Crystal Structures of the Semireduced and Inhibitor-bound Forms of Cyclic Nucleotide Phosphodiesterase from Arabidopsis thaliana

Abstract

The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 Å. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1؆,2؆-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the Nterminal lobe of this loop winds up and elongates the preceding ␣-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2,3-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.

Biophysical Characterization of Cyclic Nucleotide Phosphodiesterases

“…The results for the plant CPDase are in excellent agreement with the recently reported crystal structures and with the proposed catalytic mechanism (11,12). ITC experiments proved that all four residues of the tandem signature motif are essential for the catalytic activity of the plant CPDase; this is somewhat different in the yeast CPDase where only the histidine residues have been reported to be essential (4).…”

“…This hypothesis was supported by the crystal structure (11,12) and a mutagenesis study with CPDase (S. cerevisiae) (4). Based on the current results, it is absolutely clear that these four residues are essential for the catalytic reaction since variation in either one of the four positions leads to almost complete reduction of interaction enthalpy.…”

“…2C) increases significantly while the shape of the spectrum still indicates (␣ ϩ ␤)-structure. This result for CPDase (A. thaliana) is remarkable, since the crystal structure shows a conformational rearrangement of residues 100 -115 leading to an extended ␣-helical element in the semireduced state (12). However, the additionally gained percentage of helical content is not significant enough to be detected by CD.…”

“…We recently reported the crystal structures for CPDase (A. thaliana) in its oxidized (11), semi-reduced, and inhibitor-bound forms (12). The overall structure consists of two almost symmetrical lobes formed by the non-contiguous parts of the peptide chain, where each lobe consists of a three-or four-stranded antiparallel ␤-sheet that constitutes the inner core of the protein.…”

“…In the presence of DTT, we succeeded in crystallizing the semi-reduced species of CPDase, in which Cys104 and Cys110 were in their reduced states, while the other disulfide bridge remained intact. In the semi-reduced state, the N-terminal part of the surface loop folds into ␣-helical conformation, thereby elongating the preceding helix by two more turns (12).…”