Báo cáo khóa học: Structural characterization of the human Nogo-A functional domains Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration ppt

Eur J Biochem 271, 3512–3522 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04286.x Structural characterization of the human Nogo-A functional domains Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration Minfen Li1, Jiahai Shi1, Zheng Wei1, Felicia Y H Teng2, Bor Luen Tang2 and Jianxing Song1,2 Department of Biological Sciences and 2Department of Biochemistry, National University of Singapore, Singapore The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration So far, no structural information is available for Nogo-A or any of its structural domains We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567–748) and the 66 residue Nogo-66 in Escherichia coli CD and NMR characterization indicated that Nogo-A(567–748) was only partially structured while Nogo-66 was highly insoluble Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration Survivors of severe central nervous system (CNS) injury often suffer from permanent disability Previously, it was thought that the inability of CNS neurons to regenerate was due to the absence of growth-promoting factors in CNS neurons However, recent discoveries challenge this dogma It has been shown that the failure of CNS neuronal regeneration results to a large extent from the existence of inhibitory molecules of axon outgrowth in adult CNS myelin [1] So far, three proteins have been identified that cause inhibitory effects on CNS neuronal regeneration, namely Nogo [2–4], myelin-associated glycoprotein [5] and oligodendrocyte myelin glycoprotein [6] All three molecules appear to exert their inhibitory action through the initial binding of the Nogo-66 receptor (NgR) [3], first identified as a high affinity neuronal receptor for Nogo-A [7] NgR binding leads to subsequent activation of signaling pathways that possibly involve Rho activation, and the induction of growth cone collapse [8] These discoveries raise a promising possibility to enhance axonal growth by disrupting the interaction between NgR and its ligands Of the three myelin-associated molecules above, the CNS-enriched Nogo belonging to the reticulon protein family has received intense attention recently Nogo has several splicing variants, among which Nogo-A is the largest, composed of 1192 amino acids (Fig 1) Recent studies have demonstrated that NogoA is a multidomain protein containing several discrete regions with growth inhibitory functions [4,9–11] Two major inhibitory regions have been identified The first is a stretch in the middle of the Nogo-A molecule (residues 544–725 for mouse and residues 567–748 for human Nogo-A proteins) that restricts neurite outgrowth and cell spreading and induces growth cone collapse The second is the extracellular 66 amino acid loop called Nogo-66 that is also capable of inhibiting neurite growth and inducing growth cone collapse [4,9–11] The Nogo-66 domain has been shown to be anchored on the oligodendrocyte surface and binds to the neuronal glycophosphatidylinositol-linked NgR, via its leucine-rich repeat containing domain The binding of Nogo-66 to NgR is competitively inhibited by a peptide consisting of the N-terminal 40 residues of Nogo-66, named Nogo-40 [12,13] This Nogo-40 peptide has been experimentally demonstrated to be a strikingly effective NgR antagonist capable of enhancing recovery from spinal cord injury [13] In contrast to the extensive functional studies on NogoA, thus far no structural information has been available for any region of the Nogo-A protein In the present study, we cloned and expressed the two functional regions of human Nogo-A and performed structural characterization by CD Correspondence to J Song, Department of Biochemistry, National University of Singapore; 10 Kent Ridge Crescent, Singapore 119260 Fax: +65 6779 2486, Tel.: +65 6874 1013, E-mail: bchsj@nus.edu.sg Abbreviations: CNS, central nervous system; IPTG, isopropyl thiob-D-galactoside; NgR, Nogo-66 receptor; rmsd, root mean square deviation; TFE, trifluoroethanol (Received 10 June 2004, revised July 2004, accepted 12 July 2004) Keywords: CNS neuronal regeneration; NMR spectroscopy; Nogo-40; NogoA; spinal cord injury Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur J Biochem 271) 3513 Fig Schematic representation of the domain organization of the human Nogo-A protein (A) The domain organization of human Nogo-A showing the N-terminal stretch region Nogo-A(567–748) and the extracellular 66 amino acid loop Nogo-66 with growth cone collapsing functions The black boxes indicate transmembrane domains (B) The amino acid sequence of Nogo-40, a Nogo-66 receptor antagonist that has been demonstrated to enhance CNS neuronal regeneration (C) The amino acid sequence of the N-terminal 24 residues of Nogo-40 and NMR spectroscopy While Nogo-66 is highly insoluble, the 182 residue fragment was found to be partially structured and could be further induced to form a helical structure with the introduction of mM Zn2+ We conducted further NMR studies on two truncated forms of Nogo-66: Nogo-40 and Nogo-24 Although Nogo-40 and Nogo-24 appeared to be unstructured in aqueous buffer, a detailed NMR analysis revealed that these have intrinsic helix-forming propensity This observation, together with results from secondary structure predictions, offered the rationale to study the structure of Nogo-40 after its intrinsic helix-forming propensity is stabilized by the introduction of the helix-stabilizing solvent trifluoroethanol We report here the structure of Nogo-40, a Nogo-66 Fig Expression and purification of Nogo-A(567–748) and Nogo-66 (A) Coomasie Brilliant Blue stained SDS/PAGE gel showing the expression and affinity-purification of the human Nogo-A(567–748) protein Lane 1, total cell extract before isopropyl thio-b-D-galactoside (IPTG) induction; lane 2, total cell extract after 0.5 mM IPTG induction at 20 °C overnight; lane 3, supernatant of the cell lysate after high speed centrifugation; lane 4, pellet of the cell lysate after high speed centrifugation; lane 5, Ni-agarose beads with bound Nogo-A(567–748); lane 6, protein molecular mass markers; lane 7, affinity-purified Nogo-A(567–748) protein; lane 8, protein molecular mass markers (B) Coomasie Brilliant Blue stained SDS/ PAGE gel showing the expression and affinity-purification of the Nogo-66 protein under denaturing conditions Lane 1, total cell extract before IPTG induction; lane 2, total cell extract after 0.5 mM IPTG induction at 20 °C overnight; lane 3, Ni-agarose beads with bound Nogo-66; lane 4, elution under denaturing conditions (in the presence of M urea); lane 5, elution under denaturing conditions (in the presence of M urea); lane 6, elution under denaturing conditions (in the presence of M urea); lane 7, elution under denaturing conditions (in the presence of M urea); lane 8, protein molecular mass markers Ó FEBS 2004 3514 M Li et al (Eur J Biochem 271) receptor antagonist, determined by NMR spectroscopy The obtained results may contribute to further understanding of Nogo-A function and aiding in future designs of NgR antagonists Experimental procedures Cloning and expression of the Nogo-A fragments The Nogo-A cDNA (designated KIAA 0886) was obtained from the Kazusa DNA Research Institute (KazusaKamatari, Kisarazu, Chiba, Japan) A DNA fragment encoding a 182 residue Nogo-A fragment from residues 567–748 (designated as Nogo-A(567–748); Fig 1) was generated by PCR with a pair of primers: 5¢-CG CGCGCGCGGATCCACTGGTACAAAGATTGCT-3¢ (forward) and 5¢-CGCGCGCGCCTCGAGCTAAAAT AAGTCAACTGGTTC-3¢ (reverse) A DNA fragment encoding human Nogo-66 corresponding to residues 1055–1120 of Nogo-A (Fig 1) was likewise obtained by PCR The PCR fragment encoding Nogo-A(567–748) was subsequently cloned into BamHI/XhoI restriction sites of the expression vector pET32a (Novagen) The fragment encoding Nogo-66 was cloned into the NdeI/BamHI restriction sites of pET-15b (Novagen) The DNA sequences were confirmed by automated DNA sequencing The recombinant His-tagged Nogo-A(567–748) and Nogo66 were expressed in Escherichia coli BL21 cells Briefly, the cells were cultured at 37 °C until D ¼ 0.6 Isopropyl thiob-D-galactoside was then added at a final concentration of 0.5 mM to induce the recombinant protein expression overnight at 20 °C The Nogo-A(567–748) protein was purified by Ni2+-affinity chromatography under native conditions, while the Nogo-66 protein was purified under denaturing conditions because Nogo-66 was found in the inclusion body For heteronuclear NMR experiments the Nogo-A(567– 748) and Nogo-66 proteins were prepared in 15N-labeled form using a similar expression protocol except that E coli BL21 cells were grown in minimal M9 media instead of rich (2YT) media, with the addition of [15NH4]2SO4 for 15 N-labeling Peptide synthesis and purification Nogo-40 peptide with a sequence of RIYKGVIQAIQ KSDEGHPFRAYLESEVAISEELVQKYSNS(1–40) and the Nogo-24 peptide consisting of the N-terminal 24 Fig CD and NMR characterization of Nogo-A(567–748) (A) Far-UV CD spectra of Nogo-A(567–748) collected at 20 °C in a phosphate buffer at pH 6.5 (black line) and in a Tris/HCl buffer at pH 6.5 containing mM zinc ion (grey line) (B) The 1H-15N HSQC spectrum of Nogo-A(567– 748) collected at 20 °C in a phosphate buffer at pH 6.5 Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur J Biochem 271) 3515 residues of Nogo-40 were chemically synthesized using the standard Fmoc method The peptides were purified by HPLC on a reverse-phase C18 column (Vydac), and its identity was verified by MALDI-TOF mass spectrometry and NMR resonance assignments Circular dichroism spectroscopy CD experiments were performed on a Jasco J-810 spectropolarimeter equipped with a thermal controller The far-UV CD spectra of Nogo-A(567–748), Nogo-40 and Nogo-24 were collected at 20 °C at peptide concentrations of 10–50 lM using mm path length cuvettes with a 0.1 nm spectral resolution Data from five independent scans were added and averaged NMR experiments and structure calculation NMR samples in aqueous buffer were prepared by dissolving the Nogo-40 and Nogo-24 synthetic peptides in 50 mM phosphate buffer (pH 6.5) to a final concentration of mM NMR samples for structure determination contained mM Nogo-40 in either (50 : 50, v/v) trifluoroethanol (TFE)-d3/H2O or TFE-d3/D2O in the presence of 50 mM phosphate (final pH or pD  6.5) The deuterium lock signal for the NMR spectrometers was provided by the addition of 50 lL D2O NMR experiments including two-dimensional NOESY [14], TOCSY [15], DQF-COSY and 1H-15N HSQC [16] were performed on a Bruker Avance-500 spectrometer equipped with an actively shielded cryoprobe and pulse field gradient units A mixing time of 250 ms was used for NOESY and 65 ms for TOCSY experiments Spectral processing and analysis were carried out using XWINNMR (Bruker), NMRPIPE [17] and NMRVIEW [18] software Sequence-specific assignments for Nogo-40 were achieved through identification of spin systems in the TOCSY spectra combined with sequential NOE connectivities in the NOESY spectra [19] For structural calculations, NOE connectivities were collected from NOESY spectra of Nogo-40 in TFE/H2O or TFE/D2O mixtures All NOE data were grouped into four categories: strong, medium, weak and very weak, corresponding to upper bound interproton distance ˚ restraints of 3.0, 4.0, 5.0 and 6.0 A, respectively The ˚ sum of the Van der Waals radii of 1.8 A was set to be the lower distance bound Due to resonance line broadening, overlap or small 3JHNHa, or all three, the measurement of 3JHNHa based on a DQF-COSY spectrum was on the whole unsuccessful Therefore, the backbone dihedral angles were set to center at )60 degrees for residues having both aN(i+3) NOEs and large helical conformational shifts The solution structure of Nogo-40 was calculated on a Linux-based PC station using the simulated annealing protocol [20] in the CRYSTALLOGRAPHY and NMR system [21] The structures were analyzed by INSIGHTII AND MOLMOL graphic softwares [22] Fig NMR characterization of Nogo-24 NH-NH region of a NOESY spectrum of Nogo-24 (mixing time of 250 ms) acquired in an aqueous buffer (50 mM phosphate buffer at pH of 6.5) The observed sequential NH-NH NOEs are labeled 3516 M Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig CD and NMR characterization of Nogo-40 (A) Far-UV CD spectra of Nogo-40 collected at 20 °C in the presence of methanol at different concentrations Black, 50 mM phosphate buffer (pH 6.5); pink, 20% TFE; green, 36%; cyan, 49%; dark violet, 60%; brown, 68%; dark green, 74% and blue, 80% (B) Far-UV CD spectra of Nogo-40 collected at 20 °C in the presence of TFE at different concentrations black: 50 mM phosphate buffer (pH 6.5); pink, 20% TFE solution; green, 36%; cyan, 49% and red, 60% (C) NH-aliphatic region of a NOESY spectrum (mixing time of 250 ms) of Nogo-40 acquired in a 50 mM phosphate buffer (pH 6.5) at 15 °C (D) NH-aliphatic region of a NOESY spectrum of Nogo-40 (mixing time of 250 ms) acquired in a 50 : 50 (v/v) TFE/H2O mixture at 35 °C Results Expression and structural characterization of Nogo-A(567–748) and Nogo-66 Nogo-A(567–748) and Nogo-66 were successfully cloned and expressed as His-tagged proteins As shown in Fig 2, both recombinant proteins could be affinity-purified by affinity columns either under native condition for NogoA(567–748) or under denaturing condition for Nogo-66 Attempts to refold Nogo-66 by dialysis and fast dilution were unsuccessful, indicating that Nogo-66 is highly insoluble On the other hand, the 182 residue Nogo-A(567–748) was soluble and its molecular mass as determined by MALDI-TOF MS matched that predicted from the amino acid sequence Interestingly the apparent molecular mass of Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur J Biochem 271) 3517 Fig NMR spectral assignment of Nogo-40 The NH-aH region of a NOESY spectrum of Nogo-40 (mixing time of 250 ms) acquired in a 50 : 50 (v/v) TFE/H2O mixture at 35 °C with sequential assignments indicated Several medium-range NOEs defining helical structures are labeled Nogo-A(567–748) estimated by SDS/PAGE (Fig 2A) was about  37 kDa, much larger than that expected for a 182 residue protein This anomalous behavior on SDS/PAGE has been previously observed for cloned Nogo-A fragments and was attributed to the existence of a high number of charged residues in Nogo-A [4,10] The structural properties of Nogo-A(567–748) were first investigated by CD spectroscopy As shown in Fig 3A, the CD spectrum of Nogo-A(567–748) in aqueous buffer had a maximal negative peak at  202 nm and had no significant positive signal at 198 nm, indicating that the polypeptide was not fully structured [23] However, the existence of the maximal negative signal at around 202 nm, rather than 198 nm, together with the negative shoulder signal at  225 nm, indicated that the polypeptide was also not assuming a Ôrandom coilÕ structure To explore whether Nogo-A(567–748) had any specific interaction with metal ions, we utilized CD measurements to monitor conformational changes induced by the addition of metal ions, including Ca2+, Mg2+, Cu2+, Ni2+ and Zn2+ Only Zn2+ was able to induce a significant conformational change in the polypeptide As shown in Fig 3A, the CD spectrum of Nogo-A(567–748) with dual negative signals at  206 and 221 nm in the presence of mM Zn2+ resembles that for a typical helical protein The results indicate that Zn2+ could specifically induce, to a significant degree, the polypeptide to assume a helical conformation The structural properties of the Nogo-A(567–748) were further assessed by the NMR HSQC experiment, which is very sensitive to both secondary structures and tertiary packings As shown in Fig 3B, the poor chemical dispersions of the spectrum at both 1H and 15N dimensions indicated that Nogo-A(567–748) did not have a tight sidechain packing In particular, the number of observed NMR cross peaks was only about 35, much less than expected for a 182 residue protein, thus indicating that slow conformational exchanges existed over most regions of the protein Usually, slow conformational exchange would result in significant line-broadening for HSQC peaks and make these peaks undetectable The manifested HSQC peaks in Fig 3B most likely resulted from the unstructured and flexible regions of the Nogo-A(567–748), while the peaks for the regions undergoing slow conformational changes were undetectable The results above indicated that NogoA(567–748) was partially structured, probably with some properties characteristic of molten globule states [24–27] Interestingly, upon addition of mM Zn2+, no new HSQC peaks appeared but the intensities of the existing peaks became stronger (spectrum not shown) This observation suggests that although the introduction of Zn2+ was able to 3518 M Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig The secondary structures of Nogo-24 and Nogo-40 (A) Ca proton conformational shifts of Nogo-24 (grey) and Nogo-40 (black) (B) The NOE patterns of Nogo-40 used to define its secondary structure significantly enhance the helical structure of Nogo-A(567– 748) as detected by CD, it was not sufficient to make the tertiary packing as tight as those found in a well-structured protein CD and NMR characterization of Nogo-24 and Nogo-40 The purified Nogo-66 was found to be highly insoluble in both aqueous buffer and a TFE/H2O mixture An attempt to acquire a 1H-15N HSQC spectrum of Nogo-66 was unsuccessful We therefore focused our NMR structure determination on Nogo-40, which has been shown previously to be an excellent NgR antagonist by virtue of its ability to interact with NgR without eliciting downstream inhibitory signaling [10,11] Secondary structure prediction suggested that Nogo-40 had a strong propensity to form a helical structure (data not shown) However, the preliminary CD and NMR study indicated that Nogo-40 was largely unstructured in aqueous buffers As a result, it was not possible to assign the NMR spectra of Nogo-40 under these conditions due to the severe peak overlap To gain insight into the intrinsic secondary structure preference of Nogo-40 experimentally, we dissected Nogo-40 into two fragments, namely the N- and C-terminal parts While several attempts to synthesize the C-terminal part of Nogo-40 failed, the peptide Nogo-24, comprising the N-terminal 24 residues of Nogo-66, was successfully produced The sequential assignment of Nogo24 was successfully achieved and the chemical shifts determined (data not shown) The NOE assignment shown Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur J Biochem 271) 3519 in Fig clearly indicates that sequential NH-NH NOE connectivities exist over many residues of Nogo-24, strongly indicating intrinsic helix-forming propensity in the Nogo-24 peptide, even in aqueous buffer This observation, together with the secondary structure predictions for Nogo-40, prompted us to conduct further NMR studies of Nogo-40 in the presence of TFE and methanol, which is well-known for its ability to stabilize intrinsic helixes Figure 5A shows the CD spectra of Nogo-40 in aqueous buffer and methanol/H2O mixtures The CD spectrum of Nogo-40 in the aqueous buffer has a negative peak at  198 nm, indicating that Nogo-40 had no stable conformation in aqueous buffer [23] Interestingly, with the introduction of methanol, the CD spectra of Nogo-40 undergo dramatic changes The CD spectra of Nogo-40 in the presence of methanol at a concentration of 74% or above show one positive peak at  198 nm and two negative peaks at  208 and 222 nm, respectively This observation clearly indicates that Nogo-40 adopts a wellformed helical conformation in the presence of 74% or higher percentages of methanol Similarly, as shown in Fig 5B, TFE is also able to stabilize the helical conformation of Nogo-40 It appears that 50% TFE is sufficient to stabilize a full helical conformation for the peptide NMR spectroscopy was further utilized to explore the structural properties of Nogo-40 The very narrow resonance dispersion of amide protons ( 0.7 p.p.m) and the lack of side-chain packing with aromatic ring protons in aqueous buffer (Fig 5C) demonstrate that Nogo-40 in aqueous buffer had no stable structure, which is consistent with the CD results above In contrast, the same NOESY region of Nogo-40 in the 50 : 50 (v/v) TFE/H2O mixture (Fig 5D) shows a dramatically increased dispersion of amide protons ( 1.5 p.p.m) and extensive side-chain packing with aromatic ring protons, indicating that Nogo-40 adopts a wellformed helical structure in the presence of 50% TFE NMR structure determination of Nogo-40 Based on the observations above, the structure determination of Nogo-40 by NMR spectroscopy was thus carried out in a 50 : 50 (v/v) TFE/H2O mixture Figure presents a NH-aH region of NOESY spectrum of Nogo-40 with sequential assignments labeled The aH conformational shifts (Fig 7A) suggest that Nogo-40 contains two helical fragments, one at the N-terminal part and the other over the C-terminus The medium-range NOE connectivities such as daN(i, i+2), daN(i, i+3), daN(i, i+4) and dab(i, i+3) used for identification of secondary structures, again support the observation that two helical segments exist in Nogo-40 (Fig 7B) It is also noteworthy that the helical conformational shifts already existed for Nogo-24 in aqueous buffer (Fig 7A), although were less pronounced than those for Nogo-40 in 50% TFE Fifty Nogo-40 structures were calculated from the NMR restraints detailed in Table with a simulated annealing protocol implemented by the Crystallography and NMR system Out of these, the 10 lowest-energy structures with a ˚ distance violation of less than 0.3 A and a dihedral angle violation of less than 5° were selected for further analysis The structural statistics for the 10 selected structures are also Table NMR restraints used for structure calculation and structural statistics for the 10 selected lowest-energy structures Restraints for structure determination NOE distance constraints Sequential Medium range (|i-j| £ 4) 198 122 76 Statistics for structure calculation Final energies (kcalỈmol)1) E(total) E(bond) E(angle) E(improper) E(Van der Waals) E(NOE) Root mean square deviations from idealized geometry ˚ Bond (A) Angle (degree) Improper (degree) ˚ NOE (A) ˚ Average RMSD (A) from the lowest-energy structure for backbone/heavy atoms Whole (2–39) N-terminal helix (7–12) C-terminal helix (26–37) Additional helix (20–24) 63.5 2.3 28.5 4.2 21.0 7.3 ± ± ± ± ± ± 0.002 0.400 0.285 0.027 6.6 0.3 1.9 1.1 2.5 2.5 ± ± ± ± 0.0001 0.0135 0.0350 0.0046 3.00/4.00 0.22/1.11 0.61/1.58 0.78/1.69 included in Table The low values of distance and dihedral angle energies indicate that all selected structures satisfy the experimental NMR constraints Moreover, the covalent geometry is well-respected as demonstrated by the low root mean square deviation (rmsd) values for the bond lengths ˚ (0.0019 A) and the valence angles (0.4°) All 10 structures of Nogo-40 contain two helices, one over residues 7–12 and another over residues 26–37 Superimposition of the 10 structures over either helix (Fig 8A,B) gives low rmsd values (Table 1), indicating that both helices are well defined However, due to the absence of NOEs between N- and C-terminal helices, their relative orientation cannot be determined A more detailed examination of the 10 selected structures shows that there are two populations among the 10 structures Five of these structures, as represented in Fig 8C, contain only two helices (one from residues to 12 and another from 26 to 37) However, another set of five structures, as represented in Fig 3D, has an additional helix over residues 20–24 Indeed, conformational shifts shown in Fig 7A and medium-range NOEs in Fig 7B indicate a helical conformation over residues 20–24 Possibly due to the existence of side-chain–side-chain NOEs among residues His17, Phe19, Tyr22 and Leu23, the helix over residue 20–25 is distorted to some extent and consequently became undetectable in five of the 10 selected structures Figure 8E shows a representation of the electrostatic potential associated with the contact surface of the Nogo-40 structure The most interesting observation here is that the N- and C-terminal parts of Nogo-40 have opposite electrostatic potential surfaces More specifically, the N-terminal nine residues of 3520 M Li et al (Eur J Biochem 271) Ó FEBS 2004 Fig Solution structure of Nogo-40 (A) The 10 lowest-energy structures superimposed over the N-terminal helix over residues 7–12 (B) The same 10 lowest-energy structures superimposed over the C-terminal helix over residues 26–37 (C) Ribbon representation of one conformational ensemble of Nogo-40 structure with only two helices formed (D) Ribbon representation of another conformational ensemble of Nogo-40 structure with an additional helix over residues 20–24 (E) Representation of the electrostatic potential associated with the contact surface of the Nogo-40 solution structure Two distinctive surfaces are observed: the N-terminal surface is largely positive (blue) while the C-terminal part is negative (red) Nogo-40 constitute a large positive surface (blue) while the C-terminal residues make up a large negative surface (red) Discussion The discovery that the molecular interaction between Nogo66 and NgR poses inhibitory effects on the CNS neuronal regeneration makes the Nogo-66–NgR interface an extremely promising target for design of molecules to treat CNS injuries However, it has been extensively speculated that in addition to the Nogo-66 loop, other regions of Nogo-A might also play critical roles in inhibiting CNS neuronal regeneration [7–11] Indeed, a recent study showed that Nogo-A, the longest member of the Nogo transcripts encoding for more than 1000 amino acid residues, has at least two discrete regions with neuronal growth inhibitory effects [4,11] As no previous structural study has been reported for Nogo-A, we carried out a detailed CD and NMR investigation in an attempt to gain structural insights into these two functional regions Our results revealed that although Nogo-A(567–748) is functionally active, it is only partially structured either due to the loss of the stabilizing contacts provided by other parts of the Nogo-A protein or is a member of so called natively unstructured proteins, which only become well-structured upon binding to their interacting partners or cognate receptors [28,29], or even both Interestingly, the observation that the Zn2+ was able to specifically induce the formation of helical structures in Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur J Biochem 271) 3521 Nogo-A(567–748) might constitute an interesting clue for future functional studies of Nogo-A On the other hand, the recent identification of Nogo-40 as a potent NgR antagonist suggests a promising starting point for the design of potential therapeutic agents to enhance CNS neuronal regeneration Knowledge of the three-dimensional structure of Nogo-40 is necessary for both understanding the endogenous Nogo-66–NgR interaction and for the rational design of other NgR-binding antagonists Although Nogo-40 is highly disordered in aqueous buffer, close NMR examination indicates that it has an intrinsic propensity to assume helical conformations This provides a key rationale for the use of TFE, which represents a common practice in stabilizing the structure of a polypeptide with intrinsic helical propensity to enable their further analysis [29] The NMR structure of Nogo-40 reveals that the N- and C-terminal segments of Nogo-40 have opposite electrostatic potential surfaces, thus providing an important clue for understanding the Nogo-40–NgR interaction Recently, the determination of the crystallographic structure of the NgR ectodomain led to the speculation that one potential Nogo-66 binding site on NgR has characteristics of a negative cavity, consisting of residues Asp111, Asp114, Ser113 and Asp138 [30,31] As shown in Fig 8E, the C-terminal part of Nogo-40 is highly negatively charged, making it unlikely as a candidate for binding to this acidic NgR cavity On the other hand, it is highly probable that the N-terminal positive part is responsible for its binding to the NgR negative cavity This is in complete agreement with previous findings that deletion of the first five residues at the N-terminal end of Nogo-66 greatly diminished NgR binding, and deletion of the first 10 residues abolished NgR binding [10] It has also been shown that residues 30–33 of Nogo-66 (containing residues Glu31 and Glu32) are important for NgR binding Given the fact that both N- and C-terminal residues of Nogo-40 were required for NgR binding, it would be logical to speculate that the C-terminal part of Nogo-40 may bind to a positively charged surface on NgR, which is not revealed by the current NgR structure Alternatively, it is also possible that this part of Nogo-40 may even bind to other molecules such as the recently identified NgR coreceptor p75NTR in the formation of a multicomponent complex In summary, our study represents the first structural insights into the two functional regions of Nogo-A critical for inhibiting CNS neuronal regeneration The results showed that the region consisting of Nogo-A(567– 748) is only partially structured but can be induced to form a helical structure via interaction with Zn2+ Furthermore, the determination of the Nogo-40 solution structure offers a starting point for further understanding the interaction between NgR and Nogo-40, and for future designs of molecules to enhance CNS neuronal regeneration using NMR methodology as demonstrated previously [32–35] Acknowledgements This work is supported by the NMRC grant R183-000-092-214, the BMRC grant R-183-000-097-305 and the BMRC Young Investigator Award R-154-000-217-305 to J Song and BMRC grant R-183-000- 098-305 to B.L Tang The authors acknowledge J Lefebvre for peptide synthesis, H Zhang, Y.H Han for accessing NMR spectrometer and X.H Wu at the Protein and Proteomics Center (PPC), National University of Singapore for MALDI-TOF mass spectrometric analysis References Woolf, C.J & Bloechlinger, S (2002) Neuroscience It takes more than two to Nogo Science 297, 1132–1134 Oertle, T & Schwab, M.E (2003) Nogo and its paRTNers Trends Cell Biol 13, 187–194 McGee, A.W & Strittmatter, S.M (2003) The Nogo-66 receptor: focusing myelin inhibition of axon regeneration Trends Neurosci 26, 193–198 Schwab, M.E (2004) Nogo and axon regeneration Curr Opin Neurobiol 14, 118–124 Liu, B.P., Fournier, A., GrandPre, T & Strittmatter, S.M (2002) Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor Science 297, 1190–1193 Wang, K.C., Koprivica, 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Cate, R., Strittmatter, S.M & Nikolov, D.B (2003) Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins EMBO J 22, 3291–3302 32 Diercks, T., Coles, M & Kessler, H (2001) Applications of NMR in drug discovery Curr Opin Chem Biol 5, 285–291 33 Pellecchia, M., Sem, D.S & Wuthrich, K (2002) NMR in drug discovery Nat Rev Drug Discov 1, 211–219 34 Song, J & Ni, F (1998) NMR for the design of functional mimetics of protein–protein interactions: one key is in the building of bridges Biochem Cell Biol 76, 177–188 35 Homans, S.W (2004) NMR spectroscopy tools for structureaided drug design Angew Chem Int Ed Engl 43, 290–300 Supplementary material The following material is available from http:// www.blackwellpublishing.com/products/journals/suppmat/ EJB/EJB4286/EJB4286sm.htm Tables S1 and S2 Chemical shifts of Nogo-24 in a 25 mM phosphate buffer (pH 6.8) at 298 K, and chemical shifts of Nogo-40 in a 50/50 % (TFE/H2O) mixture at 308 K  ... characterization of the Nogo -A functional domains (Eur J Biochem 271) 3513 Fig Schematic representation of the domain organization of the human Nogo -A protein (A) The domain organization of human. .. fragments The Nogo -A cDNA (designated KIAA 0886) was obtained from the Kazusa DNA Research Institute (KazusaKamatari, Kisarazu, Chiba, Japan) A DNA fragment encoding a 182 residue Nogo -A fragment... (designated as Nogo -A( 567–748); Fig 1) was generated by PCR with a pair of primers: 5¢-CG CGCGCGCGGATCCACTGGTACAAAGATTGCT-3¢ (forward) and 5¢-CGCGCGCGCCTCGAGCTAAAAT AAGTCAACTGGTTC-3¢ (reverse) A DNA