Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily

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 β-sheet and two α-helices. The β-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. 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 β-sheet and two α-helices. The β-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. The abundance of the enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP 1The 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.; 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. 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. 1Sprinkle T.J. Crit. Rev. Neurobiol. 1989; 4: 235-301PubMed Google Scholar, 2Tsukada Y. Kurihara T. Myelin: Biology and Chemistry. CRC Press, Inc., Boca Raton, FL1992Google Scholar, 3Vogel U.S. Thompson R.J. J. Neurochem. 1988; 50: 1667-1677Crossref PubMed Scopus (118) Google Scholar). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4Ballestero R.P. Wilmot G.R. Leski M.L. Uhler M.D. Agranoff B.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8621-8625Crossref PubMed Scopus (27) Google Scholar, 5Ballestero R.P. Wilmot G.R. Agranoff B.W. Uhler M.D. J. Biol. Chem. 1997; 272: 11479-11486Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and the catalytic active site of CNP has been investigated (6Lee J. Gravel M. Gao E. O'Neill R.C. Braun P.E. J. Biol. Chem. 2001; 276: 14804-14813Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). CNP and RICH share catalytic features with three other groups of enzymes: fungal/plant RNA ligases involved in tRNA splicing (7Tyc K. Kellenberger C. Filipowicz W. J. Biol. Chem. 1987; 262: 12994-13000Abstract Full Text PDF PubMed Google Scholar, 8Xu Q. Teplow D. Lee T.D. Abelson J. Biochemistry. 1990; 29: 6132-6138Crossref PubMed Scopus (49) Google Scholar), bacterial and archaeal RNA ligases (9Arn E. Abelson J. Simons R. Grunberg-Manago M. RNA Structure and Function. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 695-726Google Scholar) 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) (10Culver G.M. McCraith S.M. Zillmann M. Kierzek R. Michaud N. LaReau R.D. Turner D.H. Phizicky E.M. Science. 1993; 261: 206-208Crossref PubMed Scopus (80) Google Scholar, 11Genschik P. Hall J. Filipowicz W. J. Biol. Chem. 1997; 272: 13211-13219Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12Hofmann A. Zdanov A. Genschik P. Ruvinov S. Filipowicz W. Wlodawer A. EMBO J. 2000; 19: 6207-6217Crossref PubMed Scopus (50) Google Scholar, 13Hofmann A. Grella M. Botos I. Filipowicz W. Wlodawer A. J. Biol. Chem. 2002; 277: 1419-1425Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14Hofmann A. Tarasov S. Grella M. Ruvinov S. Nasr F. Filipowicz W. Wlodawer A. Biochem. Biophys. Res. Commun. 2002; 291: 875-883Crossref PubMed Scopus (11) Google Scholar) and, most recently, 2′-5′ RNA ligase from Thermus thermophilus (15Kato M. Shirouzu M. Terada T. Yamaguchi H. Murayama K. Sakai H. Kuramitsu S. Yokoyama S. J. Mol. Biol. 2003; 329: 903-911Crossref PubMed Scopus (32) Google Scholar) have been determined. Members of this enzyme superfamily occur across a vast range of organisms ranging from bacteria to mammals. It has been suggested (16Nasr F. Filipowicz W. Nucleic Acids Res. 2000; 28: 1676-1683Crossref PubMed Scopus (42) Google Scholar) 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 (17Mazumder R. Iyer L.M. Vasudevan S. Aravind L. Nucleic Acids Res. 2002; 30: 5229-5243Crossref PubMed Scopus (124) Google Scholar). 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 (18Braun P.E. Sandillon F. Edwards A. Matthieu J.M. Privat A. J. Neurosci. 1988; 8: 3057-3066Crossref PubMed Google Scholar, 19Trapp B.D. Bernier L. Andrews S.B. Colman D.R. J. Neurochem. 1988; 51: 859-868Crossref PubMed Scopus (173) Google Scholar, 20Dyer C.A. Benjamins J.A. J. Neurosci. Res. 1989; 24: 201-211Crossref PubMed Scopus (110) Google Scholar, 21De Angelis D.A. Braun P.E. J. Neurosci. Res. 1994; 39: 386-397Crossref PubMed Scopus (55) Google Scholar, 22De Angelis D.A. Braun P.E. J. Neurochem. 1996; 66: 2523-2531Crossref PubMed Scopus (22) Google Scholar, 23Gravel M. Peterson J. Yong V.W. Kottis V. Trapp B. Braun P.E. Mol. Cell. Neurosci. 1996; 7: 453-466Crossref PubMed Scopus (107) Google Scholar, 24Yin X. Peterson J. Gravel M. Braun P.E. Trapp B.D. J. Neurosci. Res. 1997; 50: 238-247Crossref PubMed Scopus (75) Google Scholar, 25Kim T. Pfeiffer S.E. J. Neurocytol. 1999; 4: 281-293Crossref Google Scholar). 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 (26Bifulco M. Laezza C. Stingo S. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1807-1812Crossref PubMed Scopus (128) Google Scholar). A second isoform (CNP2) has also been identified, which contains a unique 20-amino acid N-terminal domain that targets the protein to mitochondria (27McFerran B. Burgoyne R. J. Cell Sci. 1997; 110: 2979-2985PubMed Google Scholar). Also, recent studies on CNP-null mutant mice revealed that the absence of CNP causes axonal swelling and neuronal degeneration (28Lappe-Siefke C. Goebbels S. Gravel M. Nicksch E. Lee J. Braun P.E. Griffiths I.R. Nave K.A. Nat. Genet. 2003; 33: 366-374Crossref PubMed Scopus (798) Google Scholar). 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. 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) (29Higuchi R. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA1990: 177-183Google Scholar). 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 (6Lee J. Gravel M. Gao E. O'Neill R.C. Braun P.E. J. Biol. Chem. 2001; 276: 14804-14813Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). NMR Spectroscopy—NMR resonance assignments of the catalytic fragment of CNP were determined previously (30Kozlov Lee J. Gravel M. I. Braun P.E. K. J. 2002; PubMed Scopus Google Scholar). NMR were at K. NMR were protein in phosphate 1 and at to or to were Overhauser effect correlation for the structure were from of and of using the at the High NMR NMR were with M.A. J. 1997; 8: Scholar) and and with C. M. P. K. J. 1995; PubMed Scopus Google Scholar). Structure the structure a of nuclear Overhauser were from and of CNP-CF 164-378) at assignments were using M. H. J. Mol. Biol. 1997; PubMed Scopus Google Scholar), and the structure was using in G.M. P. J. M. R.J. L.M. T. Biol. PubMed Scopus Google Scholar). The structure for was generated using to the CPDase and was in with a of NMR was used to the protein J.A. R. J.M. J. 1996; 8: PubMed Scopus Google Scholar). The have been in the Protein and the NMR assignments have been in the number CNP-CF with and were from and used any RNA was and purified by Protein of The and composition of the oligonucleotide was by NMR were by heteronuclear single quantum correlation of inhibitors to CNP-CF 164-378) on a The contained MES, 1 and at and CNP-CF at K. from to on the affinity and of the The from to were by upon of the substrate because the binding in The of the NMR was the titrations and as for residues were to a binding using the to the of binding. CNP-CF were using the enzyme described previously J. Neurochem. PubMed Scopus (128) Google Scholar). CNP-CF was determined by the of at using a The was by of CNP-CF to 1 of containing 2′,3′-cyclic and (Roche The of cyclic was from to The were from the enzyme and were to the the of CNP-CF by and and were in the 1 of CNP-CF in the and absence of inhibitors were at least for each CNP to the of 2H determined the structure of the catalytic fragment of rat brain CNP the of a 2′,3′-cyclic nucleotide 3′-phosphodiesterase The previously reported resonance assignments (30Kozlov Lee J. Gravel M. I. Braun P.E. K. J. 2002; PubMed Scopus Google Scholar) were used to from and The structures of were to the The structural are in I. were used to the CNP-CF This is the number of in resolution NMR structures and from the number of and a of because of the large protein mass The of CNP-CF to also the protein in NMR for the CNP catalytic for structure range range from from of structures from and and for residues in most in in in a new The structure a bilobal arrangement of two modules, each consisting of a four-stranded β-sheet and two on the of the The of and and and the second one of and and and The of the a large in the of CNP-CF a backbone for residues from to with low for this this and to The structure has a similarity to CPDase from A. thaliana (12Hofmann A. Zdanov A. Genschik P. Ruvinov S. Filipowicz W. Wlodawer A. EMBO J. 2000; 19: 6207-6217Crossref PubMed Scopus (50) Google Scholar) and to 2′-5′ RNA ligase from T. thermophilus (15Kato M. Shirouzu M. Terada T. Yamaguchi H. Murayama K. Sakai H. Kuramitsu S. Yokoyama S. J. Mol. Biol. 2003; 329: 903-911Crossref PubMed Scopus (32) Google Scholar). CNP-CF in an that the β-sheet containing role of this is to the and on the of the CNP domain and to the CNP site at the The structural similarity of CNP-CF that CNP to the superfamily of phosphodiesterases containing catalytic tetrapeptide HX(T/S)X motifs. CNP-CF has a cavity This that a for for the CPDase The and in CPDase with each other of and and the size of the catalytic The in CPDase is with a of and in CNP-CF are and positively that they are and may involved in with a charged the 2′-5′ RNA ligase has and an similar to that of This RNA to the catalytic site (15Kato M. Shirouzu M. Terada T. Yamaguchi H. Murayama K. Sakai H. Kuramitsu S. Yokoyama S. J. Mol. Biol. 2003; 329: 903-911Crossref PubMed Scopus (32) Google Scholar). of CNP the active site of CNP, we CNP-CF with previously to CNP in 1Sprinkle T.J. Crit. Rev. Neurobiol. 1989; 4: 235-301PubMed Google Scholar). These and The titrations were by correlation and of as a function of were These as a and identify amino acid residues by binding of the catalytic fragment of CNP with in that it binds to CNP and was present in the structure determined by The and were for and and are of the tetrapeptide which are for the catalytic activity. This that the phosphate binds in the active The catalytic and the phosphate The with the regions of sequence in the catalytic domains of CNP from were The upon phosphate binding were at at The for this is of the catalytic or the This change the and with to the phosphate In of the CNP enzymatic was at and at this of or the substrate remains to determined. The binding of in a pattern of similar to that upon binding of phosphate The for the binding to CNP-CF. The titrations also us to identify residues by binding. in the and and These residues are to the tetrapeptide and in substrate by with the with either or to the upon substrate binding. this is conserved and upon binding that either the function as a or that it the substrate for The also us to binding of CNP inhibitors. In the binding the in from the to the bound on the of These using the binding to the The from NMR titrations are in The show that has the affinity for the CNP catalytic by and This in the a of the in the or may an of the CNP catalytic at a second are to the of and that a phosphate binding for CNP-CF with inhibitors as determined by NMR and enzyme in a new of the enzymatic role of the conserved tetrapeptide we mutated each and the kinetic of the mutant enzymes using cyclic as substrate with (6Lee J. Gravel M. Gao E. O'Neill R.C. Braun P.E. J. Biol. Chem. 2001; 276: 14804-14813Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), of and in a in any effect on that conserved are for but for substrate binding. of or by and The mutant also an in the mutant a minimal These that is for substrate is for studies with yeast CPDase that of residues in the tetrapeptide has on enzymatic and suggested that the residues play a role in substrate (16Nasr F. Filipowicz W. Nucleic Acids Res. 2000; 28: 1676-1683Crossref PubMed Scopus (42) Google for the CNP-CF cyclic for CNP-CF is cyclic The for CNP-CF is in a new of the site in CNP in a in and in of other residues on such as and the kinetic These with the NMR that that is for the of The other mutants and kinetic that these residues are for CNP activity. a in the CNP titrations in the residues These residues are in and from the active This a second binding site or a conformational change that these residues with in the active these we the by NMR for two and using and affinity inhibitors were for a conformational change that from a single binding It is that two binding for In the most residues are and These amino acid are common in and we that the conformational change is of the N-terminal of in the of bound The CNP of the CNP catalytic fragment is the of aromatic and and positively charged and residues in the of the active site The of these residues are on the N-terminal and with an abundance of positively charged and aromatic residues are common a K. Rev. Biophys. PubMed Scopus Google Scholar). The of these with a large binding that RNA may a CNP many from the 2H phosphodiesterase superfamily are involved in and some of RNA (17Mazumder R. Iyer L.M. Vasudevan S. Aravind L. Nucleic Acids Res. 2002; 30: 5229-5243Crossref PubMed Scopus (124) Google Scholar). this we CNP-CF with an RNA The of binding was similar to that of phosphate and other inhibitors but its affinity determined from the NMR because the a binding The with RNA were with or binding. were used to the for the and the oligonucleotide the inhibitors and the N-terminal fragment of CNP has some to domains 1990; PubMed Scopus Google Scholar), we also and with a of It was previously reported that RNA is a CNP T.J. Biochem. Biophys. Res. Commun. 1987; PubMed Scopus Google Scholar). show that oligonucleotide has a to the CNP with a of for the is the of in the CNP-CF which produce from RNA and CNP activity. on we that RNA produce any with CNP-CF those identified for The that CNP binds RNA is with the is the function of the N-terminal of revealed similarity to containing This to the that CNP may a 1990; PubMed Scopus Google Scholar), although for this has been E. Braun and D. of CNP with RICH at the the of and Structure of the N-terminal domain and/or CNP may to the role of CNP in it is most that other members of this protein superfamily are involved in RNA a physiological substrate for The structural features of the catalytic domain such as the RNA of each the abundance of positively charged and aromatic residues on the β-sheet and the large of the binding cavity are with this that affinity for the CNP catalytic we that CNP to or to a RNA that a or phosphate for or that the N-terminal of CNP may binding or binding activity. In the of CNP, it is to into other such as the localization of this enzyme. CNP are at their Angelis D.A. Braun P.E. J. Neurosci. Res. 1994; 39: 386-397Crossref PubMed Scopus (55) Google Scholar). The isoform of CNP, contains a sequence at the (27McFerran B. Burgoyne R. J. Cell Sci. 1997; 110: 2979-2985PubMed Google Scholar). CNP is also to with tubulin (26Bifulco M. Laezza C. Stingo S. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1807-1812Crossref PubMed Scopus (128) Google Scholar), to the that CNP have a role in as in oligodendrocytes S. E. Neurobiol. 8: PubMed Scopus Google Scholar, H. W. B. C. E. Cell 2001; PubMed Scopus Google Scholar, E. C. S. H. J. Neurocytol. 1999; 28: PubMed Google Scholar). such as myelin protein and R.P. 4: PubMed Scopus Google Scholar, H. W. Neurochem. Res. 1996; PubMed Scopus Google Scholar) and P. S. R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar) are specifically at the of the some of and localization In are such as phosphate and in J. 2002; Scopus (22) Google Scholar) upon which CNP structural and enzyme studies of CNP the function of this highly conserved protein. Edwards for and the High NMR for and of the

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