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

doi:10.1111/j.1432-1033.2004.04286.x

Eur. J. Biochem. 271, 3512–3522 (2004) (cid:1) FEBS 2004

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 1Department of Biological Sciences and 2Department of Biochemistry, National University of Singapore, Singapore

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.

Keywords: CNS neuronal regeneration; NMR spectroscopy; Nogo-40; NogoA; spinal cord injury.

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 fam- ily, 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 sub- cloned 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

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 path- ways that possibly involve Rho activation, and the induc- tion of growth cone collapse [8]. These discoveries raise a promising possibility to enhance axonal growth by disrupt- ing 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 that residues 567–748 for human Nogo-A proteins) 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 Nogo- A, 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 thio- b-D-galactoside; NgR, Nogo-66 receptor; rmsd, root mean square deviation; TFE, trifluoroethanol. (Received 10 June 2004, revised 6 July 2004, accepted 12 July 2004)

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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

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 4 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

Fig. 1. 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.

Fig. 2. 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 (cid:2)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 (cid:2)C overnight; lane 3, Ni-agarose beads with bound Nogo-66; lane 4, elution 1 under denaturing conditions (in the presence of 8 M urea); lane 5, elution 2 under denaturing conditions (in the presence of 8 M urea); lane 6, elution 3 under denaturing conditions (in the presence of 8 M urea); lane 7, elution 4 under denaturing conditions (in the presence of 8 M urea); lane 8, protein molecular mass markers.

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receptor antagonist, determined by NMR spectroscopy. The obtained results may contribute to further understand- ing of Nogo-A function and aiding in future designs of NgR antagonists.

Experimental procedures

Cloning and expression of the Nogo-A fragments

restriction sites of pET-15b (Novagen). The DNA sequences were confirmed by automated DNA sequencing. The recombinant His-tagged Nogo-A(567–748) and Nogo- 66 were expressed in Escherichia coli BL21 cells. Briefly, the cells were cultured at 37 (cid:2)C until D ¼ 0.6. Isopropyl thio- b-D-galactoside was then added at a final concentration of 0.5 mM to induce the recombinant protein expression overnight at 20 (cid:2)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 [15NH4]2SO4 for (2YT) media, with the addition of 15N-labeling.

Peptide synthesis and purification

The Nogo-A cDNA (designated KIAA 0886) was obtained from the Kazusa DNA Research Institute (Kazusa- Kamatari, 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

Nogo-40 peptide with a sequence of RIYKGVIQAIQ KSDEGHPFRAYLESEVAISEELVQKYSNS(1–40) and the Nogo-24 peptide consisting of the N-terminal 24

Fig. 3. CD and NMR characterization of Nogo-A(567–748). (A) Far-UV CD spectra of Nogo-A(567–748) collected at 20 (cid:2)C in a phosphate buffer at pH 6.5 (black line) and in a Tris/HCl buffer at pH 6.5 containing 4 mM zinc ion (grey line). (B) The 1H-15N HSQC spectrum of Nogo-A(567– 748) collected at 20 (cid:2)C in a phosphate buffer at pH 6.5.

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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

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 software. (Bruker), NMRPIPE [17] and NMRVIEW [18] 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].

CD experiments were performed on a Jasco J-810 spectro- polarimeter equipped with a thermal controller. The far-UV CD spectra of Nogo-A(567–748), Nogo-40 and Nogo-24 were collected at 20 (cid:2)C at peptide concentrations of 10–50 lM using 1 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

lower distance bound. Due

to resonance

set

NMR samples in aqueous buffer were prepared by dissol- ving the Nogo-40 and Nogo-24 synthetic peptides in 50 mM phosphate buffer (pH 6.5) to a final concentration of 1 mM. NMR samples for structure determination contained 1 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 (cid:1) 6.5). The deuterium lock signal for the NMR spectrometers was provided by the addition of 50 lL D2O.

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 line broadening, overlap or small 3JHNHa, or all three, the measurement of 3JHNHa based on a DQF-COSY spec- the trum was on the whole unsuccessful. Therefore, backbone dihedral at to center angles were )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 struc- tures were analyzed by INSIGHTII AND MOLMOL graphic softwares [22].

NMR experiments including two-dimensional NOESY [14], TOCSY [15], DQF-COSY and 1H-15N HSQC [16]

Fig. 4. 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.

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Fig. 5. CD and NMR characterization of Nogo-40. (A) Far-UV CD spectra of Nogo-40 collected at 20 (cid:2)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 (cid:2)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 (cid:2)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 (cid:2)C.

Results

Expression and structural characterization of Nogo-A(567–748) and Nogo-66

affinity columns either under native condition for Nogo- A(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 insol- uble. 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

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

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specifically induce, to a significant degree, the polypeptide to assume a helical conformation.

Nogo-A(567–748) estimated by SDS/PAGE (Fig. 2A) was about (cid:1) 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 (cid:1) 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 (cid:1) 225 nm, indicated that the polypeptide was also not assuming a (cid:1)random coil(cid:2) structure. To explore whether Nogo-A(567–748) had any specific interaction with metal ions, we utilized CD measurements to monitor conforma- tional 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 (cid:1) 206 and 221 nm in the presence of 4 mM Zn2+ resembles that for a typical helical protein. The results indicate that Zn2+ could

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 disper- sions of the spectrum at both 1H and 15N dimensions indicated that Nogo-A(567–748) did not have a tight side- chain 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 conform- ational 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 Nogo- A(567–748) was partially structured, probably with some properties characteristic of molten globule states [24–27]. Interestingly, upon addition of 4 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

Fig. 6. 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 (cid:2)C with sequential assignments indicated. Several medium-range NOEs defining helical structures are labeled.

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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

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 dissec- ted 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 Nogo- 24 was successfully achieved and the chemical shifts determined (data not shown). The NOE assignment shown

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 previ- ously to be an excellent NgR antagonist by virtue of its ability to interact with NgR without eliciting downstream inhibitory signaling [10,11].

Fig. 7. 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.

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Table 1. NMR restraints used for structure calculation and structural statistics for the 10 selected lowest-energy structures.

Restraints for structure determination

NOE distance constraints

in Fig. 4 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.

198 122 76 Sequential Medium range (|i-j| £ 4)

Statistics for structure calculation

Final energies (kcalÆmol)1)

E(total) E(bond) E(angle) E(improper) E(Van der Waals) E(NOE) 63.5 ± 6.6 2.3 ± 0.3 28.5 ± 1.9 4.2 ± 1.1 21.0 ± 2.5 7.3 ± 2.5

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 (cid:1) 198 nm, indicating that Nogo-40 had no stable confor- mation 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 (cid:1) 198 nm and two negative peaks at (cid:1) 208 and 222 nm, respectively. This observation clearly indicates that Nogo-40 adopts a well- formed 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 conforma- tion of Nogo-40. It appears that 50% TFE is sufficient to stabilize a full helical conformation for the peptide.

Root mean square deviations from idealized geometry Bond (A˚ ) Angle (degree) Improper (degree) NOE (A˚ ) 0.002 ± 0.0001 0.400 ± 0.0135 0.285 ± 0.0350 0.027 ± 0.0046

NMR spectroscopy was further utilized to explore the structural properties of Nogo-40. The very narrow reson- ance dispersion of amide protons ((cid:1) 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 ((cid:1) 1.5 p.p.m) and extensive side-chain packing with aro- matic ring protons, indicating that Nogo-40 adopts a well- formed helical structure in the presence of 50% TFE.

included in Table 1. 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(cid:2)).

NMR structure determination of Nogo-40

Based on the observations above, the structure determin- ation of Nogo-40 by NMR spectroscopy was thus carried out in a 50 : 50 (v/v) TFE/H2O mixture. Figure 6 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.

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 exam- ination of the 10 selected structures shows that there are two populations among the 10 structures. Five of these struc- tures, as represented in Fig. 8C, contain only two helices (one from residues 7 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 confor- mation 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 represen- tation 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

Fifty Nogo-40 structures were calculated from the NMR restraints detailed in Table 1 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(cid:2) were selected for further analysis. The structural statistics for the 10 selected structures are also

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) 3.00/4.00 0.22/1.11 0.61/1.58 0.78/1.69

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Nogo-40 constitute a large positive surface (blue) while the C-terminal residues make up a large negative surface (red).

Fig. 8. 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).

Discussion

The discovery that the molecular interaction between Nogo- 66 and NgR poses inhibitory effects on the CNS neuronal regeneration makes the Nogo-66–NgR interface an extre- mely 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 inter- acting 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

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Nogo-A(567–748) might constitute an interesting clue for future functional studies of Nogo-A.

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

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The NMR structure of Nogo-40 reveals that the N- and C-terminal segments of Nogo-40 have opposite electro- static potential surfaces, thus providing an important clue for understanding the Nogo-40–NgR interaction. Recently, the determination of the crystallographic struc- ture of the NgR ectodomain led to the speculation that one potential Nogo-66 binding site on NgR has charac- teristics 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 structure via interaction with Zn2+. form a helical 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].

16. Sattler, M., Schleucher, J. & Griesinger, C. (1999) Heteronuclear multidimensional NMR experiments for the structure determi- nation of proteins in solution employing pulsed field gradients. Prog. NMR Spectrosc. 34, 93–158.

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Supplementary material

available

following material

26. Song, J., Jamin, N., Gilquin, B., Vita, C. & Menez, A. (1999) A gradual disruption of tight side-chain packing: 2D, 1H-NMR characterization of acid-induced unfolding of CHABII. Nat. Struct. Biol. 6, 129–134.

The from http:// is 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.

27. Bai, P., Song, J., Luo, L. & Peng, Z.Y. (2001) A model of dynamic side-chain–side-chain interactions in the alpha-lactalbumin molten globule. Protein Sci. 10, 55–62.

28. Wright, P.E. & Dyson, H.J. (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J. Mol. Biol. 293, 321–331.