Abstract
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.
Highlights
The abundance of the enzyme 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase (CNP1; EC 3.1.4.37) in the central nervous system of all mammals and some other vertebrates such as amphibians and birds has long been an enigma
Cyclic-nucleotide 3-phosphodiesterase (CNP) and RICH share catalytic features with three other groups of enzymes: fungal/plant RNA ligases involved in tRNA splicing [7, 8], bacterial and archaeal RNA ligases [9] that ligate tRNA half-molecules containing 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini, and plant and yeast cyclic phosphodiesterases (CPDases) that hydrolyze ADP-ribose 1Љ,2Љ-cyclic phosphate to yield ADP-ribose 1Ј-phosphate [10, 11]
CNP Belongs to the Superfamily of 2H Phosphodiesterases—We determined the structure of the catalytic fragment of rat brain CNP (CNP-CF), the first of a vertebrate-specific 2Ј,3Јcyclic nucleotide 3Ј-phosphodiesterase (Fig. 1)
Summary
Protein Expression and Purification—The catalytic fragment of CNP (CNP-CF, residues 164 –378) was subcloned into pET15b (Novagen, Madison, WI) and expressed in the Escherichia coli expression host BL21(DE3) (Stratagene) as a His-tagged fusion protein. The resulting 219amino acid protein contained four extraneous residues from the His tag. NMR Spectroscopy—NMR resonance assignments of the catalytic fragment of CNP were determined previously [30]. NMR samples were 1–2 mM protein in 50 mM sodium phosphate buffer, 0.15 M NaCl, 1 mM dithiothreitol, and 0.1 mM sodium azide at pH 6.0. Structure Calculation—For the structure determination, a set of 1925 nuclear Overhauser effects (NOEs) were collected from 15N- and 13C-edited NOESY spectra of CNP-CF (amino acids 164 –378) acquired at 800 MHz. Automated NOE assignments were made using ARIA [33], and the structure was refined using standard protocols in CNS Version 1.1 [34]. Titrations were monitored by 15N-1H heteronuclear single quantum correlation spectroscopy (HSQC) following addition of inhibitors to 15Nlabeled CNP-CF (amino acids 164 –378) on a Bruker Avance 600-MHz spectrometer. CNP-CF activity data in the presence and absence of inhibitors were replicated at least twice for each inhibitor
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
