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Originally published In Press as doi:10.1074/jbc.M305176200 on August 28, 2003

J. Biol. Chem., Vol. 278, Issue 46, 46021-46028, November 14, 2003
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Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily*

Guennadi Kozlov{ddagger}, John Lee{ddagger}, Demetra Elias{ddagger}, Michel Gravel{ddagger}, Pablo Gutierrez{ddagger}, Irena Ekiel§, Peter E. Braun{ddagger}, and Kalle Gehring{ddagger}

From the {ddagger}Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada and the §Health Sector, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada

Received for publication, May 16, 2003 , and in revised form, July 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The abundance of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP1; EC 3.1.4.37 [EC] ) in the central nervous system of all mammals and some other vertebrates such as amphibians and birds has long been an enigma. This derives from the continuing failure to identify a physiological substrate for this enzyme. CNP has an apparent specificity for nucleoside 2',3'-cyclic phosphate, which it cleaves to 2'-nucleotide end products, none of which (with the exception of NADP/NADPH) are found in metabolite pools. The last 4 decades of research have failed to attribute a function to this protein, although many possibilities have been considered (extensively reviewed in Refs. 1-3). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4, 5), and the catalytic active site of CNP has been investigated (6).

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 2',3'-cyclic phosphate and 5'-hydroxyl termini, and plant and yeast cyclic phosphodiesterases (CPDases) that hydrolyze ADP-ribose 1'',2''-cyclic phosphate to yield ADP-ribose 1'-phosphate (at least one of these latter enzymes also hydrolyzes nucleoside 2',3'-cyclic phosphates) (10, 11). These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12-14) and, most recently, 2'-5' RNA ligase from Thermus thermophilus (15) have been determined.

Members of this enzyme superfamily occur across a vast range of organisms ranging from bacteria to mammals. It has been suggested (16) that all four classes of enzymes originated from a common ancestor because they all have two similarly spaced histidine-containing tetrapeptides; their catalytic domains have a similar size of ~200 residues with similar pattern of predicted secondary structural elements; and they all catalyze hydrolysis of either 2',3'-cyclic phosphates to 2'-phosphates or 1'',2''-cyclic phosphate to 1''-phosphate. Recently, new members of this superfamily have been identified (17).

Investigations of CNP have provided a variety of observations concerning the relationship of CNP to the cytoskeleton and its localization to discrete regions of oligodendrocytes and paranodal compartments of the myelin sheath, adjacent to the axon (18-25). CNP comprises ~4% of the central nervous system total myelin protein and is most abundant in oligodendrocytes. Recently, it has been reported that CNP binds to tubulin and that it may play a role in anchoring microtubules to the plasma membrane as well as in regulating tubulin polymerization (26). A second isoform (CNP2) has also been identified, which contains a unique 20-amino acid N-terminal domain that targets the protein to mitochondria (27). Also, recent studies on CNP-null mutant mice revealed that the absence of CNP causes axonal swelling and neuronal degeneration (28). These observations underline the importance of this enzyme in brain and point to a multifaceted role for CNP in myelinogenesis and the maintenance of the myelin-axonal interface.

Here, we describe the structure of the brain CNP catalytic domain as determined by NMR and show that it is highly similar to the plant CPDase and the archaebacterial RNA ligase despite low overall similarity in amino acid sequence. This work brings us a step closer to understanding the function of CNP and its evolutionarily conserved enzymatic activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 protein was purified by immobilized metal affinity chromatography on a Ni2+-loaded chelating Sepharose column (Amersham Biosciences). Isotopically labeled CNP-CF was prepared from cells grown on minimal M9 medium containing [15N]ammonium chloride and/or [13C]glucose (Cambridge Isotopes Laboratory, Andover, MA). For the backbone assignments, partially deuterated triple-labeled (2H, 15N, 13C) CNP-CF was produced by expressing the protein in 90% D2O- and 10% H2O-containing minimal M9 medium. The N-terminal His tag was cleaved from CNP-CF by overnight dialysis with thrombin (Amersham Biosciences) at 1 unit/mg of fusion protein at room temperature. Benzamidine-Sepharose and Ni2+-loaded chelating Sepharose were used to remove thrombin and the His tag peptide from CNP-CF. The resulting 219-amino acid protein contained four extraneous residues from the His tag. The sequence composition of purified CNP-CF was confirmed by mass spectrometry.

CNP catalytic fragment mutants were created by overlap extension PCR using the Expand High Fidelity PCR system (Roche Diagnostics) (29). The mismatched oligonucleotide sequences used to generate the mutants were as follows (only the sense oligonucleotides are listed): T232A (ACA to GCA), 5'-GTG CTG CAC TGT GCA ACC AAA TTC TGT-3'; D237V (GAC to GTC), ACC AAA TTC TGT GTC TAC GGG AAG GCC-3'; G276A (GGG to GCA), 5'-CCC AAG ACA GCT GCA GCC CAG GTG GTG-3'; A308G (GCT to GGA), 5'-CCA GGG AGC CGA GGA CAT GTC ACC CTA-3'; T311A (ACC to GCG), 5'-AGC CGA GCT CAC GTC GCG CTA GGC-3'; Q322A (CAG to GCC), 5'-GTG CAG CCA GTG GCC ACA GGC CTT GAC-3'; G324A (GGC to GCG), 5'-CCA GTG CAG ACA GCG CTT GAC CTC TTA-3'; L327A (CTC to GCG), 5'-ACA GGC CTT GAC GCG TTA GAG ATT TTA-3'; and Y376A (TAC to GCT), 5'-TTC ACG GGG GCT TAT GGG TGA GGA TCC ATT AT-3. The boldface underlined sequences correspond to the mutated codons. The authenticity of the substitutions and the absence of any undesired mutations were confirmed by sequence analysis. The CNP-CF histidine mutants (H230L and H309L) were generated as previously described (6).

NMR Spectroscopy—NMR resonance assignments of the catalytic fragment of CNP were determined previously (30). All NMR experiments were recorded at 310 K. 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. Attempts to use Pf1 phage or compressed polyacrylamide to obtain residual dipolar coupling constraints were not successful. Nuclear Overhauser effect correlation spectroscopy (NOESY) constraints for the structure determination were obtained from 15N-edited NOESY (mixing time of 100 ms) and 13C-edited NOESY (mixing time of 100 ms) three-dimensional experiments using the Varian Inova 800-MHz spectrometer at the Canadian National High Field NMR Centre (NANUC). NMR spectra were processed with GIFA (31) and XWINNMR Version 2.5 (Bruker Biospin) and analyzed with XEASY (32).

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). The starting structure for ARIA was generated using MODELLER according to the CPDase fold and was in agreement with a set of manually assigned NOEs. PROCHECK NMR was used to check the protein stereochemical geometry (35). The coordinates have been deposited in the Protein Data Bank (code 1N4T [PDB] ), and the NMR assignments have been deposited in the BioMagResBank Database (accession number 5202 [BMRB] ).

CNP-CF Titrations with Inhibitors—2'-AMP, 3'-AMP, 5'-AMP, NAD, and pyrophosphate (Na2H2P2O7) were purchased from Sigma and used without any additional purification. An RNA oligoadenylate hexanucleotide (A6) was chemically synthesized and purified by University Core DNA & Protein Services (University of Calgary, Calgary, Canada). The purity and composition of the oligonucleotide was verified by one-dimensional NMR spectroscopy.

Titrations were monitored by 15N-1H heteronuclear single quantum correlation spectroscopy (HSQC) following addition of inhibitors to 15N-labeled CNP-CF (amino acids 164-378) on a Bruker Avance 600-MHz spectrometer. The sample contained 50 mM MES, 0.15 M NaCl, 1 mM dithiothreitol, and 0.1 mM sodium azide at pH 6.0 and ~0.4-0.5 mM CNP-CF at 310 K. Inhibitor concentrations varied from 0.1-0.2 to 7-60 mM depending on the affinity and solubility of the inhibitor. The A6 hexanucleotide concentration varied from 0.06 to 0.9 mM. HSQC spectra were assigned by monitoring chemical shift changes upon addition of the substrate because the binding takes place in fast exchange. The pH of the NMR samples was verified during the titrations and adjusted as needed. Chemical shift changes for individual residues were fitted to a one-site binding equation using the computer program GraFit (Version 3.0, Leatherbarrow) to determine the Kd of binding.

CNP-CF Inhibition Assays—CNP-CF activity assays were performed using the spectrophotometric coupled enzyme assay described previously (36). CNP-CF activity was determined by monitoring the formation of NADPH at 340 nm ({epsilon} = 6.22 mM-1 cm-1) using a Cary UV-visible spectrophotometer (Varian). The assay was initiated by adding 25 ng of CNP-CF to 1 ml of assay buffer containing 50 mM MES (pH 6.0), 30 mM MgCl2, 2',3'-cyclic NADP (Sigma), 5 mM D-glucose 6-phosphate, and 5 µg D-glucose-6-phosphate dehydrogenase (Roche Diagnostics). The concentration of cyclic NADP was varied from 0.05 to 2.0 mM. The initial velocity values were obtained from the Cary WinUV enzyme kinetics application and were fitted to the Michaelis-Menten equation. To test the inhibition of CNP-CF by AMP analogs, 1.5, 0.75, and 0.5 mM 5'-AMP, 2'-AMP, and 3'-AMP, respectively, were included in the 1 ml of assay buffer. CNP-CF activity data in the presence and absence of inhibitors were replicated at least twice for each inhibitor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 2',3'-cyclic nucleotide 3'-phosphodiesterase (Fig. 1). The previously reported resonance assignments (30) were used to assign NOEs from 15N- and 13C-edited three-dimensional NOESY experiments. The 20 lowest energy structures of 60 calculated were chosen to represent the final ensemble. The structural statistics are shown in Table I. On average, 10.9 constraints per residue were used to calculate the CNP-CF structure. This is below the typical number of 15-20 constraints per residue in high resolution NMR structures and results from the number of unresolved overlapping NOEs and a lower sensitivity of NOESY experiments because of the relatively large protein molecular mass (24.3 kDa). The tendency of CNP-CF to aggregate also limited the protein concentration in NMR samples.



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  		FIG. 1.
Structure of the catalytic fragment of CNP. a, the backbone superposition of the 20 lowest energy structures. The superposition was done using regions Phe169-His195 and Val228-Ile372. b, ribbon representation of CNP-CF showing locations of conserved residues from tetrapeptide HX(T/S)X motifs. C-term and N-term, C and N termini, respectively. c, very similar topology of CNP-CF (upper), A. thaliana CPDase (middle), and T. thermophilus RNA ligase (lower) from the 2H phosphodiesterase superfamily.

 


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  		TABLE I
Structural statistics for the CNP catalytic fragment

 
The structure shows a bilobal arrangement of two modules, each consisting of a four-stranded antiparallel {beta}-sheet and two antiparallel {alpha}-helices located on the outer part of the modules (Fig. 1b). The first lobe consists of strands {beta}1, {beta}2, {beta}6, and {beta}7 and helices {alpha}2 and {alpha}3, whereas the second one consists of strands {beta}3, {beta}4, {beta}5, and {beta}8 and helices {alpha}1 and {alpha}4. The internal face of the modules forms a large cavity. Intense peaks in the 15N-1H HSQC spectrum of CNP-CF indicate a flexible backbone for residues from Gly208 to Lys214. Coupled with low NOE density for this region, this loop between helix {alpha}1 and strand {beta}2 appears to be mobile.

The structure has a striking similarity to CPDase from A. thaliana (12) and to 2'-5' RNA ligase from T. thermophilus (15). Topologically, CNP-CF differs only in an extra C-terminal strand {beta}8 that extends the antiparallel {beta}-sheet containing strand {beta}3 (Fig. 1c). One possible role of this {beta}-strand is to place the N and C termini on the opposite sides of the CNP domain and to position the CNP C-terminal isoprenylation site at the membrane. The structural similarity of CNP-CF provides direct evidence that CNP belongs to the superfamily of phosphodiesterases containing dual catalytic tetrapeptide HX(T/S)X motifs.

CNP-CF has a more open cavity than CPDase. This difference suggests that a larger natural ligand could exist for CNP. Sequence comparison (Fig. 2) provides possible explanations for the more closed CPDase structure. The turns between strands {beta}3-{beta}4 and {beta}6-{beta}7 in CPDase make hydrophobic contacts with each other via side chains of Phe84 and Leu168 and limit the size of the catalytic cavity. The {beta}3-{beta}4 turn in CPDase is very hydrophobic, with a triplet of phenylalanine residues, Phe82, Phe83, and Phe84, whereas both turns in CNP-CF are hydrophilic and positively charged, suggesting that they are solvent-exposed and may be involved in interactions with a negatively charged ligand. Interestingly, the 2'-5' RNA ligase has hydrophilic turns and an open conformation similar to that of CNP. This conformation likely allows RNA to access the catalytic site (15).



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  		FIG. 2.
The catalytic domains of rat and human CNPs and the phosphodiesterase domain of the RICH70 protein from goldfish (g) show low sequence homology to CPDase from A. thaliana (A. th.) and RNA ligase from T. thermophilus (T. th.). The secondary structural elements refer to rat CNP-CF. The conserved catalytic residues are indicated in boldface.

 
Binding of CNP Inhibitors—To obtain more information about the active site of CNP, we titrated 15N-labeled CNP-CF with several compounds previously shown to inhibit CNP activity (reviewed in Ref. 1). These included orthophosphate, pyrophosphate, 2'-AMP, 3'-AMP, 5'-AMP, {beta}-NAD, and NADP. The titrations were monitored by 1H-15N correlation spectroscopy, and shifts of amide signals as a function of ligand addition were recorded. These shifts act as a fingerprint and identify amino acid residues affected by binding (Fig. 3a).



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  		FIG. 3.
a, chemical shift perturbation plots of the 15N-labeled catalytic fragment of CNP upon titration with KH2PO4 (upper left), the A6 oligonucleotide (upper right), 2'-AMP (lower left), and 3'-AMP (lower right). The same regions of CNP-CF were affected with a similar magnitude of changes. b, Lineweaver-Burk plot showing hydrolysis of 2',3'-cyclic NADP (cNADP) by CNP-CF in the absence of AMP analogs (x) and in the presence of 1.5 mM 5'-AMP ({square}), 0.75 mM 2'-AMP ({diamond}), and 0.5 mM 3'-AMP ({triangleup}). The plots exhibit apparent Km values of 230 µM in the absence of cAMP and 591, 700, and 775 µM in the presence of 5'-AMP, 2'-AMP, and 3'-AMP, respectively. The unaltered Vmax value of ~38 µM/min indicates that the AMP analogs act as competitive inhibitors.

 
Titration of the catalytic fragment of CNP with orthophosphate resulted in chemical shift changes, indicating that it binds to CNP and was present in the structure determined by NMR. The biggest 1H and 15N amide chemical shift changes were observed for Thr232 (0.56), Thr311 (0.53), Gly324 (0.24), Val228 (0.21), Ala308 (0.17), His230 (0.16), Gly305 (0.15), and Thr323 (0.15). Thr232, Thr311, and His230 are part of the tetrapeptide motifs, which are important for the catalytic activity. This shows that the phosphate group binds in the active site. The catalytic threonines, Thr232 and Thr323, likely coordinate the phosphate moiety through hydrogen bonds. The chemical shift changes correlate with the regions of highest sequence conservation in the catalytic domains of CNP from different species (Fig. 4b).



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  		FIG. 4.
Sequence conservation and chemical shift perturbation analysis. Shown are C-{alpha} traces of CNP-CF (residues 164-378) colored according to residue flexibility measured by signal intensity in an HSQC spectrum (red, intense; white, weak) (a), phylogenetic conservation (blue, >80% identity; light blue, {approx}50%; white, <30%) (b), and amide chemical shift changes ({Delta}{delta}) upon A6 RNA binding (red, {Delta}{delta} > 0.2; pink, {Delta}{delta} {approx} 0.1; white, {Delta}{delta} < 0.05) (c). Amide resonances for Thr232, Thr311, and Gly324 showed the largest changes. The figures were generated with GRASP (47). N-term, N terminus.

 
Interestingly, CNP-inhibitor interactions were pH-dependent. The chemical shift changes upon phosphate binding were much smaller at pH 6.5 (and above) than at pH 6.0 (data not shown). The likely reason for this is deprotonation of the catalytic histidines or the phosphoryl group. This would change the electrostatic charges and interfere with hydrogen bonding to the phosphate ion. In support of this, the CNP enzymatic activity was optimal at pH 5.5-6.5 and decreased at higher pH (data not shown). Whether this reflects protonation of active-site histidine(s) or the substrate phosphoryl group remains to be determined.

The binding of AMPs resulted in a pattern of chemical shift changes very similar to that observed upon binding of phosphate (Fig. 3a). The relatively larger shifts observed for AMPs reflect the stronger binding to CNP-CF. The AMP titrations also allowed us to identify additional residues affected by binding. Located in the loop between strand {beta}2 and helix {alpha}2, Cys236 and Asp237 showed minor chemical shift changes. These residues are relatively close to the tetrapeptide motifs and could participate in substrate recognition by interacting with the mobile loop Gly208-Lys214. Speculatively, Asp237 could interact with either Lys212 or Lys214 to close the mobile loop upon substrate binding. However, this loop is poorly conserved among mammalian CNPs (Fig. 4b) and shows relatively small changes upon inhibitor binding (Fig. 3a), suggesting that either the loop does not function as a flap or that it needs the proper substrate for specific loop-ligand interactions.

The titration experiments also allowed us to compare binding affinities of CNP inhibitors. In the weak binding (fast exchange) regime, the signals in HSQC spectra gradually move from the unbound position to the fully bound position depending on the amount of inhibitor added. These changes can be fitted using the binding equation to estimate the dissociation constant (Kd). The dissociation constants obtained from NMR titrations are shown in Table II. The results show that 3'-AMP has the highest affinity for the CNP catalytic domain, followed by 2'-AMP and 5'-AMP. This might reflect differences in the pKa of the phosphoryl group in the different AMPs or may be an inherent property of the CNP catalytic site. Studies at a second pH are needed to resolve the issue. Comparison of NAD and NADP shows that a terminal phosphate significantly improves binding affinity.


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  		TABLE II
Affinity constants for CNP-CF interactions with inhibitors as determined by NMR titration (Kd) and enzyme assays (Ki)

 
Kinetic Properties of Active-site Mutants—To define the enzymatic role of the conserved tetrapeptide motif residues, we mutated each residue individually and measured the kinetic parameters of the mutant enzymes using cyclic NADP as substrate (Table III). Consistent with our previous results (6), mutation of His230 and His309 resulted in a 15,000-fold reduction in kcat without any effect on Km, suggesting that both conserved histidines are critical for catalysis, but not for substrate binding. Mutation of Thr232 or Thr311 significantly decreased kcat by 100- and 700-fold, respectively. The Thr232 mutant also exhibited an 8-fold increase in Km, whereas the Thr311 mutant showed a minimal 3-fold increase. These results suggest that Thr232 is more critical for substrate binding, whereas Thr311 is more important for catalysis. Parallel studies with yeast CPDase showed that mutation of Ser/Thr residues in the tetrapeptide motifs has minor effects on enzymatic activity and suggested that the residues play a larger role in substrate recognition (16).


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  		TABLE III
Kinetic parameters for the CNP-CF mutants

 
Interestingly, mutation of the non-active site residue Gly324 (helix {alpha}4) in CNP resulted in a 5-fold increase in Km and 37-fold decrease in kcat. Mutations of other proximal residues on helix {alpha}4 such as Gln322 and Leu327 did not affect the kinetic parameters. These results agree with the NMR titration data (see below) that suggest that Gly324 is important for the conformation of helix {alpha}4. The other remaining mutants (D237V, G276A, A308G, and Y376A) displayed wild-type kinetic parameters, indicating that these residues are not particularly important for CNP activity.

Inhibitor Binding Causes a Conformational Change in the CNP Catalytic Domain—NMR titrations showed ligand-induced chemical shift changes in the residues around Gly324 (Fig. 3a). These residues are located in helix {alpha}4 and relatively far from the active site. This could represent a second binding site or a minor conformational change that affects these residues through contacts with strand {beta}5 in the active site.

To distinguish between these possibilities, we compared the dissociation constants obtained by NMR for two residues, Thr232 and Gly324, using both weak (orthophosphate) and high affinity (2'-AMP) inhibitors (Table II). Identical Kd values were observed for both residues, indicating a concerted conformational change that arises from a single binding site. It is very unlikely that two distinct binding sites would demonstrate identical Kd values for both substrates. In helix {alpha}4, the most affected residues are Thr323 and Gly324. These amino acid types are less common in {alpha}-helices, and we speculate that the conformational change observed is elongation of the N-terminal part of helix {alpha}4 in the presence of bound substrate.

The CNP Catalytic Fragment Weakly Binds Hexanucleotide RNA—An interesting feature of the CNP catalytic fragment is the presence of several aromatic (Tyr168, Phe172, Phe235, and Tyr352) and positively charged (Lys214, Lys234, and Arg307) residues in the vicinity of the active site (Fig. 5). The majority of these residues are located on the N-terminal lobe (strands {beta}1, {beta}2, and {beta}7). {beta}-Sheets with an abundance of positively charged and aromatic residues are common RNA-binding surfaces (for a review, see Ref. 37). The presence of these residues, coupled with a large binding cavity, hints that RNA may be a CNP substrate. Furthermore, many proteins from the 2H phosphodiesterase superfamily are involved in RNA-processing pathways, and some of them bind RNA (17). To test this hypothesis, we titrated CNP-CF with an RNA hexanucleotide (A6). The fingerprint of A6 binding was very similar to that of phosphate and other inhibitors (Fig. 3a), but its affinity could not be determined from the NMR data because the data did not fit a simple one-site binding (data not shown). The chemical shift changes with A6 RNA were smaller than with 3'-AMP or 5'-AMP, suggesting weaker binding.



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  		FIG. 5.
Vicinity of the catalytic site of CNP-CF. The region around the conserved HX(T/S)X motifs is abundant with aromatic and positively charged residues. The proposed location of the bound phosphate ion is shown in gray.

 
Enzyme assays were used to determine the inhibition constants (Ki) for the AMPs and the A6 oligonucleotide (Table II). All the inhibitors showed competitive inhibition (increased Km and unchanged Vmax). Because the N-terminal fragment of CNP has some homology to ATP-binding domains (38), we also tested ATP and observed very weak inhibition, with a Ki of ~13 mM. It was previously reported that poly(A) RNA is a potent CNP inhibitor (39). Surprisingly, our data show that A6 oligonucleotide has a rather weak ability to inhibit the CNP activity, with a Ki of ~0.8 mM. One possible explanation for the previous result is the presence of RNase activity in the CNP-CF preparation, which would produce mononucleotides from added RNA and so inhibit CNP activity. Based on our results, we conclude that single-stranded RNA does not produce any specific interactions with CNP-CF above those identified for mononucleotides. The possibility that CNP binds double-stranded RNA is currently under study.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Compared with the C terminus, little is known about the function of the N-terminal half of CNP. Sequence analysis revealed similarity to numerous proteins containing P-loop ATPase domains. This led to the suggestion that CNP may be a polynucleotide kinase (38), although experimental evidence for this has been unsuccessfully sought.2 Comparison of CNP with RICH shows divergence at the N terminus, suggesting the existence of shared (C-terminal) and divergent (N-terminal) functions. Structure determination of the N-terminal domain and/or full-length CNP may help to define the global role of CNP in oligodendrocytes, where it is most abundant.

Given that other members of this protein superfamily are involved in RNA-processing pathways, could RNA be a physiological substrate for CNP? The structural features of the catalytic domain such as the RNA recognition motif-like fold of each lobe, the abundance of positively charged and aromatic residues on the {beta}-sheet surface, and the large curvature of the binding cavity are consistent with this hypothesis. Although our data demonstrate that single-stranded oligo(A) possesses weak, millimolar affinity for the CNP catalytic domain, we cannot rule out 1) that CNP might bind only to double-stranded nucleic acids or to a specific RNA sequence, 2) that a 3'- or 5'-terminal phosphate might be important for binding, or 3) that the N-terminal portion of CNP may regulate binding or affect binding activity.

In considering the possible cellular functions of CNP, it is important to take into account other information such as the cellular localization of this enzyme. Both CNP isoforms are membrane-associated via isoprenylation at their C termini (21). The larger isoform of CNP, CNP2, contains a mitochondrial targeting sequence at the N terminus (27). CNP is also known to interact with tubulin (26), leading to the speculation that CNP might have a role in mRNA transport as observed in oligodendrocytes (40-42). Proteins such as myelin basic protein and carbonic anhydrase (43, 44) and tau (45) are specifically synthesized at the periphery of the myelin-forming processes; some type of specific mRNA transport and localization machineries must exist. In addition, there are signaling molecules such as nicotinic acid-adenine dinucleotide phosphate and cADP-ribose (reviewed in Ref. 46) upon which CNP might potentially act. Future structural and enzyme studies of CNP will hopefully clarify the cellular function of this highly conserved yet enigmatic protein.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1N4T [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a Canadian Institutes of Health Research grant (to K. G. and P. E. B.) and the Canadian Foundation for Innovation. Preliminary studies were supported by the Ontario Research and Development Challenge Fund and Multiple Sclerosis Society of Canada. NANUC is funded by the Canadian Institutes of Health Research, the Natural Science and Engineering Research Council of Canada, and the University of Alberta. This is National Research Council of Canada Publication 46157. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-7287; Fax: 514-398-7384; E-mail: kalle.gehring{at}bri.nrc.ca.

1 The abbreviations used are: CNP, 2',3'-cyclic nucleotide 3'-phosphodiesterase; CNP-CF, CNP catalytic fragment; RICH, regeneration-induced CNP homolog; CPDase, cyclic phosphodiesterase; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect correlation spectroscopy; HSQC, heteronuclear single quantum correlation spectroscopy; MES, 4-morpholineethanesulfonic acid. Back

2 P. E. Braun and D. Lasco, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Aled Edwards for constant interest and helpful discussions. We acknowledge the Canadian National High Field NMR Centre (NANUC) for assistance and use of the facilities.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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