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 Li
1
, Jiahai Shi
1
, Zheng Wei
1
, Felicia Y. H. Teng
2
, Bor Luen Tang
2
and Jianxing Song
1,2
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 multido main protein containing at le ast two regions
responsible for inhibiting central ner vous 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

a high affinity neuronal r eceptor 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 t he reticulon protein
family has received intense attention recently. Nogo has
several splicing variants, among which N ogo-A is the
largest, composed of 1192 a mino acids (Fig. 1). Recent
studies have demonstr ated 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 m ouse and
residues 5 67–748 f or huma n Nogo-A proteins) that
restricts neurite outgrowth and cell spreading and induces
growth cone co llapse. The second is the e xtracellular 6 6
amino acid loop called Nogo-66 that is also capable of
inhibiting neurite g rowth and inducing growth c one
collapse [4,9–11]. The Nogo-66 domain has been shown
to be anchored on the oligodendrocyte surface and binds
to the neuronal g lycophosphatidylinositol-linked NgR, via
its leucine-rich r epeat containing do main. The binding of
Nogo-66 to N gR 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 e ffective
NgR antagonist capable of enhancing recovery from spinal

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 doma in organization o f the hum an Nogo-A protein. (A) T he do main o rganizat ion of human Nogo-A s howing
the N -terminal stretch region Nogo-A(567–748) and the extracellular 66 amino a cid loop Nogo-66 w ith growth cone collapsing f unctions. 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 se quence 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 e xtract after 0.5 m
M
IPTG in duction at 20 °C o vernight ; l ane 3, supernatant of the cell lysate after h igh speed centrifugation; lane 4,
pellet of the c ell lysate after high s peed centrifugation; lane 5, N i-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 marke rs. (B) Coomasie Brilliant Blue stained SDS/
PAGE gel showing the expression an d affinity-purificatio n of the Nogo-66 protein under denaturing conditions. Lane 1, total cell e xtract 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 u nder d enaturing c ondition s (in th e prese nce of 8
M
urea); lane 5, elutio n 2 u nder d enaturin g con ditions (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

b-
D
-galactoside was then added at a final concentration of
0.5 m
M
to induce the recombinant protein expression
overnight at 20 °C. The Nogo-A(567–748) protein was
purified by Ni
2+
-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
15
N-labeled
form using a similar expression protocol except t hat E. coli
BL21 cells were grown in minimal M9 media instead of rich
(2YT) media, with t he addition of [
15
NH
4
]
2
SO
4
for
15
N-labeling.

added and average d.
NMR experiments and structure calculation
NMR samples in aqueous buffer were p repared by dissol-
ving the Nogo-40 and Nogo-24 synthetic peptides in 50 m
M
phosphate buffer (pH 6.5) to a final concentration of
1m
M
. NMR samples for structure determination contained
1m
M
Nogo-40 in e ither (50 : 50, v/v) trifluoroethanol
(TFE)-d
3
/H
2
O or TFE-d
3
/D
2
O in the presence of 50 m
M
phosphate (final p H or pD  6.5). The deu terium lock
signal for the NMR spectrometers was provided by the
addition of 50 lLD
2
O.
NMR e xperiments including two-dimensional NOESY
[14], TOCSY [15], DQF-COSY and
1

sum of t he Van d er Waals r adii of 1.8 A
˚
was set to be
the lower distance bound. Due to resonance line
broadening, overlap or small
3
J
HNHa
, or all thre e, the
measurement of
3
J
HNHa
basedonaDQF-COSYspec-
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 c alculated on a Linux-b ased
PC station using the simulated annealing protocol [20] in
the
CRYSTALLOGRAPHY
and
NMR
system [21]. The struc-
tures were analyzed by
INSIGHTII AND M OLMOL
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

2
Omixtureat35°C.
3516 M. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
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 w as attributed to the e xistence 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 o f Nogo-A(567–748) in aqueous buffer had a
maximal negative peak at  202 nm and had no significant
positive signal a t 198 nm, indicating that the polypeptide
was not fully structured [23]. However, the existence of t he
maximal negative signal at around 202 nm, rather than
198 nm, together with the negative shoulder signal at
 225 nm, indicated that the polypeptid e was also not
assuming a Ôrandom coilÕ structure. To explore whether
Nogo-A(567–748) h ad any specific interact ion with metal
ions, we utilized CD measurements to monitor conforma-
tional changes induced by the addition o f metal ions,
including Ca
2+
,Mg
2+
,Cu
2+
,Ni
2+

ational exchanges existed over most regions of the protein.
Usually, slow conformational exchange w ould 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 N ogo-A(567–748), while the p eaks for the
regions undergoing slow conformational changes were
undetectable. The results above indicated that Nogo-
A(567–748) was partially structured, p robably with some
properties characteristic of m olten g lobule s tates [ 24–27].
Interestingly, upon addition of 4 m
M
Zn
2+
,nonewHSQC
peaks appeared but the intensities of the existing peaks
became s tronger (spectrum not shown). This observation
suggests that although the introduction of Zn
2+
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 m edium-range NOEs defining h elical structures are labeled.
Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur. J. Biochem. 271) 3517
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 f ound in a well-structured
protein.
CD and NMR characterization of Nogo-24 and Nogo-40

NOE patterns of Nogo-40 u sed to define its secondary structure.
3518 M. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004
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.
Figure 5A shows t he CD spectra of Nogo-40 in aqueous
buffer and methanol/H
2
O 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 meth anol, 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, r espectively. This
observation clearly indicates that Nogo-40 adopts a well-
formed helical conformation in the presence of 74% or
higher percentages of methanol. S imilarly, 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 p eptide.
NMR s pectroscopy was further utilized to explore the
structural properties of Nogo-40. The very narrow reson-

TFE.
Fifty Nogo-40 structures were calculated from the NMR
restraints detailed i n Table 1 with a simulated a nnealing
protocol implem ented by the Crystallography and NMR
system. O ut o f these, the 10 lowest-energy structures with a
distance violation of less than 0.3 A
˚
and a dihedral angle
violationoflessthan5° 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 i ndicate that all s elected structures satisfy the
experimental NMR c onstraints. 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 a ngles (0.4°).
All 10 s tructures o f N ogo-40 contain t wo 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 v alues (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 r epresented in F ig. 8C, contain only two helices
(one from residues 7 to 12 and a nother f rom 2 6 t o 37).
However, another set of five structures, as represented in

from idealized geometry
Bond (A
˚
) 0.002 ± 0.0001
Angle (degree) 0.400 ± 0.0135
Improper (degree) 0.285 ± 0.0350
NOE (A
˚
) 0.027 ± 0.0046
Average RMSD (A
˚
) from the
lowest-energy structure for
backbone/heavy atoms
Whole (2–39) 3.00/4.00
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
Ó FEBS 2004 NMR characterization of the Nogo-A functional domains (Eur. J. Biochem. 271) 3519
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 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. H owever, it has been extensively speculated that in
addition to the N ogo-66 loop, other regions of N ogo-A
might a lso play c ritical r oles in inhibiting CNS neuronal
regeneration [7–11]. Indeed, a recent study showed that

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 k ey rationale for the use of TFE, which
represents a common practice in stabilizing the structure o f
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 deter mination of t he crystallographic s truc-
ture of the N gR ectodomain l ed 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 p art 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 t he N-terminal positive p art i s r esponsible
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 a bolished NgR b inding [10]. I t has also been
shown that residues 30–33 of Nogo-66 (con taining residues
Glu31 and Glu32) are important for NgR binding. Given
the fact t hat both N- and C-terminal residues of Nogo-40

University of Singapore for MALDI-TOF mass spectrometric analysis.
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Supplementary material
The following material is available from http://
www.blackwellpublishing.com/products/journals/suppmat/
EJB/EJB4286/EJB4286sm.htm
Tables S1 and S 2. 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.
3522 M. Li et al.(Eur. J. Biochem. 271) Ó FEBS 2004