Sequence–Structure and Structure–Function Analysis in Cysteine-rich Domains Forming the Ultrastable Nematocyst Wall

doi:10.1016/j.jmb.2007.02.026 J. Mol. Biol. (2007) 368, 718–728

Sequence–Structure and Structure–Function Analysisin Cysteine-rich Domains Forming theUltrastable Nematocyst Wall

Sebastian Meier1⁎, Pernille Rose Jensen1, Patrizia Adamczyk2

Hans Peter Bächinger3, Thomas W. Holstein2, Jürgen Engel4

Suat Özbek2 and Stephan Grzesiek1⁎
1Department of StructuralBiology, Biozentrum,University of Basel,Klingelbergstrasse 70,CH-4056 Basel, Switzerland2Institute of Zoology,Department for MolecularEvolution and Genomics,University of Heidelberg,Im Neuenheimer Feld 230,D-69120 Heidelberg, Germany3Shriners Hospital for Childrenand Department of Biochemistryand Molecular Biology,Oregon Health and ScienceUniversity, Portland,OR 97239, USA4Department of BiophysicalChemistry, Biozentrum,University of Basel,Klingelbergstrasse 70,CH-4056 Basel, Switzerland
Present address: S. Meier, InstitutCopenhagen, Universitetsparken 13Abbreviations used: CRD, cystein

cysteine-rich octarepeat domain of Nof NOWA; Mcol1hN, N-terminal CRHydra; RDC, residual dipolar coupliTris(2-carboxyethyl)-phosphine; GSHquantum correlation; indel, insertionE-mail addresses of the correspon

0022-2836/$ - see front matter © 2007 E

The nematocyst wall of cnidarians is a unique biomaterial that withstandsextreme osmotic pressures, allowing an ultrafast discharge of the nemato-cyst capsules. Assembly of the highly robust nematocyst wall is achieved bycovalent linkage of cysteine-rich domains (CRDs) from two main proteincomponents, minicollagens and nematocyst outer wall antigen (NOWA).The bipolarminicollagens have different disulfide patterns and topologies intheir N and C-terminal CRDs. The functional significance of this polarity hasbeen elusive. Here, we show by NMR structural analysis that allrepresentative cysteine-rich domains of NOWA are structurally related toN-terminal minicollagen domains. Natural sequence insertions in NOWACRDs have very little effect on the tightly knit domain structures, nor do theypreclude the efficient folding to a single native conformation. The differentfolds in NOWA CRDs and the atypical C-terminal minicollagen domain onthe other hand can be directly related to different conformational preferencesin the reduced states. Ultrastructural analysis in conjunction with aggrega-tion studies argues for an association between the similar NOWA andN-terminal minicollagen domains in early stages of the nematocyst wallassembly, which is followed by the controlled association between theunusual structures of C-terminal minicollagen domains.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: molecular evolution; conformational diversity; NMR structure;cysteine-rich; dihedral angle preference
*Corresponding authors
e of Molecular Biology and Physiology, August Krogh Building, University of, DK-2100 Copenhagen, Denmark.e-rich domain; NOWA, nematocyst outer wall antigen; CROD,OWA; NW1r, first CRD of NOWA; NW6r, sixth CRD of NOWA; NW8r, eighth CRDD of minicollagen 1 from Hydra; Mcol1hC, C-terminal CRD of minicollagen 1 fromng; NOE, nuclear Overhauser effect; TCEP,, reduced glutathione; GSSG, oxidized glutathione; HSQC, heteronuclear single/deletion.ding authors: [email protected]; [email protected]

lsevier Ltd. All rights reserved.

mailto:[email protected]

mailto:[email protected]

719Structure and Function of Nematocyst CRDs

Introduction

Nematocysts are the characteristic explosive orga-nelles of the phylum cnidaria. They comprise acylindrical capsule of about 10 μm diameter thatencloses an attached tubular coil structure andtoxins. Upon mechanical stimulation by a preyorganism or predator, a lid in the capsule wallopens and the tube together with the stored toxins isreleased in an ultrafast extrusion process.1 Thisprocess is driven by the extreme osmotic pressure(150 bar; 1 bar=105 Pa) within the capsule.2 The wallstructure of the Hydra nematocyst is a uniquebiological polymer formed mainly by two proteinspecies, nematocyst outer wall antigen (NOWA)and minicollagens, which share a common cys-teine-rich motif.3 These short cysteine-rich domains(CRDs) contain a conserved pattern of six cysteineresidues spaced by three residues. Minicollagensexhibit a symmetrical domain organization with acentral collagen sequence flanked by polyprolinestretches and a CRD at the N and C termini of themolecule (Figure 1(a)).4 The best-characterizedminicollagen in the nematocyst wall has beentermed minicollagen 1, and serves as a model forthe protein family. NOWA is a 90 kDa glycoproteinwith an N-terminal sperm-coating protein (SCP)domain, a central C-type lectin domain (CTLD) andan eightfold CRD repeat at its C terminus (Figure1(a)). The capsule structure has been shown to be

Figure 1. Domain organization of NOWA and mini-collagen 1. (a) Schematic modular arrangement of signalpeptide (SP), sperm-coating protein (SCP) domain, C-typelectin domain (CTLD) and cysteine-rich octarepeatdomain (CROD) in NOWA; CRDs are indicated as circlesand coloured according to sequence similarity; CRDs inminicollagen1 are known to form different structures. (b)Sequence alignment of the eight CRDs in the NOWAoctarepeat domain and comparison to N and C-terminalCRDs of Hydraminicollagen 1 (Mcol1hN and Mcol1hC).10

highly sensitive to reducing agents, indicating thatthe wall proteins are involved in the formation of acysteine-linked network.5 As minicollagens are stillsoluble in the early stages of nematocyst develop-ment6 and their cysteine residues form intramole-cular disulfide bridges,7 it has been proposed thatisomerization to intermolecular disulfide bridgeswould constitute the final maturation step inmorphogenesis, which involves wall compactionand polymerization.8

We have shown recently that recombinantlyexpressed NOWA as well as its cysteine-rich octare-peat domain (CROD) spontaneously form disulfide-linked globular aggregates that resemble the globu-lar building units of the nematocyst wall.3 TheseNOWAglobules are extremely heterogeneous in sizeranging from 15 nm to 45 nm in diameter and formseveral densely packed layers along the wall profile.Ultrastructural analysis of the wall architecturerevealed that the individual globules are intercon-nected via rod-like protrusions that probably repre-sent minicollagen. NOWA appears early duringnematocyst morphogenesis and forms a thin layer atthe inside of the nematocyst vesiclemembrane.9 Mini-collagens, which are expressed at later stages,gradually attach to this layer in a pre-assemblyprocess,which is followedbypolymerizationbetweenthe bipolar minicollagens. Thus, NOWA acts as apositional organizer of the nematocyst superstructure,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 disulfidelinks from identical cysteine patterns (Figure1(b)).10,11 This clearly serves as a rare example forthe divergent evolution of protein folds in closelyrelated domains.We have demonstrated that the twofolds overlap in sequence space and can be inter-converted by changes of a few amino acids.12 Theability of single cysteine-rich sequences to populatetwo different tertiary structures is reminiscent of theconformational plasticity of prion proteins andamyloidogenic proteins. Moreover, this ability hasgiven strong indications that complex features,including protein tertiary structures, can developby smooth evolutionary transitions. While we haveobtained strong indications that the structuralpolarity of N and C-terminal CRD folds is conservedin minicollagens, the functional significance of thispolarity in the formation of the nematocyst wall hasbeen elusive, mainly due to lack of structuralinformation on the NOWA CROD.Here, we present the structure of all representative

cysteine-rich modules in the NOWA CROD, thuscompleting the structural data on minicollagens andNOWA, the key components of the nematocyst wall.Despite sequence insertions of varying length intothe tightly knit disulfide rich domains, all NOWACRDs form an identical, prototypical structure. Thisfold is similar to the Mcol1hN domain structure anddifferent from the atypical Mcol1hC fold. NMRanalysis of isotope-enriched recombinant proteinshows that the different CRD folds in Mcol1hC and

720 Structure and Function of Nematocyst CRDs

NOWA can be directly ascribed to different con-formational sampling in the reduced states. We findthat CRDs consistently show a rapid folding reactionin accordance with the fast cross-linking of thenematocyst wall. Distinct association properties ofthe different CRD domains in conjunction withelectron microscopic analysis suggest that NOWAnanoparticles are deposited early in the nematocystwall formation and are crosslinked by short mini-collagen polymers in a controlled fashion to form ahighly stable composite biomaterial.

Results and Discussion

Recombinant expression of cysteine-richdomains

The pattern of six cysteine residues in little morethan 20 amino acids of NOWA cysteine-richdomains resembles the pattern CXXXCXXXCXXXC-XXXCC in the closely related CRDs of minicollagen1 (Figure 1). While the first repeat in the cysteine-richoctarepeat domain (NWr1) has the same length asthe Mcol1hN and Mcol1hC domains, the repeatsNWr2 to NWr7 carry an insertion of two amino acidsbetween the first two cysteine residues and NWr8carries an insertion of three amino acids. For amodular study of the entire NOWA protein domainsNWr1, NWr6 (representing the highly homologousrepeats NWr2-NWr7), and NWr8 were recombi-nantly produced in Escherichia coli. The domainswere expressed as fusion proteins C-terminal to theGB1 domain of protein G,13 with a thrombincleavage site in the linker between protein G andthe cysteine-rich domain. This resulted in goodexpression yields of about 20 mg of soluble fusionprotein per litre of minimal medium. Cleavage,purification and oxidative refolding of the isolated,uniformly 15N-labeled domains yielded >5 mg endproduct per litre of minimal medium for NWr1,NWr6 and NWr8. Mass spectrometry validated thechemical identity and purity of the CRDs, andshowed that the domains were fully oxidized withthree disulfide bonds formed (Table 1).

Structure of NOWA CRDs

While six cysteine residues can be fully oxidized to15 different disulfide bond topologies, the 1H-15N

Table 1. Molecular mass of recombinantly expressed[U-15N]CRDs

Molecular mass (Da)

Mass spectroscopy Theoretical

NWr1 3109.3 3109.6NWr6 2802.9 2802.8NWr8 2930.6 2931.0

Theoretical values are given for fully 15N-labelled, fully oxidizeddomains.

heteronuclear single quantum correlation (HSQC)spectra show only one folded spectral species foreach of the purified CRDs (Figure 2). Otherprominent spectral species are absent, and anypopulation of minor forms is smaller than 10% ofthe main structural species according to the HSQCpeak intensities. Thus, the NOWA CRDs fold veryeffectively towards their native structure. Prelimin-ary structural information can be obtained from theassigned HSQC spectra, as a central canonical βIturn between the third and fourth cysteine inMicol1hC (PDB accession number 1SP7) gives riseto a characteristic 15N upfield shifts at turn positioni+2.14 This upfield shift is not observed in any of theNOWA CRDs at the respective positions E480, Q662and A738 (Figure 2), indicating that the fold of allNOWA domains is different from that of Mcol1hC.For a detailed analysis of the structural determi-

nants of the different CRD folds, the structures ofthe three NOWA domains NWr1, NWr6 and NWr8were determined de novo with very high precision(Table 2). The RMSD of all heavy atoms in thefolded core is less than 0.6 Å for the solutionstructures of NWr1, NWr6 and NWr8. All NOWACRD structures have the same fold (Figure 3). Thisis in contrast to the structural polarity of mini-collagens with different structures of Mcol1hN andMcol1hC domains, and indicates different assem-bly functions for NOWA and minicollagens. Thecysteine pattern 1-4, 2-6 and 3-5 and a central cisproline turn between cysteine 3 and cysteine 4coincide with the Mcol1hN structure (PDB acces-sion number 1ZPX). However, the overall left-handed fold of the NOWA domains and Mcol1hNdiffers completely from the Mcol1hC fold, which isright-handed. In particular, the NOWA cysteinepattern differs fundamentally from the Mcol1hCstructure (1-5,2-4,3-6; Figures 3 and 4) and none ofthe NOWA turns, which bring the disulfide-bonded 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, thedifferent structures of prototypical NOWA CRDsand the atypical Mcol1hC must be a consequenceof different local turn propensities of the inter-cysteine residues.12 The sequential arrangement ofturns in the prototypical NWr1 domain starts witha canonical βII turn from residues 470–473between the first two cysteine residues, C469 andC473, followed by a short 310 helix formed by twoconsecutive βIII turns (474–478), which containsthe third cysteine, C477. The conserved prolineresidue 479 is in cis conformation and part of aβVIa turn from residues 478–481, which continuesinto a γ turn (480–482). Both turns position thefourth cysteine, C481. The chain terminates in asingle α-helical turn (482–486) that contains theremaining cysteine residues C485 and C486. Allhydrogen bonds and a scheme of the turn anddisulfide topology are given in Figure 4. The aminoacid insertions between the first two cysteineresidues in the NWr6 and NWr8 domains are

Figure 2. Assigned 1H-15N HSQC spectra of the oxidized recombinant octarepeat domains NWr1, NWr6 and NWr8from NOWA. N-terminal artefactual serine residues of the thrombin cleavage sites are marked with an asterisk (*).


incorporated into the NOWA fold in α-helicalconformations directly following the first cysteineresidue. As the α-helical pitch is small in compa-rison with an extended conformation, these turnscan accommodate the increase in chain lengthwithout 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 homologouspositions: while NWr8 retains the canonical βIIturn before the second cysteine, NWr6 adopts a βIturn at this location. Presumably, the mutation G→Sabolishes the high positional potential for the for-mation of a βII turn and induces a preference for theformation of a βI turn (Figure 1).15

Table 2. Statistics of the NWr1, NWr6 and NWr8 NMR struc

RMSD from experimental distance constraintsa (Å) 0.07RMSD from dihedral constraintsb (deg.) 1.2RMSD from scalar coupling constraintsc 0.5Deviation from the idealized covalent geometry

Bond lengths (Å) 0.00Bond angles (deg.) 1.3Impropersd (deg.) 1.3

Coordinate precisione (Å)Backbone non-hydrogen atomsAll non-hydrogen atoms

Non-Gly, non-Pro residues in Ramachandran plotf

Core regions (%)Allowed regions (%)Generously allowed regions (%)Disallowed regions (%)

The statistics were obtained for a subset of the ten lowest energy strprotocol. The number of constraints is given in the footnotes. Coordincore residues excluding flexible N and C termini.

a NOEs comprise a non-redundant set of 77/61/75 intraresidual NONOEs (1<|i–j| ≤5) and 42/32/17 long range NOEs (|i–j|>5).

b The dihedral angle constraints comprise 8/4/5 ϕ and 6/9/7 ψ aninputs for TALOS,36 as well as 7/6/6 χ1 angles obtained from ROESY

c 3JHNHA scalar couplings were obtained with a quantitative Jmeasuconstraints were obtained for NWr1 andwere includedwith the ISAC pof the non-flexible NWr1 core residues were incorporated.

d The improper torsion angle restraints serve to maintain planaritye The coordinate precision is defined as the average RMS distance be

coordinates. Values are reported for core residues.f Values are calculated with the program PROCHECK-NMR39 for c

Structural consequences of insertion, deletionand substitution events during the evolution ofnematocyst CRDs

The cysteine-rich domains fromminicollagens andNOWA form an attractive natural framework for thestudy of sequence–structure relationships as well asstructural evolution resulting from insertion/dele-tion (indel) or substitution events. Despite the highlevel of similarity of NWr2–NWr7, which points to arather recent gene duplication event, CRDs ofdifferent proteins have little sequence similaritydue to extensive neutral drift. On the other hand,local sequence variations between the cysteineresidues suffice to induce different folds in Mcol1hC

tures

NWr1 NWr6 NWr8

3±0.001 0.060±0.001 0.071±0.0011±0.03 0.90±0.02 1.19±0.039±0.02 0.97±0.02 0.32±0.01

86±0.001 0.0061±0.001 0.0048±0.00173±0.01 0.775±0.01 0.665±0.0148±0.01 0.660±0.01 0.539±0.01

0.15 0.12 0.200.26 0.60 0.60

94.7 100 90.65.3 0.0 9.40.0 0.0 0.00.0 0.0 0.0

uctures out of 100 calculated with a CNS31 simulated annealingate precision and Ramachandran plot quality are reported for the

Es, 153/90/86 sequential NOEs (|i–j|=1), 148/46/45 short-range

gles obtained from 1H, 15N and 13C chemical shift assignments aspeak intensities, mainly for the six cysteine residues.rement to give (15/14/19) restraints. Additional dipolar couplingrotocol,37 resulting in an NMR quality factor of 0.16.38 Only RDCs

and chirality.tween the individual simulated annealing structures and themean

ore residues.

Figure 3. Solution structures of cysteine-rich domains. The structures of the first (PDB 2HM3), sixth (PDB 2NX6) andeighth (PDB 2NX7) cysteine-rich domains of NOWA are shown in comparison to the sequence-related C-terminalcysteine-rich domain of minicollagen 1 (PDB 1SP7). The ten lowest energy conformers out of 100 structures calculated inCNS31 are depicted with cystine shown in yellow. The topology and disulfide pattern are conserved upon natural aminoacid insertions between the first two cysteine residues in NOWA domains. The closely related Mcol1hC domain differs incysteine pattern, topology and one proline cis-trans isomerization.


relative to the NOWA domains and Mcol1hN.12

While indels are often major contributors to proteinevolution expected to promote more drastic struc-tural changes than substitutions,16,17 the sequenceinsertions into a tightly knit disulfide-rich structurein longer NOWA domains have very little effect onthe structure, nor do they preclude the efficientfolding to a single native conformation (Figure 2). Asthese insertions into CRDs assume no obviousstructural role, they may result from neutral evolu-

tionary drift, rather than performing central func-tions in the assembly of the nematocyst wall. Suchstructurally neutral indels may, however, have acompensatory function in relaxing structural tensionupon an accumulation of amino acid substitutions inorder to retain the original NOWA CRD fold.18

C-terminal of the insertion between cysteine residues1 and 2, the structures of the NOWA repeats arevirtually identical (Figure 3). This lack of conse-quences for the overall structure and disulfide

Figure 4. A stereo representa-tion of the NOWA CRD domainstructures.12 The structure, disul-fide pattern and turn topology isconserved in the NOWA fold uponnatural insertions into the structureat the indicated location.

Figure 5. NMR spectroscopic analysis of residualstructure in reduced CRD domains. (a) Assigned 1H-15NHSQC spectra of reduced Mcol1HC and NWr1. (b)Distinct conformational preferences of NWr1 andMcol1HC in their reduced states are monitored viaresidual dipolar couplings in weakly aligned samples.


pattern is highly unexpected, given the high depen-dence of disulfide loop closing kinetics and stability onthe number of intercysteine residues.19 As sequenceinsertion into other loops of nematocyst CRDs is notknown, insertions may be tolerated only directly C-terminal of the first cysteine residue, even before thefirst turn of the NWr1 structure. This indicates thatthe first cysteine is not part of the core of the domainand suggests a special role of the disulfide bond 1–4,which will be particularly accessible in the N-terminaldomain of minicollagens.

Determinant residues in the folding ofnematocyst CRDs to different structures

The predominance of the NOWA fold relative tothe Mcol1hC fold indicates that particular mutationshave been used in evolution to destabilize and“design out” the more common NOWA structurein C-terminal minicollagen domains, thus populat-ing a novel target structure.12,20 Residual dipolarcouplings (RDCs) are very well suited to monitordirectly the different conformational propensities inlargely unfolded reduced CRD sequences thatultimately give rise to different structures forNOWA and Mcol1hC cysteine-rich domains. RDCsreport on the time-average and ensemble-averageorientation of internuclear bond vectors relative tothe magnetic field in weakly aligned NMR samples.Random coil polymers yield non-zero RDCs due tothe anisotropy of the linear chain, but are expected toshow a smooth profile of RDCs along the chain in theabsence of sequence-specific interactions.21 Like-wise, abrupt changes of RDCs between sequentialamino acids in largely unfolded states must arisefrom sequence-specific interactions resulting inspecific conformational preferences, since the persis-tence length of polypeptide chains is about five toseven amino acid residues.To monitor different conformational preferences

that determine the different structures in the oxi-dized domains, we compared 1H-15N RDCs re-corded on recombinantly expressed 15N-labeledMcol1hC and NWr1 in the reduced state (Figure 5).While the 1H-15N HSQC spectra of reduced CRDsexhibit a small signal dispersion characteristic forunfolded states (Figure 5(a)), RDCs confirm thepersistence of local conformational preferences in thereduced CRDs (Figure 5(b)). The sequential RDCpattern and thus the structural sampling in reducedNWr1 and Mcol1hC differ markedly between thefirst two cysteine residues and between the fourthand fifth cysteine residues, thus rationalizing theimportance of these sites for the structural differ-ences in the native, oxidized form. In agreementwiththis direct conformational analysis, a mutationalapproach showed that only two central substitutionsin the NWr1 sequence are sufficient to induce theformation of the Mcol1hC instead of the NOWAdomain structure:12 one mutation (G11V in thenumbering used in Figures 4 and 5) that disfavoursthe native βII-turn conformation between the firstand second cysteine residues as well as a proline

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 aβI turn in the Mcol1hC structure but assumes a cisconformation in NOWA and Mcol1hN CRDs.Previous studies had suggested that differentpropensities for the formation of cis-peptide bondsat P18 explain the structural differences between thedifferent CRD folds. Notably though, favouring thetrans over the cis proline conformation with a (4R)-fluoroproline analogue did not suffice to switch thestructure of the prototypical domain structure,22

most likely due to the only weakly increasedstabilization of the trans form by 0.2 kcal/molrelative to unsubstituted proline. Inspection of CRDsequences further suggests that sequences assumingthe prototypical CRD fold are not optimized for anespecially high Xaa-Pro cis prolyl propensity. TheAla-Pro sequences found in NWr2-8 and Mcol1N infact have a cis prolyl propensity near or below theaverage both in model peptides and in theBrookhaven Protein Data Base.23 Despite a sub-stantially higher propensity for cis-peptide bondformation between Tyr17 and Pro18, the NWr1peptide structure has been transformed to theatypical Mcol1C fold with a trans-peptide bond.12

Figure 6. Kinetics and thermodynamics of NWr1folding. (a) Oxidative refolding of NWr1 at 280 K moni-tored via 1DNMR spectra. (b) Temperature-dependence ofNWr1 refolding in 40 mM oxidized glutathione at pH 7.5fit to mono-exponentials for data from 295 K, 308 K and320 K. Refolding at 280 K is fit to the sum of two mono-exponentials, where the faster folding reaction accountsfor 20% of the native signal and has a rate nearly 20-foldfaster than the main reaction. (c) The resultant Arrheniusplot of the main slow-folding reactions shown in (b) givesan activation energy of 20.4 kcal/mol for the rate-limitingproline trans-cis isomerization. (d) Uncooperative equili-brium unfolding of NWr1 at low [GSH]2/ [GSSG] at 298 K,pH 7.0; at higher [GSH], all three disulfide bonds openupon titration with GSH.


In naturally evolved C-terminal minicollagenCRDs, Pro18 is usually preceded by β-branchedamino acids,12 which are known to have particu-larly low propensities for the formation ofcis-peptide bonds N-terminal of proline in thePDB.23 Thus, it would seem that the cis prolylbond is not enforced in the prototypical CRDs but israther disfavoured in the atypical C-terminal CRDs.The cis proline conformation in oxidized NWr1,

NWr6 and NWr8 is evidenced from the absenceof strong sequential Hαi–1-Hδi nuclear Overhausereffects (NOEs) and the presence of strong sequentialHαi–1-Hαi NOEs between the proline and itspreceding residue. This central proline is notconserved in the second NOWA repeat NWr2, thuspresumably destabilizing the folded state andindicating a special role of the domain withinNOWA. Accordingly, our attempts to refold andpurify this domain in high yields after recombinantexpression have failed, but have resulted in acomplex mixture of several spectral species (notshown), presumably due to the stochastic formationof disulfide bonds.A temperature series of oxidative refolding reac-

tions on NWr1 (Figure 6) was recorded to monitorthe role of the cis proline βVIa turn in the formationof the NOWA CRD domain structure. NWr1 wasfully reduced with Tris(2-carboxyethyl)-phosphine(TCEP) and oxidative refolding was started by theaddition of oxidized glutathione (GSSG) to a finalconcentration of 40 mM at pH 7.5. One-dimensionalproton NMR spectra were recorded to follow therefolding reaction in real time and averaged relativeintensities of well-separated backbone resonanceswere obtained (Figure 6(a)). At low temperatures(<295 K), a fast phase is observed in addition to aslower main reaction. A fit of the normalized data(Figure 6(b)) to:

I ¼ f *ð1� expð�t=s1ÞÞ þ ð1� f Þð1� expð�t=s2ÞÞ

reveals a fast phase with τ1=7.5 min for a fractionf=0.20 of the molecules, and a main reaction withτ2=166.4 min (Figure 6(b)). The fraction of fast-folding peptide is in excellent agreement with thepopulation of cis-prolyl estimated from nearestneighbour effects in model peptides23 (f=0.24 forTyr-Pro sequences such as in NWr1). This suggestsstrongly that peptidyl-prolyl trans-cis isomerizationis rate-limiting for the folding of the domain atphysiological temperatures, while disulfide bondsclose faster due to the presence of excess GSSG. Asanticipated in this case, the refolding reaction provesto be highly temperature-dependent (Figure 6(b)).Arrhenius analysis of the slow-phase time constantsshows that the main folding reaction encounters anactivation energy of 20.4 kcal/mol (Figure 6(c)) asexpected for proline trans-cis isomerization.24–27

While the activation energy of proline isomerizationthus is unchanged upon the GSSG-catalyzed forma-tion of disulfide-bridged cycles, the folding reactionproceeds remarkably fast, within a fewminutes nearroom temperature (Figure 5(c) and (d)). This corro-

borates the notion from MD22 and NMR studies oncyclic disulfide-bridged peptides25 that peptidyl-prolyl cis-trans and trans-cis isomerization may be


accelerated upon oxidative collapse, which thusmayfunction as a built-in catalyst in the oxidative foldingof prototypical CRD domains.

Implications for the formation of the nematocystwall superstructure

The cooperativity of unfolding and the redoxstability of disulfide bonds were investigated withan equilibrium reductive unfolding analysis onNWr1 at pH 7.0 after incubation with increasingratios [GSH]2/ [GSSG] for 12 h in airtight ShigemiNMR tubes (GSH is reduced glutathione). Theunfolding reaction was followed by 1D protonNMR spectroscopy. Signals of the native andunfolded state were integrated to yield the ratio[NWr1unfold]/ [NWr1fold]. The overall transitionmidpoint [NWr1unfold]/ [NWr1fold]=1 is found at[GSH]2/ [GSSG]=156 mM. Using a standard redoxpotential for glutathione E'o(glutathione) of−230mVat 25 °C28 the Nernst relation gives:

E oVðNOWAÞ ¼E oVðglutathioneÞ�RT=2F lnð½GSH��2=½GSSG��Þmidpoint ¼ �206 mV

Thus, the disulfide bonds in the NWr1 domain areonly slightly more stable towards the reductant thanthe disulfide bonds in the Mcol1hC structure (E'o(Mcol1hC)=−185 mV).14 A Hill analysis of theunfolding curve (see Materials and Methods fordetails) showed that unfolding results from anopening of all three disulfide bonds at [GSH]2/[GSSG]>200 mM, but that unfolding is not coopera-tive at lower [GSH]2/ [GSSG] (Figure 6(d)). Thepresence of one less stable disulfide bond is identicalwith findings for Mcol1hC14 and may be a pre-requisite for the reshuffling of CRD disulfide bridgesfrom intra- to intermolecular during the formationof the nematocyst wall.Rapid re-oxidation of NWr1 by 1% (v/v) H2O2

and subsequent mass spectrometric analysis showsthe presence of dimers and gives indications forhigher-order aggregates (Figure 7). IndividualNOWA cysteine-rich domains thus bear an intrinsic

Figure 7. Mass spectrometric evidence for oligomer-ization of NWr1 domains. (a) and (b) Mass spectrum ofNWr1 (theoretical mass=3109.6 Da, deconvoluted in (a))upon reduction at 2 mM peptide and fast re-oxidationwith 1% (v/v) H2O2. (b) The isotope ladder in the rawspectrum shows an NWr1 dimer with z=3 and m=6218Da. The background between signals in (b) furtherindicates the presence of higher aggregates.

ability for oligomerization, which is in agreementwith the observation of spontaneous crosslinking ofthe NOWACROD.3 The homogeneity of CRD struc-tures in NOWA and the ability of the NOWA cys-teine-rich octarepeat domain to self-aggregatefurther argue for a homo-association mechanismbetween similar CRD folds during the formation ofthe nematocyst wall superstructure. As the struc-tures of NOWA domains and Mcol1hN coincide, aprimordial appearance of this domain structureduring evolution is likely. The Mcol1hC domainstructure may have appeared later. As compared tothe NWr1 dimerization, Mcol1hC homodimeriza-tion as well as NWr1-Mcol1hC heterodimerizationtendencies are reduced.12 This lower associationtendency of Mcol1hC may provide the basis for acontrolled and most likely catalysed aggregationstep during nematocyst wall assembly.Presumably, the initial wall assembly occurs in the

absence of minicollagens as NOWA and minicolla-gens are secreted by separate pathways.9 The inter-action between NOWA and minicollagens thus isconstricted to the nematocyst membrane, to whichNOWA is directed by the association of its basic Cterminus with acidic lipids (data not shown). Thefact that there is only one known isoform of NOWAsupports a primordial function in the assembly ofthe capsule wall. Variations in minicollagen se-quences might then have induced different capsulemorphologies and functions.12 Future work willfocus on the molecular pattern of the resultingheteropolymers (Figure 8) and the structure ofdimeric CRD complexes.

Materials and Methods

Protein synthesis

The 15N-labeled NWr1 (NOWA cysteine-rich domainrepeat 1), NWr6 and NWr8 proteins were expressed at30 °C as C-terminal fusions to the protein G B1domain13 in M9 medium containing 15NH4Cl as a solenitrogen source. The fusion proteins were purified byaffinity chromatography on an IgG Sepharose fast-flowcolumn (Pharmacia) and cleaved by thrombin (Pharma-cia). The cleaved CRD peptides were purified on a C8reverse-phase HPLC column (Vydac; 10 μm filmthickness; 250 mm×22 mm). Subsequently, the cleavedpeptides were subjected to oxidative folding in refold-ing buffer as described,10 and purified by preparativeHPLC. The final yield was 5–10 mg of CRD peptidesper litre of M9 medium. The recombinantly expressedCRDs have sequences

NWr1: GSTGTCP470 SGCSGDCYPE480

CKPGCCGQVN490 LNNWr6: GSSSCP650 QFPSCSPSCA660

PQCSQQCCQQ670 PNWr8: GSA720 QNPCSLQQPG730 CSSACAPACR740

LSCCSLG

where the first two residues (GS) are cloning artifactsfrom the thrombin cleavage site. HPLC, mass spectro-scopy and NMR spectra of both the synthetic peptide

Figure 8. Model for the assembly of a NOWA/minicollagen network to form the nematocyst wall superstructure. Theinsert shows an electron micrograph of the nematocyst wall surface with NOWA particles interconnected by rod-likeprotrusions formed probably by minicollagen (the scale bar represents 40 nm). NOWA is deposited at the membrane viabasic C-terminal residues (not shown). A pre-assembly between NOWA CRDs and minicollagen N-terminal CRDs (redsquares) is indicated by the high homo-oligomerization propensity of the NOWA and minicollagen N-terminal CRD foldonly. The invention of a novel fold in minicollagen C-terminal CRDs may allow a controlled step, which leads to acatalyzed intercysteine linkage between C-terminal CRDs (blue squares) and to final wall maturation.


and the expressed peptides indicate an estimated purityabove 85 % in all cases. Mass spectroscopy and NMRspectra show the presence of three intact disulfide bonds.Isotope-labelled CRDs were lyophilized and dissolved in5 mM phosphate (pH 5.5), 2 mM NaN3, 95% H2O/ 5%2H2O, to a concentration of 0.7 mM peptide. In addition,solid phase synthesis of unlabeled NWr1 peptide of thesequence

TCP470 SGCSGDCYPE480 CKPGCCGQVN490 LN

was carried out by an N-(9-fluorenyl)methoxycarbonyl(Fmoc) strategy. The peptide was purified by preparativeHPLC both before and after oxidative folding as des-cribed.10 Unlabelled NWr1 was dissolved to a finalconcentration of 3.5 mM in 5 mM phosphate (pH 5.5),2 mM NaN3, 95% H2O/ 5% 2H 2O,.

Mass spectrometry

Recombinant peptides were characterized by electro-spray ionization-mass spectrometry using a BrukermicroTOF mass spectrometer. Experimental and theore-tical masses for uniformly 15N-labeled, fully oxidizeddomains are given in Table 1.

NMR spectroscopy

The different uniformly 15N-labelled cysteine-richdomains were assigned using 2D as well as 3D-15NseparatedNOE spectroscopy (NOESY) and total correlatedspectroscopy (TOCSY) in conjunction with 1H-15N HSQCspectra and 1H-13C HSQC spectra at natural 13C isotopeenrichment. 3JHNHA scalar couplings were measured withan HNHA experiment. All spectra were recorded on aBruker DRX600 spectrometer equipped with a TXI probeor on a Bruker DRX800 spectrometer equipped with a TCIcryogenic probe. Spectra were processed with NMRPipe29

and analysed with PIPP.30 Structures were calculated inCNS31 from the experimental data summarized in Table 1.All structure representations were generated with theprogram MOLMOL.32

Structural study on reduced CRDs

Mcol1hC and NWr1 were reduced by the addition of5 mM TCEP and were assigned with 15N separatedNOESY and TOCSY spectra after adjusting the pH to 5.5.RDCs were measured in strained (10 %, w/v) polyacry-lamide gels33,34 in the presence of 5 mM TCEP at 200 μMpeptide 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 phosphatebuffer (pH 7.5), was fully reduced with 3 mM TCEP atpH 7.5. Oxidative refolding was initiated by addingGSSG in 20 mM phosphate buffer (pH 7.5) to a final[GSSG]= 40 mM. Oxidative refolding was carried out at280 K, 295 K, 308 K and 320 K. An equilibrium unfoldingtitration was performed on synthetic NWr1 by titratingvarious amounts of a stock solution of 300 mM GSH,20 mM phosphate buffer (pH 7.0) to a solution of 300 μMCRD peptide, 8 mM GSSG, 20 mM phosphate buffer (pH7.0). After equilibrating for 12 h at 25 °C in airtightShigemi tubes, the relative amount of folded andunfolded CRD was determined from the loss of nativesignal and the increase of reduced CRD in 1D protonNMR spectra. Similarly, the ratio of reduced andoxidized glutathione was determined from the respectivesignals to yield the redox potential of the solution. Theunfolding reaction:

NWr1ox þ nð2GSHÞ X NWr1red þ nGSSG

proceeds with an opening of n disulfide bonds upontitration with GSH. The equilibrium of the form Keq=[NWr1red]/[ NWr1ox]·( [GSSG]/[GSH]2)n yields the coop-erativity of reduction n as the slope of a Hill plot of log([NWr1red]/[NWr1ox]) versus log([GSH]2/[GSSG]).35

Protein Data Bank accession number

The atomic coordinates of the ten lowest energy CNSconformers of NW1r, NW6r and NW8r have been


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 ofmass spectra. This work was supported by SNFgrant 31-109712 to S.G. by a grant from the GermanScience Foundation (DFG) to T.W.H. and S.Ö. andby 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

Documents

Sequence–Structure and Structure–Function Analysis in Cysteine-rich Domains Forming the Ultrastable Nematocyst Wall