Sequence–Structure and Structure–Function Analysis ... - Biozentrum

doi:10.1016/j.jmb.2007.02.026 J. Mol. Biol. (2007) 368, 718–728
Sequence–Structure and Structure–Function Analysis
in Cysteine-rich Domains Forming the
Ultrastable Nematocyst Wall
Sebastian Meier 1 ⁎, Pernille Rose Jensen 1 , Patrizia Adamczyk 2
Hans Peter Bächinger 3 , Thomas W. Holstein 2 , Jürgen Engel 4
Suat Özbek 2 and Stephan Grzesiek 1 ⁎
1 Department of Structural
Biology, Biozentrum,
University of Basel,
Klingelbergstrasse 70,
CH-4056 Basel, Switzerland
2 Institute of Zoology,
Department for Molecular
Evolution and Genomics,
University of Heidelberg,
Im Neuenheimer Feld 230,
D-69120 Heidelberg, Germany
3 Shriners Hospital for Children
and Department of Biochemistry
and Molecular Biology,
Oregon Health and Science
University, Portland,
OR 97239, USA
4 Department of Biophysical
Chemistry, Biozentrum,
University of Basel,
Klingelbergstrasse 70,
CH-4056 Basel, Switzerland
*Corresponding authors
The nematocyst wall of cnidarians is a unique biomaterial that withstands
extreme osmotic pressures, allowing an ultrafast discharge of the nematocyst
capsules. Assembly of the highly robust nematocyst wall is achieved by
covalent linkage of cysteine-rich domains (CRDs) from two main protein
components, minicollagens and nematocyst outer wall antigen (NOWA).
The bipolar minicollagens have different disulfide patterns and topologies in
their N and C-terminal CRDs. The functional significance of this polarity has
been elusive. Here, we show by NMR structural analysis that all
representative cysteine-rich domains of NOWA are structurally related to
N-terminal minicollagen domains. Natural sequence insertions in NOWA
CRDs have very little effect on the tightly knit domain structures, nor do they
preclude the efficient folding to a single native conformation. The different
folds in NOWA CRDs and the atypical C-terminal minicollagen domain on
the other hand can be directly related to different conformational preferences
in the reduced states. Ultrastructural analysis in conjunction with aggregation
studies argues for an association between the similar NOWA and
N-terminal minicollagen domains in early stages of the nematocyst wall
assembly, which is followed by the controlled association between the
unusual structures of C-terminal minicollagen domains.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: molecular evolution; conformational diversity; NMR structure;
cysteine-rich; dihedral angle preference
Present address: S. Meier, Institute of Molecular Biology and Physiology, August Krogh Building, University of
Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark.
Abbreviations used: CRD, cysteine-rich domain; NOWA, nematocyst outer wall antigen; CROD,
cysteine-rich octarepeat domain of NOWA; NW1r, first CRD of NOWA; NW6r, sixth CRD of NOWA; NW8r, eighth CRD
of NOWA; Mcol1hN, N-terminal CRD of minicollagen 1 from Hydra; Mcol1hC, C-terminal CRD of minicollagen 1 from
Hydra; RDC, residual dipolar coupling; NOE, nuclear Overhauser effect; TCEP,
Tris(2-carboxyethyl)-phosphine; GSH, reduced glutathione; GSSG, oxidized glutathione; HSQC, heteronuclear single
quantum correlation; indel, insertion/deletion.
E-mail addresses of the corresponding authors: smeier@aki.ku.dk; stephan.grzesiek@unibas.ch
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

Structure and Function of Nematocyst CRDs
719
Introduction
highly sensitive to reducing agents, indicating that
the wall proteins are involved in the formation of a
Nematocysts are the characteristic explosive organelles
of the phylum cnidaria. They comprise a soluble in the early stages of nematocyst develop-
cysteine-linked network. 5 As minicollagens are still
cylindrical capsule of about 10 μm diameter that ment 6 and their cysteine residues form intramolecular
disulfide bridges, 7 it has been proposed that
encloses an attached tubular coil structure and
toxins. Upon mechanical stimulation by a prey isomerization to intermolecular disulfide bridges
organism or predator, a lid in the capsule wall would constitute the final maturation step in
opens and the tube together with the stored toxins is morphogenesis, which involves wall compaction
released in an ultrafast extrusion process. 1 This and polymerization. 8
process is driven by the extreme osmotic pressure We have shown recently that recombinantly
(150 bar; 1 bar=10 5 Pa) within the capsule. 2 The wall expressed NOWA as well as its cysteine-rich octarepeat
domain (CROD) spontaneously form disulfide-
structure of the Hydra nematocyst is a unique
biological polymer formed mainly by two protein linked globular aggregates that resemble the globular
building units of the nematocyst wall. 3 These
species, nematocyst outer wall antigen (NOWA)
and minicollagens, which share a common cysteine-rich
motif. 3 These short cysteine-rich domains ranging from 15 nm to 45 nm in diameter and form
NOWA globules are extremely heterogeneous in size
(CRDs) contain a conserved pattern of six cysteine several densely packed layers along the wall profile.
residues spaced by three residues. Minicollagens Ultrastructural analysis of the wall architecture
exhibit a symmetrical domain organization with a revealed that the individual globules are interconnected
via rod-like protrusions that probably repre-
central collagen sequence flanked by polyproline
stretches and a CRD at the N and C termini of the sent minicollagen. NOWA appears early during
molecule (Figure 1(a)). 4 The best-characterized nematocyst morphogenesis and forms a thin layer at
minicollagen in the nematocyst wall has been the inside of the nematocyst vesicle membrane. 9 Minicollagens,
which are expressed at later stages,
termed minicollagen 1, and serves as a model for
the protein family. NOWA is a 90 kDa glycoprotein gradually attach to this layer in a pre-assembly
with an N-terminal sperm-coating protein (SCP) process, which is followed by polymerization between
domain, a central C-type lectin domain (CTLD) and the bipolar minicollagens. Thus, NOWA acts as a
an eightfold CRD repeat at its C terminus (Figure positional organizer of the nematocyst superstructure,
1(a)). The capsule structure has been shown to be providing a scaffold for minicollagen polymerization.
It has been shown recently that the N and
C-terminal CRDs of minicollagen 1 from Hydra
(Mcol1hN and Mcol1hC) form different disulfide
links from identical cysteine patterns (Figure
1(b)). 10,11 This clearly serves as a rare example for
the divergent evolution of protein folds in closely
related domains. We have demonstrated that the two
folds overlap in sequence space and can be interconverted
by changes of a few amino acids. 12 The
ability of single cysteine-rich sequences to populate
two different tertiary structures is reminiscent of the
conformational plasticity of prion proteins and
amyloidogenic proteins. Moreover, this ability has
given strong indications that complex features,
including protein tertiary structures, can develop
by smooth evolutionary transitions. While we have
obtained strong indications that the structural
polarity of N and C-terminal CRD folds is conserved
in minicollagens, the functional significance of this
polarity in the formation of the nematocyst wall has
been elusive, mainly due to lack of structural
information on the NOWA CROD.
Here, we present the structure of all representative
cysteine-rich modules in the NOWA CROD, thus
Figure 1. Domain organization of NOWA and minicollagen
1. (a) Schematic modular arrangement of signal NOWA, the key components of the nematocyst wall.
completing the structural data on minicollagens and
peptide (SP), sperm-coating protein (SCP) domain, C-type Despite sequence insertions of varying length into
lectin domain (CTLD) and cysteine-rich octarepeat
the tightly knit disulfide rich domains, all NOWA
domain (CROD) in NOWA; CRDs are indicated as circles
and coloured according to sequence similarity; CRDs in CRDs form an identical, prototypical structure. This
minicollagen1 are known to form different structures. (b) fold is similar to the Mcol1hN domain structure and
Sequence alignment of the eight CRDs in the NOWA different from the atypical Mcol1hC fold. NMR
octarepeat domain and comparison to N and C-terminal analysis of isotope-enriched recombinant protein
CRDs of Hydra minicollagen 1 (Mcol1hN and Mcol1hC). 10 shows that the different CRD folds in Mcol1hC and

720 Structure and Function of Nematocyst CRDs
NOWA can be directly ascribed to different conformational
sampling in the reduced states. We find
that CRDs consistently show a rapid folding reaction
in accordance with the fast cross-linking of the
nematocyst wall. Distinct association properties of
the different CRD domains in conjunction with
electron microscopic analysis suggest that NOWA
nanoparticles are deposited early in the nematocyst
wall formation and are crosslinked by short minicollagen
polymers in a controlled fashion to form a
highly stable composite biomaterial.
Results and Discussion
Recombinant expression of cysteine-rich
domains
The pattern of six cysteine residues in little more
than 20 amino acids of NOWA cysteine-rich
domains resembles the pattern CXXXCXXXCXXXC-
XXXCC in the closely related CRDs of minicollagen
1(Figure 1). While the first repeat in the cysteine-rich
octarepeat domain (NWr1) has the same length as
the Mcol1hN and Mcol1hC domains, the repeats
NWr2 to NWr7 carry an insertion of two amino acids
between the first two cysteine residues and NWr8
carries an insertion of three amino acids. For a
modular study of the entire NOWA protein domains
NWr1, NWr6 (representing the highly homologous
repeats NWr2-NWr7), and NWr8 were recombinantly
produced in Escherichia coli. The domains
were expressed as fusion proteins C-terminal to the
GB1 domain of protein G, 13 with a thrombin
cleavage site in the linker between protein G and
the cysteine-rich domain. This resulted in good
expression yields of about 20 mg of soluble fusion
protein per litre of minimal medium. Cleavage,
purification and oxidative refolding of the isolated,
uniformly 15 N-labeled domains yielded >5 mg end
product per litre of minimal medium for NWr1,
NWr6 and NWr8. Mass spectrometry validated the
chemical identity and purity of the CRDs, and
showed that the domains were fully oxidized with
three disulfide bonds formed (Table 1).
Structure of NOWA CRDs
While six cysteine residues can be fully oxidized to
15 different disulfide bond topologies, the 1 H- 15 N
Table 1. Molecular mass of recombinantly expressed
[U- 15 N]CRDs
Molecular mass (Da)
Mass spectroscopy
Theoretical
NWr1 3109.3 3109.6
NWr6 2802.9 2802.8
NWr8 2930.6 2931.0
Theoretical values are given for fully 15 N-labelled, fully oxidized
domains.
heteronuclear single quantum correlation (HSQC)
spectra show only one folded spectral species for
each of the purified CRDs (Figure 2). Other
prominent spectral species are absent, and any
population of minor forms is smaller than 10% of
the main structural species according to the HSQC
peak intensities. Thus, the NOWA CRDs fold very
effectively towards their native structure. Preliminary
structural information can be obtained from the
assigned HSQC spectra, as a central canonical βI
turn between the third and fourth cysteine in
Micol1hC (PDB accession number 1SP7) gives rise
to a characteristic 15 N upfield shifts at turn position
i+2. 14 This upfield shift is not observed in any of the
NOWA CRDs at the respective positions E480, Q662
and A738 (Figure 2), indicating that the fold of all
NOWA domains is different from that of Mcol1hC.
For a detailed analysis of the structural determinants
of the different CRD folds, the structures of
the three NOWA domains NWr1, NWr6 and NWr8
were determined de novo with very high precision
(Table 2). The RMSD of all heavy atoms in the
folded core is less than 0.6 Å for the solution
structures of NWr1, NWr6 and NWr8. All NOWA
CRD structures have the same fold (Figure 3). This
is in contrast to the structural polarity of minicollagens
with different structures of Mcol1hN and
Mcol1hC domains, and indicates different assembly
functions for NOWA and minicollagens. The
cysteine pattern 1-4, 2-6 and 3-5 and a central cis
proline turn between cysteine 3 and cysteine 4
coincide with the Mcol1hN structure (PDB accession
number 1ZPX). However, the overall lefthanded
fold of the NOWA domains and Mcol1hN
differs completely from the Mcol1hC fold, which is
right-handed. In particular, the NOWA cysteine
pattern differs fundamentally from the Mcol1hC
structure (1-5,2-4,3-6; Figures 3 and 4) and none of
the NOWA turns, which bring the disulfidebonded
cysteine side-chains into spatial proximity,
coincides with the Mcol1hC domain.
Due to the absence of long-range side-chain
interactions apart from the disulfide bridges, the
different structures of prototypical NOWA CRDs
and the atypical Mcol1hC must be a consequence
of different local turn propensities of the intercysteine
residues. 12 The sequential arrangement of
turns in the prototypical NWr1 domain starts with
a canonical βII turn from residues 470–473
between the first two cysteine residues, C469 and
C473, followed by a short 3 10 helix formed by two
consecutive βIII turns (474–478), which contains
the third cysteine, C477. The conserved proline
residue 479 is in cis conformation and part of a
βVIa turn from residues 478–481, which continues
into a γ turn (480–482). Both turns position the
fourth cysteine, C481. The chain terminates in a
single α-helical turn (482–486) that contains the
remaining cysteine residues C485 and C486. All
hydrogen bonds and a scheme of the turn and
disulfide topology are given in Figure 4. The amino
acid insertions between the first two cysteine
residues in the NWr6 and NWr8 domains are

Structure and Function of Nematocyst CRDs
721
Figure 2. Assigned 1 H- 15 N HSQC spectra of the oxidized recombinant octarepeat domains NWr1, NWr6 and NWr8
from NOWA. N-terminal artefactual serine residues of the thrombin cleavage sites are marked with an asterisk (*).
incorporated into the NOWA fold in α-helical
conformations directly following the first cysteine
residue. As the α-helical pitch is small in comparison
with an extended conformation, these turns
can accommodate the increase in chain length
without changing the overall structure.
Thus, the NWr6 and NWr8 structures differ only
locally from the NWr1 structure. Notably though,
turns are not strictly conserved at homologous
positions: while NWr8 retains the canonical βII
turn before the second cysteine, NWr6 adopts a βI
turn at this location. Presumably, the mutation G→S
abolishes the high positional potential for the formation
of a βII turn and induces a preference for the
formation of a βI turn (Figure 1). 15
Structural consequences of insertion, deletion
and substitution events during the evolution of
nematocyst CRDs
The cysteine-rich domains from minicollagens and
NOWA form an attractive natural framework for the
study of sequence–structure relationships as well as
structural evolution resulting from insertion/deletion
(indel) or substitution events. Despite the high
level of similarity of NWr2–NWr7, which points to a
rather recent gene duplication event, CRDs of
different proteins have little sequence similarity
due to extensive neutral drift. On the other hand,
local sequence variations between the cysteine
residues suffice to induce different folds in Mcol1hC
Table 2. Statistics of the NWr1, NWr6 and NWr8 NMR structures
NWr1 NWr6 NWr8
RMSD from experimental distance constraints a (Å) 0.073±0.001 0.060±0.001 0.071±0.001
RMSD from dihedral constraints b (deg.) 1.21±0.03 0.90±0.02 1.19±0.03
RMSD from scalar coupling constraints c 0.59±0.02 0.97±0.02 0.32±0.01
Deviation from the idealized covalent geometry
Bond lengths (Å) 0.0086±0.001 0.0061±0.001 0.0048±0.001
Bond angles (deg.) 1.373±0.01 0.775±0.01 0.665±0.01
Impropers d (deg.) 1.348±0.01 0.660±0.01 0.539±0.01
Coordinate precision e (Å)
Backbone non-hydrogen atoms 0.15 0.12 0.20
All non-hydrogen atoms 0.26 0.60 0.60
Non-Gly, non-Pro residues in Ramachandran plot f
Core regions (%) 94.7 100 90.6
Allowed regions (%) 5.3 0.0 9.4
Generously allowed regions (%) 0.0 0.0 0.0
Disallowed regions (%) 0.0 0.0 0.0
The statistics were obtained for a subset of the ten lowest energy structures out of 100 calculated with a CNS 31 simulated annealing
protocol. The number of constraints is given in the footnotes. Coordinate precision and Ramachandran plot quality are reported for the
core residues excluding flexible N and C termini.
a NOEs comprise a non-redundant set of 77/61/75 intraresidual NOEs, 153/90/86 sequential NOEs (|i–j|=1), 148/46/45 short-range
NOEs (15).
b The dihedral angle constraints comprise 8/4/5 ϕ and 6/9/7 ψ angles obtained from 1 H, 15 N and 13 C chemical shift assignments as
inputs for TALOS, 36 as well as 7/6/6 χ 1 angles obtained from ROESY peak intensities, mainly for the six cysteine residues.
c 3 J HNHA scalar couplings were obtained with a quantitative J measurement to give (15/14/19) restraints. Additional dipolar coupling
constraints were obtained for NWr1 and were included with the ISAC protocol, 37 resulting in an NMR quality factor of 0.16. 38 Only RDCs
of the non-flexible NWr1 core residues were incorporated.
d The improper torsion angle restraints serve to maintain planarity and chirality.
e The coordinate precision is defined as the average RMS distance between the individual simulated annealing structures and the mean
coordinates. Values are reported for core residues.
f Values are calculated with the program PROCHECK-NMR 39 for core residues.

722 Structure and Function of Nematocyst CRDs
Figure 3. Solution structures of cysteine-rich domains. The structures of the first (PDB 2HM3), sixth (PDB 2NX6) and
eighth (PDB 2NX7) cysteine-rich domains of NOWA are shown in comparison to the sequence-related C-terminal
cysteine-rich domain of minicollagen 1 (PDB 1SP7). The ten lowest energy conformers out of 100 structures calculated in
CNS 31 are depicted with cystine shown in yellow. The topology and disulfide pattern are conserved upon natural amino
acid insertions between the first two cysteine residues in NOWA domains. The closely related Mcol1hC domain differs in
cysteine pattern, topology and one proline cis-trans isomerization.
relative to the NOWA domains and Mcol1hN. 12
While indels are often major contributors to protein
evolution expected to promote more drastic structural
changes than substitutions, 16,17 the sequence
insertions into a tightly knit disulfide-rich structure
in longer NOWA domains have very little effect on
the structure, nor do they preclude the efficient
folding to a single native conformation (Figure 2). As
these insertions into CRDs assume no obvious
structural role, they may result from neutral evolutionary
drift, rather than performing central functions
in the assembly of the nematocyst wall. Such
structurally neutral indels may, however, have a
compensatory function in relaxing structural tension
upon an accumulation of amino acid substitutions in
order to retain the original NOWA CRD fold. 18
C-terminal of the insertion between cysteine residues
1 and 2, the structures of the NOWA repeats are
virtually identical (Figure 3). This lack of consequences
for the overall structure and disulfide
Figure 4. A stereo representation
of the NOWA CRD domain
structures. 12 The structure, disulfide
pattern and turn topology is
conserved in the NOWA fold upon
natural insertions into the structure
at the indicated location.

Structure and Function of Nematocyst CRDs
723
pattern is highly unexpected, given the high dependence
of disulfide loop closing kinetics and stability on
the number of intercysteine residues. 19 As sequence
insertion into other loops of nematocyst CRDs is not
known, insertions may be tolerated only directly C-
terminal of the first cysteine residue, even before the
first turn of the NWr1 structure. This indicates that
thefirstcysteineisnotpartofthecoreofthedomain
and suggests a special role of the disulfide bond 1–4,
which will be particularly accessible in the N-terminal
domain of minicollagens.
Determinant residues in the folding of
nematocyst CRDs to different structures
The predominance of the NOWA fold relative to
the Mcol1hC fold indicates that particular mutations
have been used in evolution to destabilize and
“design out” the more common NOWA structure
in C-terminal minicollagen domains, thus populating
a novel target structure. 12,20 Residual dipolar
couplings (RDCs) are very well suited to monitor
directly the different conformational propensities in
largely unfolded reduced CRD sequences that
ultimately give rise to different structures for
NOWA and Mcol1hC cysteine-rich domains. RDCs
report on the time-average and ensemble-average
orientation of internuclear bond vectors relative to
the magnetic field in weakly aligned NMR samples.
Random coil polymers yield non-zero RDCs due to
the anisotropy of the linear chain, but are expected to
show a smooth profile of RDCs along the chain in the
absence of sequence-specific interactions. 21 Likewise,
abrupt changes of RDCs between sequential
amino acids in largely unfolded states must arise
from sequence-specific interactions resulting in
specific conformational preferences, since the persistence
length of polypeptide chains is about five to
seven amino acid residues.
To monitor different conformational preferences
that determine the different structures in the oxidized
domains, we compared 1 H- 15 N RDCs recorded
on recombinantly expressed 15 N-labeled
Mcol1hC and NWr1 in the reduced state (Figure 5).
While the 1 H- 15 N HSQC spectra of reduced CRDs
exhibit a small signal dispersion characteristic for
unfolded states (Figure 5(a)), RDCs confirm the
persistence of local conformational preferences in the
reduced CRDs (Figure 5(b)). The sequential RDC
pattern and thus the structural sampling in reduced
NWr1 and Mcol1hC differ markedly between the
first two cysteine residues and between the fourth
and fifth cysteine residues, thus rationalizing the
importance of these sites for the structural differences
in the native, oxidized form. In agreement with
this direct conformational analysis, a mutational
approach showed that only two central substitutions
in the NWr1 sequence are sufficient to induce the
formation of the Mcol1hC instead of the NOWA
domain structure: 12 one mutation (G11V in the
numbering used in Figures 4 and 5) that disfavours
the native βII-turn conformation between the first
and second cysteine residues as well as a proline
Figure 5. NMR spectroscopic analysis of residual
structure in reduced CRD domains. (a) Assigned 1 H- 15 N
HSQC spectra of reduced Mcol1HC and NWr1. (b)
Distinct conformational preferences of NWr1 and
Mcol1HC in their reduced states are monitored via
residual dipolar couplings in weakly aligned samples.
substitution of a hydrogen bond donor (K21P)
between the fourth and fifth cysteine residues.
A central, conserved proline (P18 in Figures 4 and
5) between the third and fourth cysteine induces a
βI turn in the Mcol1hC structure but assumes a cis
conformation in NOWA and Mcol1hN CRDs.
Previous studies had suggested that different
propensities for the formation of cis-peptide bonds
at P18 explain the structural differences between the
different CRD folds. Notably though, favouring the
trans over the cis proline conformation with a (4R)-
fluoroproline analogue did not suffice to switch the
structure of the prototypical domain structure, 22
most likely due to the only weakly increased
stabilization of the trans form by 0.2 kcal/mol
relative to unsubstituted proline. Inspection of CRD
sequences further suggests that sequences assuming
the prototypical CRD fold are not optimized for an
especially high Xaa-Pro cis prolyl propensity. The
Ala-Pro sequences found in NWr2-8 and Mcol1N in
fact have a cis prolyl propensity near or below the
average both in model peptides and in the
Brookhaven Protein Data Base. 23 Despite a substantially
higher propensity for cis-peptide bond
formation between Tyr17 and Pro18, the NWr1
peptide structure has been transformed to the
atypical Mcol1C fold with a trans-peptide bond. 12

724 Structure and Function of Nematocyst CRDs
In naturally evolved C-terminal minicollagen
CRDs, Pro18 is usually preceded by β-branched
amino acids, 12 which are known to have particularly
low propensities for the formation of
cis-peptide bonds N-terminal of proline in the
PDB. 23 Thus, it would seem that the cis prolyl
bond is not enforced in the prototypical CRDs but is
rather disfavoured in the atypical C-terminal CRDs.
The cis proline conformation in oxidized NWr1,
NWr6 and NWr8 is evidenced from the absence
of strong sequential Hα i–1 -Hδ i nuclear Overhauser
effects (NOEs) and the presence of strong sequential
Hα i–1 -Hα i NOEs between the proline and its
preceding residue. This central proline is not
conserved in the second NOWA repeat NWr2, thus
presumably destabilizing the folded state and
indicating a special role of the domain within
NOWA. Accordingly, our attempts to refold and
purify this domain in high yields after recombinant
expression have failed, but have resulted in a
complex mixture of several spectral species (not
shown), presumably due to the stochastic formation
of disulfide bonds.
A temperature series of oxidative refolding reactions
on NWr1 (Figure 6) was recorded to monitor
the role of the cis proline βVIa turn in the formation
of the NOWA CRD domain structure. NWr1 was
fully reduced with Tris(2-carboxyethyl)-phosphine
(TCEP) and oxidative refolding was started by the
addition of oxidized glutathione (GSSG) to a final
concentration of 40 mM at pH 7.5. One-dimensional
proton NMR spectra were recorded to follow the
refolding reaction in real time and averaged relative
intensities of well-separated backbone resonances
were obtained (Figure 6(a)). At low temperatures
(

Structure and Function of Nematocyst CRDs
725
accelerated upon oxidative collapse, which thus may
function as a built-in catalyst in the oxidative folding
of prototypical CRD domains.
Implications for the formation of the nematocyst
wall superstructure
The cooperativity of unfolding and the redox
stability of disulfide bonds were investigated with
an equilibrium reductive unfolding analysis on
NWr1 at pH 7.0 after incubation with increasing
ratios [GSH] 2 / [GSSG] for 12 h in airtight Shigemi
NMR tubes (GSH is reduced glutathione). The
unfolding reaction was followed by 1D proton
NMR spectroscopy. Signals of the native and
unfolded state were integrated to yield the ratio
[NWr1 unfold ]/ [NWr1 fold ]. The overall transition
midpoint [NWr1 unfold ]/ [NWr1 fold ]=1 is found at
[GSH] 2 / [GSSG] =156 mM. Using a standard redox
potential for glutathione E' o (glutathione) of −230 mV
at 25 °C 28 the Nernst relation gives:
EVðNOWAÞ¼E o
VðglutathioneÞ o
RT=2F lnð½GSHŠŠ 2
=½GSSGŠŠÞ midpoint
¼ 206 mV
Thus, the disulfide bonds in the NWr1 domain are
only slightly more stable towards the reductant than
the disulfide bonds in the Mcol1hC structure (E' o
(Mcol1hC) =−185 mV). 14 A Hill analysis of the
unfolding curve (see Materials and Methods for
details) showed that unfolding results from an
opening of all three disulfide bonds at [GSH] 2 /
[GSSG]>200 mM, but that unfolding is not cooperative
at lower [GSH] 2 / [GSSG] (Figure 6(d)). The
presence of one less stable disulfide bond is identical
with findings for Mcol1hC 14 and may be a prerequisite
for the reshuffling of CRD disulfide bridges
from intra- to intermolecular during the formation
of the nematocyst wall.
Rapid re-oxidation of NWr1 by 1% (v/v) H 2 O 2
and subsequent mass spectrometric analysis shows
the presence of dimers and gives indications for
higher-order aggregates (Figure 7). Individual
NOWA cysteine-rich domains thus bear an intrinsic
Figure 7. Mass spectrometric evidence for oligomerization
of NWr1 domains. (a) and (b) Mass spectrum of
NWr1 (theoretical mass =3109.6 Da, deconvoluted in (a))
upon reduction at 2 mM peptide and fast re-oxidation
with 1% (v/v) H 2 O 2 . (b) The isotope ladder in the raw
spectrum shows an NWr1 dimer with z=3 and m=6218
Da. The background between signals in (b) further
indicates the presence of higher aggregates.
ability for oligomerization, which is in agreement
with the observation of spontaneous crosslinking of
the NOWA CROD. 3 The homogeneity of CRD structures
in NOWA and the ability of the NOWA cysteine-rich
octarepeat domain to self-aggregate
further argue for a homo-association mechanism
between similar CRD folds during the formation of
the nematocyst wall superstructure. As the structures
of NOWA domains and Mcol1hN coincide, a
primordial appearance of this domain structure
during evolution is likely. The Mcol1hC domain
structure may have appeared later. As compared to
the NWr1 dimerization, Mcol1hC homodimerization
as well as NWr1-Mcol1hC heterodimerization
tendencies are reduced. 12 This lower association
tendency of Mcol1hC may provide the basis for a
controlled and most likely catalysed aggregation
step during nematocyst wall assembly.
Presumably, the initial wall assembly occurs in the
absence of minicollagens as NOWA and minicollagens
are secreted by separate pathways. 9 The interaction
between NOWA and minicollagens thus is
constricted to the nematocyst membrane, to which
NOWA is directed by the association of its basic C
terminus with acidic lipids (data not shown). The
fact that there is only one known isoform of NOWA
supports a primordial function in the assembly of
the capsule wall. Variations in minicollagen sequences
might then have induced different capsule
morphologies and functions. 12 Future work will
focus on the molecular pattern of the resulting
heteropolymers (Figure 8) and the structure of
dimeric CRD complexes.
Materials and Methods
Protein synthesis
The 15 N-labeled NWr1 (NOWA cysteine-rich domain
repeat 1), NWr6 and NWr8 proteins were expressed at
30 °C as C-terminal fusions to the protein G B1
domain 13 in M9 medium containing 15 NH 4 Cl as a sole
nitrogen source. The fusion proteins were purified by
affinity chromatography on an IgG Sepharose fast-flow
column (Pharmacia) and cleaved by thrombin (Pharmacia).
The cleaved CRD peptides were purified on a C8
reverse-phase HPLC column (Vydac; 10 μm film
thickness; 250 mm×22 mm). Subsequently, the cleaved
peptides were subjected to oxidative folding in refolding
buffer as described, 10 and purified by preparative
HPLC. The final yield was 5–10 mg of CRD peptides
per litre of M9 medium. The recombinantly expressed
CRDs have sequences
NWr1: GSTGTCP 470 SGCSGDCYPE 480
CKPGCCGQVN 490 LN
NWr6: GSSSCP 650 QFPSCSPSCA 660
PQCSQQCCQQ 670 P
NWr8: GSA 720 QNPCSLQQPG 730 CSSACAPACR 740
LSCCSLG
where the first two residues (GS) are cloning artifacts
from the thrombin cleavage site. HPLC, mass spectroscopy
and NMR spectra of both the synthetic peptide

726 Structure and Function of Nematocyst CRDs
Figure 8. Model for the assembly of a NOWA/minicollagen network to form the nematocyst wall superstructure. The
insert shows an electron micrograph of the nematocyst wall surface with NOWA particles interconnected by rod-like
protrusions formed probably by minicollagen (the scale bar represents 40 nm). NOWA is deposited at the membrane via
basic C-terminal residues (not shown). A pre-assembly between NOWA CRDs and minicollagen N-terminal CRDs (red
squares) is indicated by the high homo-oligomerization propensity of the NOWA and minicollagen N-terminal CRD fold
only. The invention of a novel fold in minicollagen C-terminal CRDs may allow a controlled step, which leads to a
catalyzed intercysteine linkage between C-terminal CRDs (blue squares) and to final wall maturation.
and the expressed peptides indicate an estimated purity
above 85 % in all cases. Mass spectroscopy and NMR
spectra show the presence of three intact disulfide bonds.
Isotope-labelled CRDs were lyophilized and dissolved in
5 mM phosphate (pH 5.5), 2 mM NaN 3 , 95% H 2 O/ 5%
2 H 2 O, to a concentration of 0.7 mM peptide. In addition,
solid phase synthesis of unlabeled NWr1 peptide of the
sequence
TCP 470 SGCSGDCYPE 480 CKPGCCGQVN 490 LN
was carried out by an N-(9-fluorenyl)methoxycarbonyl
(Fmoc) strategy. The peptide was purified by preparative
HPLC both before and after oxidative folding as described.
10 Unlabelled NWr1 was dissolved to a final
concentration of 3.5 mM in 5 mM phosphate (pH 5.5),
2 mM NaN 3 , 95% H 2 O/ 5% 2 H 2 O,.
Mass spectrometry
Recombinant peptides were characterized by electrospray
ionization-mass spectrometry using a Bruker
microTOF mass spectrometer. Experimental and theoretical
masses for uniformly 15 N-labeled, fully oxidized
domains are given in Table 1.
NMR spectroscopy
The different uniformly 15 N-labelled cysteine-rich
domains were assigned using 2D as well as 3D- 15 N
separated NOE spectroscopy (NOESY) and total correlated
spectroscopy (TOCSY) in conjunction with 1 H- 15 N HSQC
spectra and 1 H- 13 C HSQC spectra at natural 13 C isotope
enrichment. 3 J HNHA scalar couplings were measured with
an HNHA experiment. All spectra were recorded on a
Bruker DRX600 spectrometer equipped with a TXI probe
or on a Bruker DRX800 spectrometer equipped with a TCI
cryogenic probe. Spectra were processed with NMRPipe 29
and analysed with PIPP. 30 Structures were calculated in
CNS 31 from the experimental data summarized in Table 1.
All structure representations were generated with the
program MOLMOL. 32
Structural study on reduced CRDs
Mcol1hC and NWr1 were reduced by the addition of
5 mM TCEP and were assigned with 15 N separated
NOESY and TOCSY spectra after adjusting the pH to 5.5.
RDCs were measured in strained (10 %, w/v) polyacrylamide
gels 33,34 in the presence of 5 mM TCEP at 200 μM
peptide in 5 mM sodium phosphate buffer (pH 5.5).
Equilibrium unfolding and oxidative refolding
For oxidative refolding in the NMR spectrometer,
300 μM unlabelled NWr1 peptide in 20 mM phosphate
buffer (pH 7.5), was fully reduced with 3 mM TCEP at
pH 7.5. Oxidative refolding was initiated by adding
GSSG in 20 mM phosphate buffer (pH 7.5) to a final
[GSSG]= 40 mM. Oxidative refolding was carried out at
280 K, 295 K, 308 K and 320 K. An equilibrium unfolding
titration was performed on synthetic NWr1 by titrating
various amounts of a stock solution of 300 mM GSH,
20 mM phosphate buffer (pH 7.0) to a solution of 300 μM
CRD peptide, 8 mM GSSG, 20 mM phosphate buffer (pH
7.0). After equilibrating for 12 h at 25 °C in airtight
Shigemi tubes, the relative amount of folded and
unfolded CRD was determined from the loss of native
signal and the increase of reduced CRD in 1D proton
NMR spectra. Similarly, the ratio of reduced and
oxidized glutathione was determined from the respective
signals to yield the redox potential of the solution. The
unfolding reaction:
NWr1 ox þ nð2GSHÞ X NWr1 red þ nGSSG
proceeds with an opening of n disulfide bonds upon
titration with GSH. The equilibrium of the form K eq =
[NWr1 red ]/[ NWr1 ox ]·( [GSSG]/[GSH] 2 ) n yields the cooperativity
of reduction n as the slope of a Hill plot of log
([NWr1 red ]/[NWr1 ox ]) versus log([GSH] 2 /[GSSG]). 35
Protein Data Bank accession number
The atomic coordinates of the ten lowest energy CNS
conformers of NW1r, NW6r and NW8r have been

Structure and Function of Nematocyst CRDs
727
deposited at the RCSB Protein Data Bank (www.rcsb.org)
under PDB accession numbers 2HM3, 2NX6 and 2NX7,
respectively.
Acknowledgements
We thank M. Rogowski for the acquisition of
mass spectra. This work was supported by SNF
grant 31-109712 to S.G. by a grant from the German
Science Foundation (DFG) to T.W.H. and S.Ö. and
by a grant from the Benzon foundation to S.M.
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Edited by M. F. Summers
(Received 1 December 2006; received in revised form 30 January 2007; accepted 8 February 2007)
Available online 24 February 2007