Structural characterization of the human Nogo-A functional domainsSolution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration Minfen Li1, Jiah
Trang 1Structural 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 Tang2and Jianxing Song1,2
1 Department of Biological Sciences and 2 Department 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
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
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
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 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
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)
Trang 2and 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
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 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 m M 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 m M IPTG induction at 20 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.
Trang 3receptor antagonist, determined by NMR spectroscopy.
The obtained results may contribute to further
understand-ing of Nogo-A function and aidunderstand-ing 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
(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
restriction sites of pET-15b (Novagen) The DNA sequences were confirmed by automated DNA sequencing The recombinant His-tagged A(567–748) and
Nogo-66 were expressed in Escherichia coli BL21 cells Briefly, the cells were cultured at 37C 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 20C 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 in15N-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
15N-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 3 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 4 m M zinc ion (grey line) (B) The 1 H- 15 N HSQC spectrum of Nogo-A(567– 748) collected at 20 C in a phosphate buffer at pH 6.5.
Trang 4residues 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
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 20C 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
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 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 usingXWINNMR
(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 spec-trum 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 struc-tures were analyzed by INSIGHTII AND MOLMOL graphic softwares [22]
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 m phosphate buffer at pH of 6.5) The observed sequential NH-NH NOEs are labeled.
Trang 5Expression 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 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
Fig 5 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 m M 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 m M 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 m M 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/H 2 O mixture at 35 C.
Trang 6Nogo-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 random coil 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 206 and
221 nm in the presence of 4 mMZn2+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 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 mMZn2+, 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/H 2 O mixture at 35 C with sequential assignments indicated Several medium-range NOEs defining helical structures are labeled.
Trang 7significantly 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
previ-ously 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 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
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.
Trang 8in 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
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
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 198 nm and two
negative peaks at 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
NMR spectroscopy was further utilized to explore the
structural properties of Nogo-40 The very narrow
reson-ance 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
aro-matic ring protons, indicating that Nogo-40 adopts a
well-formed helical structure in the presence of 50% TFE
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
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 were selected for further analysis
The structural statistics for the 10 selected structures are also
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)
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
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 198
Medium range (|i-j| £ 4) 76 Statistics for structure calculation
Final energies (kcalÆmol)1)
E(Van der Waals) 21.0 ± 2.5
Root mean square deviations from idealized geometry
Angle (degree) 0.400 ± 0.0135 Improper (degree) 0.285 ± 0.0350
Average RMSD (A˚) from the lowest-energy structure for backbone/heavy atoms
N-terminal helix (7–12) 0.22/1.11 C-terminal helix (26–37) 0.61/1.58 Additional helix (20–24) 0.78/1.69
Trang 9Nogo-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
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
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).
Trang 10Nogo-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
inter-action 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
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
p75NTRin 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.
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