Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

Sci Rep. 2017 Mar 30:7:45389. doi: 10.1038/srep45389.

Abstract

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and Pi-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • Flaveria / enzymology*
  • Molecular Dynamics Simulation
  • Phosphoenolpyruvate / chemistry
  • Phosphoenolpyruvate / metabolism
  • Plant Proteins / chemistry
  • Plant Proteins / metabolism*
  • Principal Component Analysis
  • Protein Conformation
  • Pyruvate, Orthophosphate Dikinase / chemistry
  • Pyruvate, Orthophosphate Dikinase / genetics
  • Pyruvate, Orthophosphate Dikinase / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Plant Proteins
  • Recombinant Proteins
  • Phosphoenolpyruvate
  • Pyruvate, Orthophosphate Dikinase