Conformational analysis of opacity proteins from
Neisseria
meningitidis
Marien I. de Jonge
1,2
, Martine P. Bos
3
, Hendrik J. Hamstra
1
, Wim Jiskoot
4
, Peter van Ulsen
4
,
Jan Tommassen
4
, Loek van Alphen
1
and Peter van der Ley
1
1
Laboratory of Vaccine Research, National Institute of Public Health and the Environment, RIVM Bilthoven, the Netherlands;
2
Department of Medical Microbiology, University of Amsterdam/AMC, Amsterdam, the Netherlands;
3
Department of Molecular
Microbiology and
4
Department of Pharmaceutics, Utrecht University, Utrecht, the Netherlands
Opacity-associated (Opa) proteins are outer membrane
proteins which play a critical role in the adhesion of patho-

receptor; in vitro folding; conformation.
The pathogenic bacteria Neisseria meningitidis and Neisseria
gonorrhoeae express a family of genes encoding outer
membrane proteins that are structurally related but highly
polymorphic. These proteins were originally identified as
colony-opacity-associated (Opa) proteins [1]. Opa proteins
appear to play a critical role in the ÔintimateÕ adhesion of the
bacteria to epithelial and endothelial cells and to polymor-
phonuclear neutrophils [2,3]. The majority of Opa proteins
bind to carcinoembryonic antigen cell-adhesion molecules
(CEACAM, formerly called CD66) [4]. CEACAM proteins
are expressed on various epithelial and endothelial cells as
well as on some lymphoid and myeloid cells [5]. A minority of
the Opa proteins target heparan sulfate proteoglycans
(HSPG) [6,7]. Recently, several Opa proteins were found
that did not bind to any of these human receptors, suggesting
that these Opa proteins haveadditional functions or that they
recognize additional receptors [8]. Opa-receptor-mediated
adhesion can lead to invasion of the bacteria into thedifferent
cell types expressing CEACAM proteins [8,9]. Opa expres-
sion was found in mucosal as well as disease isolates, 87.5%
of meningococcal strains tested bind to CEACAM1 [10].
Although the binding specificity of the variable Opa
proteins to the conserved human receptors has been studied
extensively [11], not much is known about the binding sites
present in the Opa proteins. A two-dimensional topology
model has been proposed, in which the Opa proteins form
eight-stranded b-barrels, exposing four loops at the cell
surface [12]. The variability of the Opa proteins is mainly
concentrated in surface-exposed loops two and three. An

indicative of a high content of b-strands, consistent with the
previously proposed structural models. Refolded Opa
protein was shown to be functional by specific binding to
the N-A1 part of CEACAM1.
MATERIALS AND METHODS
Construction of the expression systems
The genes encoding OpaJ129 and OpaB128 were isolated
from H44/76 using Taq polymerase (Amersham, Piscata-
way, NJ, USA) and general opa primers (5¢-CTTCT
CTTCTCTTCCGCAGC-3¢ and 5¢-TCGGTATCGGGG
AATCAGAA-3¢), cloned into plasmid pCR2.1 (Topo TA
cloning kit, Invitrogen, Carlsbad, CA, USA) and subse-
quently sequenced using M13-forward and M13-reverse
primers (Invitrogen). Plasmids pCR2.1 containing opaJ129
and opaB128 were used to amplify the DNA sequences
encoding the mature OpaB128 and OpaJ129 proteins with
Taq polymerase. The primers used (5¢-AGCGC
CCA
TGGCAAGTGAAG-3¢ and 5¢-GGCATCGGGATCCG
GGAATCAG-3¢) were based on the DNA sequence of
opaB128 and opaJ129 of N. meningitidis strains H44/76
(unpublished) and 190/87 (GenBank accession no.
AF016285) [12]. The primers contained base substitutions
(underlined) to introduce NcoIandBamHI cleavage sites,
respectively. The PCR product was cloned in plasmid
pCR2.1. The NcoI–BamHI fragment was isolated from the
resulting plasmid and ligated into the NcoI–BamHI digested
expression vector pET11d (New England Biolabs, Inc.,
Beverly, MA, USA) downstream of the inducible T7
promoter. In the resulting construct, the codon for the first

expression from the phoE promotor. The resulting plasmids
were used for transformation of strain CE1265, which
expresses the pho regulon constitutively due to a phoR
mutation [16]. Expression of OpaB128 and OpaJ129 was
determined by assaying the binding of monoclonal anti-
bodies MN20E12.70 (M. de Jonge, G. Vidarson, H. H. van
Dijken, P. Hoogerhout, L. van Alphen, J. Dankert & P. van
der Ley, unpublished results) and 15-1-P5.5 [18] in a colony
blotting experiment [19]. The bla-opaB fusion construction,
whichresultedinE. coli surface expression of gonococcal
OpaB, is described by Belland et al.[3].Thesurface
expression was confirmed with immunofluorescence. Cells
were washed with NaCl/P
i
and after blocking overnight in
NaCl/P
i
with 3% BSA, incubated with 15-1-P5.5 [18]
(diluted 1 : 100) for 1 h, followed by an incubation with
Alexa-conjugated goat anti-(mouse IgG) (Molecular Probes
Inc., Eugene, OR, USA) (diluted 1 : 300) for 1 h. After
washing three times, cells were again fixated in NaCl/P
i
with
2% formaldehyde (Merck, Darmstadt, Germany). The
construction of the recombinant N-domains of the different
CEACAM proteins is described by Bos et al.[20].
Binding of His-tagged CEACAM fragments
to bacterial cells
The binding of His-tagged CEACAM fragments to bacter-

monoclonal Ig (1 : 10 000) (Amersham Pharmacia
Biotech GmbH, Freiburg, Germany) for the detection of
the N-terminal domains of the CEACAM proteins.
Production and purification of inclusion bodies
Cultures of the E. coli strain BL21 (DE3) containing either
pET11d-opaB128 or pET11d-opaJ129, grown overnight at
37 °C, were diluted 1 : 10 into fresh LB medium supple-
mented with 0.5% glucose (Fluka, Buchs, Switzerland) and
100 lgÆmL
)1
ampicillin. When the culture reached an
optical density of 660 nm (D
660
) of 0.6, isopropyl thio-b-
D
-galactoside (IPTG) (Boehringer Mannheim, Germany)
was added to a final concentration of 1 m
M
.After3hof
incubation at 37 °C, the cells were harvested by centrifuga-
tion at 4500 r.p.m. for 15 min at 4 °C (Centrikon T324,
Rotor A6.9, Kontron Instruments, Milan, Italy). The pellet
waswashedwith10m
M
Tris/HCl (pH 8) and centrifuged at
4500 r.p.m. for 15 min at 4 °C in the same rotor. After
resuspension in the same buffer, cells were disrupted using a
French Press (SLM-Aminco) at 9000 p.s.i. three times. The
inclusion bodies were collected by a low-speed centrifuga-
tion step at 2800 g for 10 min at 4 °C (Megafuge 1.0 R,

J. Dankert & P. van der Ley, unpublished results).
Semi-native polyacrylamide gel electrophoresis was per-
formed by using SDS-free 11% polyacrylamide gels.
Loading buffer containing either 0.1% or 2.0% SDS
(Fluka, Buchs, Switzerland) was added to the samples
which were subsequently incubated at room temperature
and 100 °C, respectively. After electrophoresis, protein
bands were visualized with Coomassie Brilliant Blue (Fluka,
Buchs, Switzerland).
Refolding and purification
To find the optimal folding conditions, buffers with
different NaCl concentrations (100–300 m
M
) and final urea
concentrations (125 m
M
)1
M
) were tested at different pH
values ranging from pH 7.2–12.0. Furthermore, different
protein dilutions (1 : 20 to 1 : 200) and n-dodecyl-N,
N-dimethyl-1-ammonio-3-propanesulfonate (SB-12, Fluka,
Buchs, Switzerland) concentrations were tested. All refold-
ing experiments were performed overnight at 4 °C.
In the optimal folding procedure, Opa (10 mgÆmL
)1
)
dissolved in 8
M
urea and 50 m

in
120 mL. To check folding and purification, SDS/PAGE
was performed under seminative and denaturing conditions.
The folded and purified proteins were stored at )20 °C.
Circular dichroism spectroscopy
Circular dichroism (CD) spectra were recorded at 25 °Cwith
a dual-beam DSM 1000 CD spectrophotometer (On-Line
Instrument Systems, Bogart, GA, USA). The subtractive
double-grating monochromator was equipped with a fixed
disk, holographic gratings, and 1.24-mm slits. For far-UV
and near-UV measurements, gratings with 2400 lines per mm
(blaze wavelength 230 nm) and 600 lines per mm (blaze
wavelength 300 nm), respectively, were used. Far-UV spec-
tra were recorded from 250 to 200 nm (cell-path length
0.5 mm). For near-UV measurements (320–250 nm), cells
with a path length of 1 cm were used. The Opa protein
concentration was 0.54 mgÆmL
)1
. The results depicted
represent the average of at least six repeated scans (step
resolution 1 nm, 1 s each step), from which the correspond-
ing buffer spectrum was subtracted. The measured CD
signals were converted to molar ellipticity [h],basedona
mean residual weight of 112 (OpaB128) or 111.5 (OpaJ129).
For the comparison between folded and denatured
protein, folded protein in buffer A was incubated for
20 min at 100 °C with 1.85% SDS.
Immunodotblotting
Opa proteins were diluted to 1 lg per 100 lLin10m
M

(Invitrogen) using NcoIandBamHI restriction, resulting in
plasmid pVB1. This vector adds the pelB signal sequence to
the CEACAM domains, which allows secretion of the
protein into the periplasm. BL21 cells containing pVB1 were
grown in LB containing 50 lgÆmL
)1
kanamycin to a D
600
of
0.6. Cells were induced with 0.2 m
M
IPTG and grown
overnight at room temperature. The induced cell pellet was
resuspended in 200 m
M
Tris/HCl pH 8.0, 0.5 m
M
EDTA,
0.5
M
sucrose. Lysozyme was added to 60 lgÆmL
)1
and
Ó FEBS 2002 Conformation of meningococcal Opa proteins (Eur. J. Biochem. 269) 5217
the suspension was diluted 2 · with 0.5 m
M
EDTA and
incubated for 10 min at room temperature. Cells were
collected by centrifugation for 2 min at 8000 g. (Eppendorf
centrifuge) and the supernatant was collected as the

either OpaB128 or OpaJ129 at the cell-surface were
incubated with N-domains of the two different CEACAM
proteins (Fig. 2, lanes 5–8). Surface expressed recombinant
gonococcal OpaB protein was included in these experiments
as a positive control (Fig. 2, lanes 1 and 2). After
incubation, the bacterial cells were collected by centrifuga-
tion and the proteins were separated by SDS/PAGE. After
blotting to nitrocellulose filters, the presence of CEACAM
in the bacterial cell pellets was evaluated with an anti-His Ig.
Like the bacteria expressing the gonococcal OpaB protein
(lane 1 and 2) the bacteria expressing OpaB128 or OpaJ129
bound to CEACAM1 (lane 5 and 7) while no binding was
found with CEACAM8 (lanes 6 and 8).
Expression system for Opa proteins
To obtain large quantities of OpaB128 and OpaJ129
protein, part of the opa sequence encoding the mature Opa
protein without the signal sequence was cloned into pET11d
under the control of the inducible T7 promoter. The
recombinant genes were expressed in the E. coli strain
BL21 (DE3) upon addition of IPTG. The Opa proteins
accumulated in the cytoplasm as inclusion bodies, which
could be separated from the other cell components by
centrifugation. After dissolving these inclusion bodies in 8
M
urea followed by an ultracentrifugation step to remove
residual membrane fragments, the Opa protein in the
supernatant was approximately 90% pure as determined
by SDS/PAGE (Fig. 3A). N-terminal amino acid sequen-
cing of the purified proteins revealed the sequence ASEDG,
Fig. 1. Western blots showing the heat-modifiability of OpaB128 and OpaJ129 expressed in N. meningitidis and E. coli. OMCs of H44/76 expres-

unpublished results) or 15-1-P5.5 [18], respectively. Semi-
native PAGE, followed by Western blotting, confirmed that
both OpaB128 and OpaJ129 migrated with an apparent
molecularmassof 23 kDa, whereas completely unfolded
OpaB128 or OpaJ129 migrates as a protein of  27 kDa
(Fig. 1). The heat-modifiability of wild type OpaB128 and
OpaJ129 was taken as marker for correct folding of the
proteins purified from the inclusion bodies. The correct
folding of OpaB128 and OpaJ129 expressed at the surface of
E. coli strain CE1265 was confirmed in the same assay
(Fig. 1).
We diluted the urea-solubilized protein solution
(10 mgÆmL
)1
) 100-fold in various buffers with different
pHs, all containing 0.5% SB12 (w/v) and incubated the
samples overnight at 4 °C. When the pH of the refolding
buffer was below 10, no or hardly any refolding was
observed. However, in 328 m
M
ethanolamine buffer with
pH 10.5 (i.e. just above the calculated pI of OpaJ129 and
OpaB128, 10.3 and 10.4, respectively) almost 50% of
OpaJ129 and > 50% of OpaB proved to be refolded
according to semi-native PAGE analysis (data not shown).
To increase the folding efficiency, several buffering
substances and final protein concentrations were tested
at different pH values, and different NaCl and urea
concentrations. Although inclusion of 200 m
M

to 7.5 after refolding. This procedure did not affect the
folding state of either OpaB128 or OpaJ129 as determined
by seminative PAGE (data not shown). The neutralized
protein solution was applied to a cation-exchange column
(SP-Sepharose HP at pH 7.5). Protein was eluted from the
column with a linear salt gradient, resulting in the elution of
either folded OpaB128 or folded OpaJ129 as a single peak.
Apparently, due to a difference in affinity, the folded protein
Fig. 3. Semi-native PAGE analysis of in vitr o folding of OpaB128 (A) and OpaJ129 (B). (A) Semi-native-PAGE analysis of in vitro folding of
unpurified OpaB128 (Coomassie stained). Lane 1 isolated inclusion bodies. Lane 2 in vitro folded protein. Lane 3 denatured protein. Lane 4 and 5,
in vitro folded and denatured OpaB after additional purification. Lane 6, molecular mass marker. Samples 2 and 4 were incubated at room
temperature in loading buffer containing 0.1% SDS, samples 1, 3 and 5 were incubated at 100 °C in loading buffer containing 2.0% SDS prior to
electrophoresis. (B) Semi-native PAGE analysis of in vitro folding of unpurified OpaJ129 (Coomassie stained). Lane 1, isolated inclusion bodies.
Lane 2, in vitro folded protein. Lane 3, denatured protein. Samples 1 and 3 were incubated as samples 1, 3 and 5 (Fig. 3A) and sample 2 was treated
as sample 2 and 4 (Fig. 3A). (C) Coomassie stained polyacrylamide gel showing in vitro folded OpaJ129, after additional purification. Purified
protein samples 1 and 2 were treated as samples 2 and 3 (Fig. 3A), respectively.
Ó FEBS 2002 Conformation of meningococcal Opa proteins (Eur. J. Biochem. 269) 5219
was purified from the residual unfolded protein as well as
from other contaminants.
Analysis of the Opa protein conformation
by circular dichroism
To test whether the in vitro folded OpaB128 and OpaJ129
had adopted the expected b-sheet conformation, CD spectra
were recorded for folded Opa protein and Opa protein that
was denatured by boiling in 1.85% SDS. The far-UV spectra
revealed a clear difference between the secondary structures
of the folded and denatured proteins (Fig. 4A). The charac-
teristic feature of the spectrum, recorded for folded OpaB128
was a minimum at 217 nm. Characteristic features of the
spectrum, recorded for folded OpaJ129, were a maximum at

bound to CEACAM1-N-A1, consistent with the binding
experiments with the OpaJ129-expressing E. coli bacteria
(Fig. 5). The binding of refolded Opa appeared to be
conformation-dependent, as no binding was found with
denatured OpaJ129.
DISCUSSION
The majority of Opa proteins have been shown to speci-
fically target members of the CEACAM receptor family
[10,26]. How this binding function can be conserved
Fig. 4. Far-UV (A) and near-UV (B) circular dichroism spectra of refolded and heat-denatured OpaB128. (A) Far-UV circular dichroism spectra of
refolded OpaB128 (interrupted line) and heat-denatured OpaB128 in 1.85% SDS containing buffer (solid line). (1) Far-UV circular dichroism
spectra of refolded OpaJ129 (interrupted line) and heat-denatured OpaJ129 in 1.85% SDS containing buffer (solid line) (2). (B) Near-UV circular
dichroism spectra of refolded OpaB128 (interrupted line) and heat-denatured OpaB128 in 1.85% SDS containing buffer (solid line). (1) Near-UV
circular dichroism spectra of refolded OpaJ129 (interrupted line) and heat-denatured OpaJ129 in 1.85% SDS containing buffer (solid line) (2).
5220 M. I. de Jonge et al.(Eur. J. Biochem. 269) Ó FEBS 2002
despite the hypervariability of the surface-exposed regions
of the Opa proteins is still an enigma. The detailed
identification of the receptor-binding Opa regions would
aid greatly in the development of new vaccines or
antimicrobials specifically targeted at blocking this essen-
tial adhesion process. The study of the molecular inter-
actions between the CEACAM receptors and Opa
proteins would be facilitated greatly by the availability
of large quantities of pure Opa proteins. This was
achieved in the present study for OpaB128 and OpaJ129,
two representative Opa proteins present in invasive
variants of N. meningitidis strain H44/76.
Previously, the isolation and purification of Opa proteins
from meningococcal strains has been described [21,27].
However, translation of the constitutively transcribed opa

predicted for Opa proteins [12,33].
The CD measurements showed a clear difference
between the structure of folded Opa and Opa denatured
by boiling in SDS. The far-UV spectrum we recorded for
folded Opa resembles that of folded OmpA from E. coli
[34] and purified P5 from H. influenzae [32]. The spectra
are indicative of a high content of b-strands, consistent
with the (proposed) structure of these outer membrane
proteins (Fig. 4A). The difference between the near-UV
CD spectra of folded and denatured Opa supports the
conclusion that denatured protein has undergone a major
conformational change. In the proposed topology model
for Opa proteins, 31% of the amino acid chain is
predicted to form a transmembrane b-barrel. The high
content of b-strands reflected in the CD spectra reported
here suggests that a significant part of the extracellular
loops may also adopt this secondary structure. It is thus
conceivable that Opa proteins form a more extended
b-barrel structure that protrudes from the outer mem-
brane into the extracellular space, similar to what was
described recently for the OmpT outer membrane prote-
ase from E. coli [35].
The pH and the salt concentration are the most critical
factors in the folding efficiency of Opa. It appeared that a
pH above the calculated pI is needed for efficient folding, as
has also been found for the OmpA protein from E. coli and
the PorA protein from N. meningitidis [36,37]. The present
study demonstrates how two different Opa proteins, with
approximately 70% homology, can be folded in vitro under
similar conditions. This method will allow us to establish a

Institute in Berlin, for the generous gift of purified OpaD and to
B. Kuipers at the RIVM in Bilthoven and W. Zollinger at the
Walter Reed Army Institute of Research in Washington for
providing us with monoclonal antibodies. We also thank
W. van Noppen at the University of Amsterdam/AMC for critically
reading the manuscript.
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