Inactivation of colicin Y by intramembrane helix–helix
interaction with its immunity protein
David S
ˇmajs
1
, Magda Dolez
ˇalova
´
1,2
, Pavel Macek
3,4
and Luka
´s
ˇZ
ˇı
´dek
3
1 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
2 Department of Food Engineering, Faculty of Technology, Toma
´s
ˇBat
ˇa University, Zlı
´n, Czech Republic
3 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic
4 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Colicin Y (Cya) belongs to the group of ion channel-
forming colicins [1] having a high sequence homology
to colicin U [2]. A similar sequence relatedness also
applies to their respective immunity proteins. The
immunity of producer bacteria to pore-forming colicins
is mediated by cognate immunity proteins that are
localized in the cytoplasmic membrane [3,4], and have
either four transmembrane helices [e.g. immunity
proteins of colicins A (Cai), U (Cui) and Y (Cyi)] [5,6]
or three transmembrane regions (e.g. immunity
proteins of colicins E1 and 5) [7,8].
The A-type pore-forming colicins comprise sequen-
tially related colicins A, B, N, S4, U and Y. In con-
trast with E1-type colicins (E1, 5, 10, Ia, Ib and K),
A-type colicins contain a hydrophobic hairpin between
helices 8 and 9 in their pore-forming domains. For
colicin A, it has been shown that the sequence that
determines the specific interaction between colicin A
and its immunity protein is localized between L530
and D577, a region containing helices 8 and 9, and the
hydrophobic hairpin between them [5]. In a previous
study [6], deletion of the hydrophobic hairpin (nine
amino acid residues) in colicin U resulted in a com-
plete loss of immunity to the hairpin-deleted version of
colicin U when tested against a colicin U immune
strain. The replacement of the nine hairpin amino acid
residues of colicin U with those present in the colicin B
sequence restored partial immunity to the colicin U
hybrid. Moreover, this replacement brought additional
immunity, specific to the hybrid colicin U, when tested
against a colicin B immune Escherichia coli strain. Fur-
thermore, the mutation of individual amino acids in
this region revealed that an F580G mutation, intro-
duced to colicin U, resulted in decreased inactivation
by the colicin U immunity protein. In addition,
an F576G mutation altered immune specificity by:
Keywords
colicin immunity; colicin Y; helix–helix
interaction; pore-forming colicin;
site-directed mutagenesis
Correspondence
D. S
ˇmajs, Department of Biology, Faculty of
Medicine, Masaryk University, Kamenice 5,
Building A6, 625 00 Brno, Czech Republic
Fax: +420 549 491 327
Tel: +420 549 497 496
E-mail: dsmajs@med.muni.cz
(Received 22 May 2008, revised 4 August
2008, accepted 29 August 2008)
doi:10.1111/j.1742-4658.2008.06662.x
The construction of hybrids between colicins U and Y and the mutagenesis
of the colicin Y gene (cya) have revealed amino acid residues important for
interactions between colicin Y and its cognate immunity protein (Cyi).
Four such residues (I578, T582, Y586 and V590) were found in helices 8
and 9 of the colicin Y pore-forming domain. To verify the importance of
these residues, the corresponding amino acids in the colicin B protein were
mutated to the residues present in colicin Y. An Escherichia coli strain with
cloned colicin Y immunity gene (cyi) inactivated this mutant, but not the
wild-type colicin B. In addition, interacting amino acid pairs in Cya and
Cyi were identified using a set of Cyi point mutant strains. These data are
consistent with antiparallel helix–helix interactions between Cyi helix T3
and Cya helix 8 of the pore-forming domain as a molecular mechanism of
colicin Y inactivation by its immunity protein.
Abbreviations
cua, colicin U gene; cui, colicin U immunity gene; Cui, colicin U immunity protein; cya, colicin Y gene; cyi, colicin Y immunity gene; Cyi,
colicin Y immunity protein.
FEBS Journal 275 (2008) 5325–5331 ª2008 The Authors Journal compilation ª2008 FEBS 5325
(a) decreasing immunity against colicin U and (b)
increasing immunity of a colicin B immune strain to
this mutated colicin U [6]. The F576 residue is local-
ized in helix 8 of the colicin U pore-forming domain,
and F580 is localized in the hydrophobic loop between
helices 8 and 9 [6]. F576 and F580 in colicin U corre-
spond to amino acid residues Y586 and V590 in the
colicin Y polypeptide, respectively. Helices 8 and 9,
together with the hydrophobic hairpin between them,
thus appear to be the sequences that determine the
interactions with the immunity proteins of other
A-type pore-forming colicins.
Mapping of the colicin Y immunity protein (Cyi)
identified five amino acid residues in Cyi important in
the recognition of Cya [9]. These amino acid residues
were localized to the Cyi transmembrane helices T3
(S104, S107, F110, A112) and T4 (A159). Colicin U
immunity protein with point mutations in these posi-
tions (mutated towards sequences present in Cyi)
resulted in a mutated immunity protein with newly
acquired immunity to colicin Y.
In this study, we map the amino acid residues of
colicin Y involved in the interaction with Cyi using
hybrid colicins and Cya with introduced point muta-
tions. In addition, colicin Y point mutants are tested
against a set of Cyi mutant strains to identify the
amino acid residues that mediate interactions between
colicin Y and Cyi.
Results and Discussion
The pore-forming domains of colicins U and Y span
approximately 200 C-terminal amino acid residues.
Within these regions, 34 residues differ between colic-
ins U and Y (83% identity, data not shown). Despite
this high degree of sequence conservation, colicin U is
recognized and inactivated by both colicin U immunity
protein (Cui) and Cyi, but colicin Y interacts only with
Cyi [9]. Most of the differences in the amino acid
sequences of the colicin U and Y pore-forming
domains are located in the last 50 amino acid residues
in these domains (data not shown).
Hybrid colicins specify regions important for
interactions with immunity proteins
Colicin U and colicin Y immune strains were prepared
by the transformation of sensitive E. coli strain
DH10B with plasmids pDS519 and pDS518, encoding
Cyi and Cui, respectively (Table 1). Incomplete immu-
nity of DH10B pDS519 to colicin Y (Table 2) proba-
bly reflects the fact that immunity to pore-forming
colicins may be overcome by high doses of the exoge-
nous colicin [10]. To construct hybrid colicins, a SpeI
restriction site was introduced into the cya gene
(encoding colicin Y) cloned into pDS527, resulting in
pDS529. The cua gene (cloned into pDS526 and
encoding colicin U) contained a natural SpeI target
site. The plasmids encoding hybrid colicins, plasmids
encoding immunity to colicins and the E. coli strains
used are presented in Table 1. The cya gene with an
introduced SpeI target site (encoded on pDS529) coded
for Cya with two replacements (A594T, A595S). This
mutant colicin [Cya (A594T, A595S)] was inactivated
by E. coli strains producing Cyi and Cui to the same
extent as was wild-type colicin Y (Table 2). The recog-
nition of colicin Y by strain DH10B pDS519 (cyi) was
different from the recognition of colicin U by strain
DH10B pDS518 (cui), as strain DH10B pDS519 (cyi)
was immune to the hybrid colicin Cya ⁄Cua (Cya: 1–
595; Cua: 586–619) to the same extent as it was to
wild-type colicin Y. The last 34 C-terminal amino
acids, which were replaced in the hybrid colicin Cya ⁄
Cua (Cya: 1–595; Cua: 586–619), did not contribute to
the Cya–Cyi interaction. On the other hand, the recog-
nition of colicin U and of the hybrid colicin Cua ⁄Cya
(Cua: 1–585; Cya: 596–629) by strain DH10B pDS518
(cui) also appeared to require (in addition to the inter-
action with the colicin region containing helix 8)
amino acid residue(s) in the last 34 C-terminal posi-
tions (Table 2). Because of this, amino acid residues in
helix 8 and the surrounding regions of Cya were
mutated to the corresponding amino acid residues in
Cua (Fig. 1), and the resulting proteins were tested for
interaction with Cyi. In addition, mapping of amino
acid residues in colicin Y was carried out because of
the availability of a previously prepared set of strains
producing mutated Cyi proteins [9].
Amino acid residues of Cya interacting with Cyi
In helix 8 of colicin Y, three amino acid residues,
capable of decreasing the immunity mediated by Cyi,
were identified when they were substituted with the
corresponding amino acids from colicin U (see
Table 2): I578, T582 and Y586. An additional residue,
V590, was identified in the hydrophobic hairpin of the
colicin Y pore-forming domain between helices 8 and
9. The relatively small decrease in immunity (by one
order of magnitude) is explained by the fact that Cyi
also confers considerable immunity to colicin U. Sur-
prisingly, the A589L replacement in Cya increased the
immunity of the DH10B pDS519 strain to this colicin.
Moreover, increased immunity to Cya (A589L) was
also found in all tested Cyi mutants (data not shown),
suggesting a nonspecific increase in immunity, perhaps
Helix–helix interaction between colicin Y and Cyi D. S
ˇmajs et al.
5326 FEBS Journal 275 (2008) 5325–5331 ª2008 The Authors Journal compilation ª2008 FEBS
as a result of the increased hydrophobicity of
Cya
A589L
. To verify the importance of the identified
residues I578, T582, Y586 and V590 with regard to the
interaction between colicin Y and Cyi, we tested
whether a related colicin, which is not recognized by
Cyi, could be mutated in these positions, and whether
this mutation could result in an acquired recognition
of this mutant colicin by Cyi. Of the four amino acid
residues, that which corresponded to the T582 position
in colicin Y was identical in colicin B (T467, Fig. 1).
Nevertheless, colicin B was not recognized by the
DH10B pDS519 strain to a significant extent (Table 2).
The corresponding amino acid residues in the colicin B
protein were mutated to the residues present in coli-
cin Y. As predicted, the three point mutations (L463I,
A471Y, F475V; Fig. 1) introduced into the colicin B
protein resulted in the inactivation of this protein by
E. coli strain DH10B pDS519, indicating that these
amino acid residues are recognized by Cyi.
Mutational analysis of Cya towards the correspond-
ing sequence of Cua allowed the identification of the
amino acid residues important for the specific interac-
tions between colicin Y and Cyi. In addition to these
residues, other amino acid residues (identical in both
the colicin U and Y sequences) are probably involved
in the formation of the binding epitope. The identifica-
tion of these residues would require mutational analy-
sis of the cya gene towards the sequences present in
other colicin genes of A-type pore-forming colicins.
A similar situation has already been described for
colicin–immunity protein interactions in enzymatic
colicins [11].
Complementarity of colicin Y mutants and strains
expressing mutated colicin Y immunity genes
The mutant colicin Y forms prepared in this study
were combined with Cyi mutants prepared for an
Table 1. Bacterial strains and plasmids used in this study.
Strain, plasmid Phenotype and ⁄or genotype Source or reference
Escherichia coli K-12
DH10B F
)
araD139 D(ara,leu)7697 DlacX74 galUgalKrpsLdeoR/80dlacZDM15
endA1 nupGmcrArecA1 mcrAD(mrr hsdRMS mcrBC)
[23]
5K pDS1 Producer of colicin U [2]
K339 Producer of colicin Y [1]
BZB2102 (Ec 433 ⁄82) Producer of colicin B (pColB-K260) A. P. Pugsley (Institut Pasteur,
Paris, France)
Plasmid
pDS518 Immunity to colicin U; pCR2.1-TOPO with cloned cui [9]
pDS519 Immunity to colicin Y; pCR2.1-TOPO with cloned cyi, Cyi: L170I
a
[9]
pDS897 pDS519, S104C [9]
pDS892 pDS519, S107T [9]
pDS921 pDS519, F110I [9]
pDS922 pDS519, A112V [9]
pDS893 pDS519, A159I [9]
pDS526 Production of colicin U; cua and cui cloned on pCR2.1-TOPO [9]
pDS527 Production of colicin Y; cya and cyi cloned on pCR2.1-TOPO [9]
pDS529 pDS527 with introduced SpeI restriction site resulting in mutated Cya
(A594T, A595S)
This work
pDS541 Production of hybrid colicin Cua ⁄Cya (Cua: 1–585; Cya: 596–629) This work
pDS542 Production of hybrid colicin Cya ⁄Cua (Cya: 1–595; Cua: 586–619) This work
pMJ232 Production of mutated colicin Y (A577S, I578F) This work
pMJ240a Production of mutated colicin Y Cya (I578F) This work
pDS961a Production of mutated colicin Y Cya (T582I) This work
pMJ231 Production of mutated colicin Y Cya (L583F, A585T) This work
pMJ233 Production of mutated colicin Y Cya (Y586F) This work
pMJ230a Production of mutated colicin Y Cya (L587A, L588M) This work
pMJ230b Production of mutated colicin Y Cya (A589L) This work
pDS947 Production of mutated colicin Y Cya (V590G) This work
pMJ229 Production of mutated colicin Y Cya (G591V, A592F) This work
pDS995 Production of colicin B; cba cloned on pCR2.1-TOPO This work
pDS996 pDS995 coding for mutated Cba (L463I, A471Y, F475V) This work
a
When compared with wild-type Cyi, mutated Cyi L170I provided the same degree of immunity to colicins Y and U [9].
D. S
ˇmajs et al. Helix–helix interaction between colicin Y and Cyi
FEBS Journal 275 (2008) 5325–5331 ª2008 The Authors Journal compilation ª2008 FEBS 5327
earlier study [9]. The results are shown in Table 3. The
effect (decreased immunity) of mutations in Cyi and
Cya should add or multiply when noninteracting pairs
of amino acids are affected. In contrast, if the affected
amino acids form interaction pairs, the degree of
immunity should remain, reflecting the mutation show-
ing lower immunity, or increase as a result of the
formation of amino acid pairs present in Cui–Cua.
Four such pairs, I578–S107, T582–S107, T582–S104
and Y586–S104, were identified. Mutation studies pro-
vide structural details that are particularly valuable for
the studied system. This system represents a very
difficult target with regard to the experimental
determination of structure (interacting membrane pro-
teins, and difficult over-expression of cyi) and com-
puter-assisted structural model determination methods
(unknown structure of one interacting partner). In
order to visualize the interactions between Cya and
Table 2. Sensitivity of E. coli DH10B, DH10B pDS518, DH10B pDS519 and DH10B pDS994 to wild-type and mutated colicin U, Y and B
proteins.
Colicin encoding
plasmid
Relevant phenotype ⁄
genotype (amino acid no.)
Sensitive
strain
E. coli DH10B
Immune E. coli
DH10B
pDS518 (cui
+
)
Immune E. coli
DH10B pDS519
(cyi
+
, L170I)
Immune E. coli
DH10B
pDS994 (cbi
+
)
pDS526 Colicin U synthesis (Cua) wt 3(5)
a
i –(2) 2(5)
pDS527 Colicin Y (Cya) wt 3(5) 3(5) –(1) –(3)
pDS529 Cya (A594T, A595S) 3(5) 3(5) –(1) nt
pDS541 Cua ⁄Cya (Cua: 1–585; Cya: 596–629) 3(5) –(2) –(2) nt
pDS542 Cya ⁄Cua (Cya: 1–595; Cua: 586–619) 3(5) 2(4) –(1) nt
pMJ232 Cya (A577S, I578F) 3(5) 3(5) –(2) nt
pMJ240a Cya (I578F) 3(5) 3(5) –(2) nt
pDS961a Cya (T582I) 3(5) 3(4) 1(2) nt
pMJ231 Cya (L583F, A585T) 3(5) 3(5) –(1) nt
pMJ233 Cya (Y586F) 3(5) 3(5) –(2) nt
pMJ230a Cya (L587A, L588M) 3(5) 3(5) –(1) nt
pMJ230b Cya (A589L) 3(5) 3(5) i nt
pDS947 Cya (V590G) 3(5) 3(5) –(2) nt
pMJ229 Cya (G591G, A592F) 3(5) 3(5) –(1) nt
pDS995 Colicin B synthesis (Cba) wt 2(3) 2(3) 1(3) i
pDS996 Cba (L463I, A471Y, F475V) 1(3) 1(3) i i
i, immune: no detectable action of colicin on indicator bacteria; nt, not tested; wt, wild-type; –, no immunity inhibition resulting in a clear
zone of colicin-mediated growth inhibition: detectable colicin action results in turbid zones of inhibition (intensity shown in parentheses).
a
The numbers indicate the highest colicin dilution resulting in clear (turbid) zones of growth inhibition on the indicator bacteria (e.g.
5=10
)5
).
Fig. 1. Sequence alignment of the 28-amino-acid long region of the
colicin Y (Cya) pore-forming domain to the corresponding regions of
colicin U (Cua) and B (Cba). Helix 8 of the colicin pore-forming
domains is highlighted. Amino acid residues important in the recog-
nition of Cya or Cua by their cognate immunity proteins are shown
in bold. The last two letters in each polypeptide chain indicate the
protein region that corresponds to the naturally occurring SpeI
restriction target site (cua) or introduced SpeI site (cya). Amino acid
residues different from the colicin Y sequence are shown in italic.
Table 3. Sensitivity of E. coli DH10B strains harbouring plasmids
pDS518, pDS519 and their derivatives with mutated cyi and cui
genes to wild-type and mutated colicins Y and wild-type
colicin U. The results shown in bold indicate identified interacting
amino acid pairs.
Plasmid
Relevant
genotype
(amino acid no.)
Colicin
Y
wt
Y
I578F
Y
T582I
Y
Y586F
Y
V590G
U
wt
– 3(5)
a
3(5) 3(5) 3(5) 3(5) 3(5)
pDS518 cui 3(5) 3(5) 3(4) 3(5) 2(5) i
pDS519 cyi (L170I) –(1) –(2) 1(2) –(2) –(2) –(2)
pDS897 cyi (L170I, S104C) 1(3) 2(3) 1(2) -(2) 2(4) –(2)
pDS892 cyi (L170I, S107T) 1(2) 1(2) 1(2) 2(3) 2(3) –(2)
pDS921 cyi (L170I, F110I) 1(2) 1(3) 1(3) 2(3) 1(3) –(2)
pDS922 cyi (L170I, A112V) 1(2) 1(3) 1(3) 1(3) 1(3) –(2)
pDS893 cyi (L170I, A159I) 1(3) 2(3) 2(3) 2(5) 2(3) –(2)
i, immune; –, no inhibition resulting in clear zones of growth inhibi-
tion.
a
The numbers indicate the highest colicin dilution resulting in clear
(turbid) zones of growth inhibition on the indicator bacteria (e.g.
5=10
)5
).
Helix–helix interaction between colicin Y and Cyi D. S
ˇmajs et al.
5328 FEBS Journal 275 (2008) 5325–5331 ª2008 The Authors Journal compilation ª2008 FEBS
Cyi, a model of antiparallel helix–helix interaction was
built based on the above-mentioned amino acid pairs
identified through mutations (Fig. 2). All interactions
between the side-chains of the pairs were modelled as
distance restraints and putative hydrogen bonds, as
described in Materials and methods. The regular spac-
ing of the identified interacting residues, by three in
Cyi (S104, S107, F110) and by four in Cya (I578,
T582, Y586, V590), is consistent with helical packing
corresponding to interactions between the i± 3 ridge
of helix T3 of Cyi and the i± 4 ridge of helix 8 of
Cya. This type of helix–helix interaction is character-
ized by an angle of 23between the helical axes, and
ensures close contact between the helices [12]. Helix–
helix interaction thus appears to be at least one of the
molecular mechanisms by which colicin Y is inacti-
vated by Cyi.
Colicin Y producer mediates partial immunity to
colicins A and S4 but not to colicins B and N
The immunity of E. coli strain DH10B, expressing cyi
(on plasmid pDS519), to several closely related colicins
is shown in Table 4. Cyi also mediates immunity to
colicin A, which shows identical amino acid residues in
three of the four positions identified in Cya (I578,
T582, Y586), and to colicin S4, which has two identi-
cal residues to colicin Y (T582, Y586). Colicin B, with
only one identical residue to colicin Y (T582), and
colicin N, with no identical residues, are not inacti-
vated by Cyi. The partial immunity of E. coli DH10B
expressing cyi to colicin U (which was the sequence
used as a target sequence for mutational analysis;
Table 2) indicates that additional amino acid residues
are important for colicin–immunity protein interac-
tions. The identification of these residues would
require further mutational analysis of Cyi.
Taken together, mutations in the pore-forming
domains of A-type colicins, containing helices 8 and 9,
can alter specific interactions with related immunity
proteins. The construction of mutated colicins recog-
nized by immunity proteins of other colicin types and
of mutated immunity proteins with new immune speci-
ficities [9] suggests that colicin–immunity protein
interactions are similar for many (if not all) A-type
pore-forming colicins. In these interactions, different
amino acids specify individual colicin–immunity pro-
tein interactions, and invariant amino acid residues
may serve as a scaffold for this recognition. This
implies that the evolution of pore-forming colicins
resembles that of colicins with nuclease activity [1,13].
Materials and methods
Bacterial strains and growth conditions
Bacterial strains producing colicins B, U and Y, bacterial
strains immune to colicins and other bacterial strains and
plasmids used in this study are shown in Table 1. Bacterial
strains were cultivated at 37 C in TY medium [8.0 g tryp-
tone (HiMedia, Mumbai, India), 5.0 g yeast extract (HiMe-
dia) and 5.0 g NaCl per litre, pH 7]. For the selection and
maintenance of the plasmids, 100 lg of ampicillin or 25 lg
AB
Fig. 2. Model of the Cya–Cyi intramembrane interaction shown
from two perpendicular views (A, B). A representative structure,
selected from a bundle of five lowest energy structures, character-
ized by the backbone atom rmsd of 2.85 A
˚to the mean structure,
is shown. The blue and red ribbons represent a-helix 8 of Cya (from
I569 to G590) and a-helix T3 of Cyi (I101 to N121), respectively.
Colicin Y residues responsible for the interaction, I578, T582 and
Y586, as well as Cyi residues S104 and S107, are shown in a ball-
and-stick representation on the ribbons. Both helices form an angle
of approximately 20between the helical axes, which ensures a
close contact between the helices [12].
Table 4. Sensitivity of E. coli DH10B and DH10B pDS519 to colic-
ins A, B, N, S4 and Y. Identical amino acid residues are shown in
bold.
Colicin
Amino acid residue
corresponding to
position in colicin Y Sensitivity
of E. coli
DH10B
Immunity of E. coli
DH10B expressing
cyi gene
578 582 586 590
YITYV3(5)
a
–(1)
AITYL 3(5) 1(3)
S4 V TYL –(5) –(3)
BLTA F 2(3) 1(3)
N L V L – 3(5) 3(5)
–, no inhibition resulting in clear zones of growth inhibition.
a
The numbers indicate the highest colicin dilution resulting in clear
(turbid) zones of growth inhibition on the indicator bacteria (e.g.
5=10
)5
).
D. S
ˇmajs et al. Helix–helix interaction between colicin Y and Cyi
FEBS Journal 275 (2008) 5325–5331 ª2008 The Authors Journal compilation ª2008 FEBS 5329