Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein

Abstract

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.

Figure 1.

Stereoview of the PDRP structure. The whole structure consists of an NTD, a CTD, and a long linker. The β-sheets and α-helices of the NTD are colored yellow and red, respectively, and magenta and cyan in the CTD, respectively. The P-loop (blue), AMP (green sticks), and Mg²⁺ ion (orange sphere) are shown, and regions missing in the electron density are indicated by black dashed lines.

Figure 2.

Structures of the NTD and CTD of PDRP. A, Cartoon representation of the NTD of PDRP. B, Cartoon representation of the CTD of PDRP. Both have a central four-stranded parallel β-sheet sandwiched by three to four α-helices. The P-loop and putative P-loop are colored blue. C, Structural alignment between the NTD and CTD. D, The omit 2Fo-Fc electron density map of bound AMP at the 2.0 σ level. E, Detailed interactions and hydrogen bonds between PDRP and AMP.

Figure 3.

Structure of the PDRP dimer. A, Structural overview of the PDRP dimer, colored by protomers, green and magenta. The l-loop and I-loop are colored blue and red, respectively. The three principal regions involved in dimerization are shown in black, yellow, and blue rectangles. B, Details of the PDRP dimeric interface within the three principal regions. C, Intramolecular (C165-C171) and intermolecular (C128-C417) disulfide bonds are shown in stick representation. In the bottom panel, nine PDRP samples were pretreated with or without reducing agent (1% β-mercaptoethanol or 100 mm DTT) prior to separation by SDS-PAGE. D, Immuno-based assays of the in vitro kinase (top) and pyrophosphorylase (bottom) activities. Western blots were probed with antiphospho-T527-PPDK (antiphosT527) or anti-PPDK antibodies.

Figure 4.

Structural homologs of the NTD and CTD of PDRP. A, Structural alignment between GluA2 (tint, PDB ID 4U5F; Chen et al., 2014a) and the NTD of PDRP. The predicted substrate binding site is indicated by a magenta rectangle. B, Structural alignment between the CTD of PDRP and shikimate kinase (green, PDB ID 1L4Y; Gu et al., 2002). The lid domain of SK is indicated by a green arrowhead, and the corresponding lid-like domain of PDRP is indicated by a red oval. C, Immuno-based assays of the in vitro kinase experiments. D, Immuno-based assays of the in vitro pyrophosphorylase experiments.

Figure 5.

Structural superimposition PDRP onto HprK/P reveals the catalytic mechanism. A, The overall dimeric structure of M. pneumoniae HprK/P (PDB ID 1KNX; Allen et al., 2003). The P-loop in the CTD of HprK/P is colored blue. B, Detailed residues involved in the pyrophosphorylase mechanism of PDRP. C, Detailed residues involved in the kinase mechanism of PDRP. Resembling HprK-P-Ser-HPr (PDB ID 1KKM; yellow; Fieulaine et al., 2002) in the dephosphorylation and HprK-Ser-HPr (PDB ID 1KKL; yellow; Fieulaine et al., 2002) in the phosphorylation reaction. PDRP contains three separate regions for the catalytic mechanism: P-loop, Asp-276 and Asp-277 of the AB-loop, and Lys-274 from AB′-loop of the other protomer. The ADP (sticks) and Mg²⁺ (orange sphere) are modeled from shikimate kinase (PDB ID 1L4Y; Gu et al., 2002). D, Immuno-based assays of the in vitro kinase (left) and pyrophosphorylase (right) experiments.