnover in the individual subunits of the PPDK dimer
Summary
Distinct conformational states resolved in crystal structures from maize and the non-plant organisms Clostridium symbiosum and Trypanosoma brucei suggest that the phosphoryl group transfer in the catalytic cycle is accompanied by a large swiveling motion of the CD from a position next to the NBD to a position facing the PBD (~110° and 45 Å)[8,9,10,11]. The analysis of essential motions in available crystal structures and unrestrained molecular dynamics simulations reveal coupled motions of the CD and the NBD for non-phosphorylated PPDK.
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