Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

Abstract

The 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2′-nucleotide products from 2′,3′-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2′-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.

Self-Assembly Characteristics of a Crystalline–Amorphous Diblock Copolymer in Nanoscale Thin Films

“…The position of the cyclic nucleotide in the active site is consistent with the earlier predicted reaction mechanism 4,14 . Based on our current data, we are able to propose a much more detailed mechanism for catalysis by CNPase (Figure 8).…”

“…We previously determined the structure of wild-type CNPase bound to the product at 2.4-Å resolution 4 . The new structure of the V321A mutant-2'-AMP complex is of much higher resolution, and has a high occupancy of the product, making it more suitable for detailed analyses.…”

“…This groove could represent the continuation of a binding site for further nucleotide units in an RNA substrate. Furthermore, the N-terminal domain, likely to be responsible for high-affinity RNA binding 4 , is localized in this direction Although NADP + is a dinucleotide, it is not a perfect analogue for an RNA oligonucleotide. Hence, we also carried out docking of dinucleotide compounds, adenylyl-3',5'-uridine-2'-monophosphate and adenylyl-3',5'-adenine-2'-monophosphate, as well as their 2',3'-cyclic derivatives, representing substrate and product into the respective CNPase active-site conformations.…”

“…In our earlier work, using a slightly shorter construct without the 22-residue C-terminal tail, purified CNPase always contained a mixture of different oligomeric states 4 . SRCD was used to confirm the folding state of CNPase, and the results indicate a similar secondary structure content for the catalytic domain and full-length CNPase; and, hence, also for the N-terminal domain (Supplementary Figure 1).…”

“…Docking experiments also showed binding of dinucleotides into this groove. It can be envisaged that an oligonucleotide would bind along an aromatic surface, lined by a strong positive electrostatic potential, towards the N-terminal domain, which has been shown to efficiently bind RNA 4 . Kinetic parameters for CNPase towards 2',3'-cAMP and 2',3'-cNADP+ were reported to be essentially identical 22 , indicating that the kinetics are mainly determined by the nucleotide moiety in the active site; however, for longer substrates, the situation may be different.…”