The Cholesterol-Dependent Cytolysin Signature Motif: A
Critical Element in the Allosteric Pathway that Couples
Membrane Binding to Pore Assembly
Kelley J. Dowd, Rodney K. Tweten*
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis
of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the
CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to
changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a
combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the
archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated
membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the
oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the
intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further
show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has
evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the
undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly
of the pore complex.
Citation: Dowd KJ, Tweten RK (2012) The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane
Binding to Pore Assembly. PLoS Pathog 8(7): e1002787. doi:10.1371/journal.ppat.1002787
Editor: Theresa M. Koehler, The University of Texas-Houston Medical School, United States of America
Received April 6, 2012; Accepted May 19, 2012; Published July 5, 2012
Copyright: ? 2012 Dowd, Tweten. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported by a grant to RKT from the National Institutes of Health, NIAID (AI037657). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Rodney-Tweten@ouhsc.edu
The cholesterol-dependent-cytolysin (CDC) family of toxins
consists of over 25 members that are produced by many different
species of Gram-positive bacterial pathogens  and contribute
in various ways to the pathogenesis of these organisms [2,3,4,5].
Members of this family exhibit high levels of homology in their
primary structures (40–70%) and in the crystal structures of their
soluble monomers [6,7,8,9]. The region within the CDC primary
structure that exhibits the highest degree of sequence identity is
an 11-residue peptide known as the undecapeptide or trypto-
phan-rich motif, which is located near the C-terminus of the
molecule in domain 4 (D4) (Fig. 1). The undecapeptide
(ECTGLAWEWWR) is the signature motif for the CDCs 
and so proteins exhibiting this peptide sequence have a high
probability of belonging to the CDC family. The pore forming
mechanism of the CDCs that use cholesterol as their receptoris
highly sensitive to changes in the primary structure of the
undecapeptide [6,10,11,12,13,14,15]. These studies suggest that
the undecapeptide plays an important role in the CDC pore-
forming mechanism, yet since Iwamoto et al.  began studying
the effects of chemically altering the undecapeptide in 1987 its
contribution to the pore forming mechanism of the CDCs has
The undecapeptide is located at the tip of D4 of the CDC
structure, as shown in the structure of the CDC produced by
Clostridium perfringens, perfringolysin O (PFO) (Fig. 1). D4 also
contains the cholesterol recognition/binding motif (CRM) and two
other short loops (L2 and L3) near the undecapeptide (reviewed in
). Upon recognition of membrane cholesterol by the
CRM,loops L2 and L3 insert into the membrane. These
interactions anchor the monomers in a perpendicular orienta-
tionto the membrane surface where the tip of D4 is anchored to
the membrane surface and the top of D3 resides about 113 A˚
above the membrane surface [18,19,20]. Although the sidechains
of several residues of loops L2 and L3 and the undecapeptide
insert into and anchor the monomers to the membrane they do
not penetrate deeply into the bilayer core [19,21].
It had been generally accepted in the field that the undecapep-
tide motif wasthe CRM of the CDCs, although this function had
never been demonstrated unambiguously. An early study by
Iwamoto et al.  showed that chemical modification of the
undecapeptide cysteine caused independent defects in both
binding and pore formation. Since that time it has been shown
that mutation of many of the undecapeptide residues often affects
both binding and pore formation [6,10,11,12,13,14,15]. We
recently showed, however, that the CRMresides in the nearby
D4 loop L1 (Fig. 1) and is comprised of a threonine-leucine pair
that is strictly conserved in all known CDCs . Upon
cholesterol binding by the CRM the nearby loops L2 and L3
and the conserved undecapeptide insert into the bilayer surface
PLoS Pathogens | www.plospathogens.org1July 2012 | Volume 8 | Issue 7 | e1002787
and anchor the monomer in a perpendicular orientation to the
membrane surface [19,21,23,24]. Membrane binding in conjunc-
tion with monomer-monomer interactions  initiates and drives
a dramatic series of secondary and tertiary structural changes in
D3, which is about 60 A˚distant from the tip of D4 (Fig. 1). These
structural changes are necessary for the assembly of the membrane
bound monomers into the large oligomeric pore complex
[23,24,25,26,27]. Soluble monomers of PFO do not exhibit these
D3 structural changes, even at the high concentrations required
for crystallization of the protein : membrane binding is
required to initiate the structural changes in D3 [20,25].
As indicated above, the pore-forming mechanism of PFO-like
CDCs is highly sensitive to mutations in the undecapeptide
[6,10,11,12,13,14,15]. Furthermore, the conformational changes
in the PFO undecapeptide, reflected by the membrane insertion of
its tryptophan residues, are conformationally coupled to the
structural changes in TMH1 required for the formation of the b-
barrel pore . This observation suggests that the undecapeptide
of PFO is involved in the allosteric coupling of membrane binding
to the initiation of the D3 structural changes that are necessary for
monomer-monomer interaction and the formation of the oligo-
meric b-barrel pore complex.
A small family of CDCs, typified by Streptococcus intermedius
intermedilysin (ILY) use human CD59 as their receptor, rather
than cholesterol [29,30,31]. The D3 structural changes in ILY can
be initiated by binding to human CD59 in membranes that are
largely, though not completely depleted of cholesterol . ILY
still requires a CRM-mediated membrane interaction with
cholesterol to maintain its anchor to the membrane surface (it
disengages from CD59 during prepore to pore conversion
[22,33]), but it remains unclear if cholesterol binding also
participates in initiation of the D3 structural changes necessary
for assembly of the oligomer pore complex. Interestingly, in
contrast to the CDCs that use cholesterol as their receptor, the
pore forming mechanism of ILY is comparatively insensitive to
mutations within its undecapeptide , which suggests that it may
not play as significant of a role in the pore forming mechanism of
In the present study we performed a detailed molecular analysis
of a point mutation in the undecapeptide of PFO that reduces its
pore-forming activity 100-fold, whereasthe analogous mutation
has no significant effect on the mechanism of ILY . In PFO this
mutant blocks all measurable structural transitions in D3 and
prevents the stable interaction of membrane-bound monomers.
We further show that the effect of this mutation on the activity of
PFO is similar to that observed for cholesterol bound native ILY in
the absence of CD59. These results show that the undecapeptide
of PFO is a critical structure within the allosteric pathway of PFO
that couples cholesterol binding to the initiation of structural
changes within D3, which lead to the formation of the b-barrel
pore. We further show that this pathway appears to be missing in
the CD59-binding ILY, so that assembly of its pore complex is
initiated by its interaction with CD59 rather than cholesterol.
Cytolytic activity of PFO mutated at Arg-468
Arg-468 is the last residue of the PFO undecapeptide
(ECTGLAWEWWR), as well as in the ILY undecapeptide
(GATGLAWEPWR). Substitution of the PFO undecapeptide at
this residue with alanine decreases its hemolytic activity 100-fold
(Table 1), whereas substitution of the analogous residue in ILY has
little effect on the activity . A series of mutants were generated
for Arg-468 of PFO to examine the effects of size, length and
charge of the residue atposition 468 on the hemolytic activity of
PFO (Table 1). Neither conservative nor non-conservative
substitutions were tolerated: all substitutions decreased hemolytic
activity $100-fold. Based on the crystal structure of PFO the only
intramolecular contacts established by Arg-468 are hydrogen
bonds between its sidechain NH1 and the CRM carbonyls
(Fig. 1B). Interestingly, this contact is lost in the ILY monomer
(Fig. 1C), which presumably results from differences in its
undecapeptide structure . We selected the PFOR468Amutant
for further studies into the defect(s) induced by substitution of the
Arg-468 residue on the PFO pore-forming mechanism.
We have shown that a conserved Thr-Leu pair in Loop 1, and
not the undecapeptide, is responsible for CDC binding to
membrane cholesterol , yet mutations within the conserved
undecapeptide were often observed to affect binding [6,13,34]. To
confirm that the loss of hemolytic activity by the PFOR468Amutant
was not due solely to a defect in binding we examined the ability of
the mutant to bind to human RBCs by flow cytometry. In order to
prevent cell lysis at high concentrations of toxin, derivatives of
native PFO and PFOR468Awere generatedin which an engineered
disulfide was introduced between residuesThr-319 in b4 and Val-
334 in b5 that prevent the rotation of b5 away from b4 in domain
3 (PFOb4b5and PFOR468ANb4b5). The engineered disulfide there-
forepreventsthe formation of a functional pore by blocking the
intermolecular interaction of b1 of one monomer with b4 of
another monomer . A third cysteine was substituted at residue
Asp-30 in both mutants, which is at the amino terminus of PFO, so
that specific fluorescent probes could be introduced into these
The CDCs are a large family of pathogenesis-associated
pore-forming toxins that are expressed by many Gram-
positive pathogens. The conserved undecapeptide motif
of the CDCs has been regarded as the signature peptide
sequence for these toxins, yet its function has remained
obscure. The studies herein show that the undecapeptide
forms a critical structural element in the allosteric pathway
that couples membrane binding to cholesterol to the
initiation of distal structural changes, which are required
for the assembly of the pore forming complex. These
studies provide the first insight into the function of this
highly conserved sequence and show that through
evolution this pathway is missing in the CD59-binding
Table 1. Cytolytic activity of PFO derivatives with mutations
in the Arg-468 residue.
Toxin EC50(M)% WT
Cytolytic activity of PFO and Arg-468 mutants is shown as the effective
concentration (EC50) of toxin required for 50% lysis of human erythrocytes
under standard assay conditions (see Materials and Methods for details).
Allosteric Control of Pore Assembly
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