Báo cáo Y học: Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction ppt

Investigations into the mechanisms used by the C-terminal anchors
of
Escherichia coli
penicillin-binding proteins 4, 5, 6 and 6b
for membrane interaction
Frederick Harris
1
, Klaus Brandenburg
2
, Ulrich Seydel
2
and David Phoenix
1
1
Department of Forensic and Investigative Science, University of Central Lancashire, Preston, UK;
2
Division of Biophysics,
Forschunginstitute Borstel, Borstel, Germany
Escherichia coli low molecular mass penicillin-binding pro-
teins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evi-
dence suggests that these proteins interact with the inner
membrane via C-terminal amphiphilic a-helices. Nonethe-
less, the membrane interactive mechanisms utilized by the
C-terminal anchors of PBP4 and PBP6b show differences to
those utilized by PBP5 and PBP6. Here, hydrophobic
moment-based analyses have predicted that, in contrast to
the PBP4 and PBP6b C-termini, those of PBP5 and PBP6
are candidates to form oblique orientated a-helices. Con-
sistent with these predictions, Fourier transform infrared
spectroscopy (FTIR) has shown that peptide homologs of
the PBP4 and PBP5 C-terminal regions, P4 and P5,

respectively, both possessed the ability to adopt a-helical
structure in the presence of lipid. However, whereas P4
appeared to show a preference for interaction with the sur-
face regions of dimyristoylglycerophosphoethanolamine
and dimyristoylglycerophosphoglycerol membranes, P5
appeared to show deep penetration of both these latter
membranes and dimyristoylglycerophosphocholine mem-
branes. Based on these results, we have suggested that in
contrast to the membrane anchoring of the PBP4 and PBP6b
C-terminal a-helices, the PBP5 and PBP6 C-terminal
a-helices may possess hydrophobicity gradients and penet-
rate membranes in an oblique orientation.
Keywords: penicillin-binding protein; C-terminal a-helix;
hydrophobicity gradient; membrane.
The Escherichia coli, low molecular mass penicillin-binding
proteins (PBPs) include PBP4, PBP5, PBP6 [1,2], PBP6b [3],
PBP7 and PBP8 [4,5]. These proteins are penicillin sensitive
DD-peptidases [6,7] that are believed to play a role in the
final stages of petidoglycan manufacture [8–11]. PBP7 and
PBP8 are soluble proteins [4,5] but it has been established
that in nonoverproducing systems, PBP4, PBP5 and PBP6
are anchored to the periplasmic face of the inner membrane
[7,12] whilst a similar membrane location has been sugges-
ted for PBP6b [3]. Nonetheless, hydropathy plot analysis for
each of these membrane-associated PBPs shows no con-
ventional hydrophobic anchor sequences, nor did there
appear to be any evidence of covalent modification and
the membrane anchoring mechanisms of these proteins
remained unclear. Deletion analysis showed that the
C-terminal region of PBP5 [13,14] and PBP6 [15] were

essential for efficient membrane interaction whilst CD
analysis showed that a peptide homolog of the PBP5
C-terminal region was able to adopt high levels of a-helical
structure [16]. Furthermore, incorporation of a proline
residue into the PBP5 C-terminal region, with its ability to
disrupt or distort a-helical structure, greatly destabilized the
membrane anchoring of the protein [17] whilst fusion of the
PBP5 C-terminal region to a periplasmic b-lactamase led to
a membrane bound form of the enzyme [18].
A number of authors have used theoretical analysis to
investigate the potential of the PBP4, PBP5, PBP6 and
PBP6b C-terminal regions for membrane interaction and
based on these analyses, it would appear that these
C-terminal regions form two distinguishable subgroups.
Both hydrophobic moment-based analyses [19,20] and
DWIH analysis [21] have predicted that the PBP5 and
PBP6 C-terminal regions would form strongly amphiphilic
a-helices and in both cases, these predictions appear to be
supported by experimental results, which found that peptide
homologs of these regions were strongly hemolytic [22] and
showed high levels of lipid monolayer penetration [23]. In
contrast, similar theoretical analyses have predicted that the
PBP4 and PBP6b C-terminal regions would form weakly
amphiphilic a-helices [19,21,24]. In the case of the PBP4
C-terminal region, these predictions could be supported by
experimental results, which found that a peptide homolog of
this region possessed no hemolytic ability [22] and low levels
of lipid monolayer penetration [23]. In addition, hydropho-
bic moment profile analysis has predicted that the PBP4
C-terminal region possesses an almost equal potential to

Correspondence to D. Phoenix, Department of Forensic and
Investigative Science, University of Central Lancashire,
Preston, PR1 2HE, UK.
Fax: + 44 1772 894964, Tel.: + 44 1772 893481,
E-mail:
Abbreviations:Myr
2
-PGro, dimyristoylglycerophosphoglycerol;
Myr
2
-PCho, dimyristoylglycerophosphocholine; Myr
2
-PEtn,
dimyristoylglycerophosphoethanolamine; FTIR, Fourier transform
infrared spectroscopy; PBPs, penicillin-binding proteins; SUV, small
unilamellar vesicle.
Enzymes: Escherichia coli PBP4 (EC 3.4.16.4; EC 3.4.99-);
E. coli PBP5 (EC 3.4.16.4); E. coli PBP6 (EC 3.4.16.4);
E. coli PBP6b (EC 3.4.16.4).
(Received 10 May 2002, revised 24 September 2002,
accepted 7 October 2002)
Eur. J. Biochem. 269, 5821–5829 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03295.x
interact with membranes via either amphiphilic b-sheet or
amphiphilic a-helical secondary structures [19]. Based on
these results, it has been suggested that PBP4 and PBP6b
may utilize mechanisms of membrane interaction, which
differ to those utilized by PBP5 and PBP6 [1,2,19,21,24].
Here, we have considered the possibility that differences
between these anchoring mechanisms may lay in the ability
of the low molecular mass PBPs to form oblique orientated

a-helices in their C-terminal regions. Recently confirmed by
experimental results [25,26], these are a class of a-helices
whose lipid interactions are predicted to involve penetration
of the membrane at a shallow angle due to a hydrophobicity
gradient along the a-helical long axis [27–30]. Using
graphical and hydrophobic moment-based analyses, we
have examined the PBP4, PBP5, PBP6 and PBP6b
C-terminal sequences to identify candidate oblique orienta-
ted a-helix forming segments. In an effort to confirm this
potential, we then used FTIR spectroscopy to investigate
the ability of the PBP4 and PBP5 C-terminal sequences to
adopt secondary structure at a lipid interface and for lipid
interaction. Conformational analyses of peptide homologs
of these sequences, P4 and P5, respectively, were performed
in the presence of vesicles formed from either: Myr
2
-PGro,
Myr
2
-PCho or Myr
2
-PEtn. FTIR spectroscopy was then
used to monitor the effects of P4 and P5 on the phase transi-
tion temperature and membrane fluidity of membranes
formed from either: Myr
2
-PGro, Myr
2
-PCho or Myr
2

-PEtn.
MATERIALS AND METHODS
The identification of candidate oblique orientated
a-helix forming segments
The primary sequence of the influenza viral fusion peptide,
HA2, a known oblique orientated a-helix former [31–33]
and those of the PBP4, PBP5, PBP6 and PBP6b C-terminal
regions (Table 1) were analyzed according to conventional
hydrophobic moment methodology [34]. The hydropho-
bicity of successive amino acids in these sequences are
treated as vectors and summed in two dimensions, assuming
anaminoacidsidechainperiodicityof100°. The resultant
of this summation, the hydrophobic moment, l
H
,provides
a measure of a-helix amphiphilicity. Our analysis used a
moving window of 11 residues and for each sequence under
investigation (Table 1), the window with the highest
hydrophobic moment was identified (Table 1). For these
windows, the mean hydrophobic moment, Æl
H
æ,andthe
corresponding mean hydrophobicity, ÆH
0
æ (Table 1), were
computed using the normalized consensus hydrophobicity
scale of Eisenberg et al. [35] and plotted on the hydrophobic
moment plot diagram [36], as modified by Harris et al. [28]
(Fig. 1).
Table 1. The primary sequences and hydrophobic moment parameters of protein segments. The C-terminal sequences of PBP4 [44], PBP5 and PBP6

[45], PBP6b [3] and the primary sequence of the HA2 fusion peptide [33] were analyzed using hydrophobic moment methodology [34]. Eleven
residue windows of maximum amphiphilicity were identified (shown in bold) and are shown, along with their corresponding Æl
H
æ and ÆH
0
æ.
Segment Primary sequence Æl
H
æÆH
0
æ
+++
) ++))
PBP4 C-terminus RRIPLVRFESRLYKDIYQNN-COO 0.75 )0.31
+
) +++ )
PBP5 C-terminus GNFFGKIIDYIKLMFHHWFG-COO 0.67 0.24
+
) +++ )
PBP6 C-terminus GGFFGRVWDFVMMKFHHWFGSWFS-COO 0.51 0.42
)) + ) +++ )
PBP6b C-terminus LVTLESVGEGSMFSRLSDYFHHKA-COO 0.59 )0.03
)) )
HA2 fusion peptide GLFGAIAGFIENGWEGMIDG 0.65 0.23
Fig. 1. The hydrophobic moment plot diagram. The conventional
hydrophobic moment plot diagram of [36] with an overlaid gray region
delineating candidate oblique orientated a-helices [28] are shown. The
sequences shown in Table 1 were plotted on the diagram according to
their Æl
H

æ and corresponding ÆH
0
æ values (Table 1). Data points rep-
resentingtheC-terminalsequencesofPBP5(2)andPBP6(3)canbe
seen to lie in the gray region, proximal to that representing the HA2
peptide (5), indicating that these C-terminal sequences may be candi-
dates for oblique orientated a-helix formation. The data points
representing the C-terminal sequences of PBP4 (1) and PBP6b (4) lie
outside this area and are unlikely to adopt such structure.
5822 F. Harris et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Materials
The peptides P4 and P5 (Table 1) were supplied by
PEPSYN, University of Liverpool, UK, produced by solid
state synthesis and purified by HPLC to a purity of greater
than 99%. The peptides were stored as 1
M
aqueous stock
solutions at 4 °C. Myr
2
-PGro, Myr
2
-PCho and Myr
2
-PEtn,
and all solvents, which were of spectroscopic grade, were
purchased from Sigma (UK).
Preparation of phospholipid small unilamellar vesicles
Small unilamellar vesicles (SUVs) were prepared accord-
ing to Keller et al. [37]. Essentially, lipid/chloroform
mixtures were dried with nitrogen gas and hydrated with

aqueous Hepes at pH 7.5 to give final phospholipid
concentrations of 50 m
M
. The resulting cloudy suspensions
were sonicated at 4 °C with a Soniprep 150 sonicator
(amplitude 10 lm) until clear suspensions resulted (30
cycles of 30 s), which were then centrifuged (15 min,
3000 g,4°C).
FTIR conformational analyses of P4 and P5
To give a final peptide concentration of 1 m
M
,eitherP4
or P5 were solubilized in either aqueous buffer (50 m
M
Hepes; pH 7) or suspensions of SUVs, which were formed
from either: Myr
2
-PGro, Myr
2
-PCho or Myr
2
-PEtn, and
were prepared as described above. Samples of solubilized
peptide were spread on a CaF
2
crystal, and the free excess
water was evaporated at room temperature. The single
band components of the P4 or P5 amide I vibrational
band (predominantly C¼O stretch) was monitored using
an FTIR Ô5-DXÕ spectrometer (Nicolet Instruments,

Madison, WI, USA).
Analysis of FTIR spectra
FTIR spectra were analyzed and for those with strong
absorption bands, the evaluation of the band parameters
(peak position, band width and intensity) was performed
with the original spectra, if necessary after the subtraction of
strong water bands. In the case of spectra with weak
absorption bands, resolution enhancement techniques
such as Fourier self-deconvolution [38] were applied after
baseline subtraction with the parameters: bandwidth,
22–28 cm
)1
, resolution enhancement factor, 1.2–1.4 and
Gauss/Lorentz ratio of 0.55. In the case of overlapping
bands, curve fitting was applied using a modified version of
the
CURFIT
procedure written by D. Moffat (National
Research Council, Ottowa, Canada). An estimation of the
number of band components was obtained from deconvo-
lution of the spectra, curve fitting was then applied within
the original spectra after the subtraction of baselines
resulting from neighboring bands. Similar to the deconvo-
lution technique, the bandshapes of the single components
are superpositions of Gaussian and Lorentzian bandshapes.
Best fits were obtained by assuming a Gauss fraction of
0.55–0.6. The
CURFIT
procedure measures the peak areas of
single band components and after statistical evaluation,

determines the relative percentages of primary structure
involved in secondary structure formation. For P4 and P5,
relative levels of a-helical structure (1650–1655 cm
)1
)and
b-sheet structures (1625–1640 cm
)1
) were computed and are
showninTable2.
FTIR analysis of phospholipid phase transition
properties
Using FTIR spectroscopy, the effects of either P4 or P5 on
the phase transition properties of phospholipid was inves-
tigated. To give a final peptide concentration of 1 m
M
,
either P4 or P5 was solubilized in suspensions of SUVs
formed from: either Myr
2
-PGro, Myr
2
-PCho or Myr
2
-
PEtn, which were prepared as described above. As controls,
SUVs formed from: either Myr
2
-PGro, Myr
2
-PCho or

Myr
2
-PEtn alone were prepared as described above. These
samples were then placed in a calcium fluoride cuvette,
separated by a 12.5-lm thick Teflon spacer and subjected to
automatic temperature scans with a heating rate of 3 °C
5min
)1
within the temperature range 0 to 60 °C. For every
3 °C interval, 50 interferograms were accumulated, apo-
dized, Fourier transformed and converted to absorbance/
temperature spectra [39] (Figs 3 and 6). These spectra
monitored changes in the b fi a acyl chain melting
behavior of phospholipids with these changes determined
as shifts in the peak position of the symmetric stretching
vibration of the methylene groups, m
s
(CH
2
), which is known
to be a sensitive marker of lipid order. The peak position of
m
s
(CH
2
) lies at 2850 cm
)1
in the gel phase and shifts at a
lipid specific temperature T
c

to 2852.0 cm
)1
)2852.5 cm
)1
in
the liquid crystalline state.
RESULTS
The identification of candidate oblique orientated
a-helix forming segments
The sequences shown in Table 1 were plotted on the
modified hydrophobic moment plot diagram (Fig. 1)
according to their Æl
H
æ and ÆH
0
æ values (Table 1). Data
points representing the PBP5 and PBP6 C-terminal
sequences are seen to lay within the shaded area, proximal
to that representing the sequence of HA2, a known oblique
Table 2. P4 and P5 secondary structural levels in the presence of lipid.
Levels of secondary structure determined for P4 and P5. FTIR con-
formational analysis of P4 and P5 were performed with each peptide
either: in aqueous solution (–) or in the presence of either: dimyristoyl
phosphatidylcholine (Myr
2
-PCho), dimyristoyl phosphatidylethanol-
amine (Myr
2
-PEtn), or dimyristoyl phosphatidylglycerol (Myr
2

-
PGro). For spectra produced (Figs 2 and 5), the peak areas of single
band components were used to determine the relative percentages of
primary structure involved in secondary structure formation.
Peptide Lipid
a-helical
structures (%)
b-sheet
structures (%)
P4 – 0 85
Myr
2
-PCho 0 85
Myr
2
-PEtn 37 63
Myr
2
-PGro 20 77
P5 – 58 42
Myr
2
-PCho 48 52
Myr
2
-PEtn 56 44
Myr
2
-PGro 43 57
Ó FEBS 2002 Membrane anchoring by E. coli PBPs 4, 5, 6 and 6b (Eur. J. Biochem. 269) 5823

orientated a-helix former. These observations indicate that
the PBP5 and PBP6 C-terminal sequences are candidate
oblique orientated a-helix forming segments. However, data
points representing the PBP4 and PBP6b C-terminal regions
are seen to lay outside the shaded area, indicating that these
sequences are unlikely to form oblique orientated a-helices
(P > 0.01 confidence).
FTIR conformational analysis of peptides
FTIR spectroscopy was used to perform conformational
analyses of P4 and P5 either in aqueous solution or in the
presence of SUVs. A typical overview spectrum for these
peptide–lipid systems is shown in Fig. 4, which represents
absorbance by the P4-Myr
2
-PCho system within the
spectral range 1800–1100 cm
)1
. The spectrum comprises
lipid vibrational bands such as the ester double bond
stretching at 1738 cm
)1
,themethylenechainscissoring
mode at 1464 cm
)1
, and the phosphate antisymmetric
stretching at 1240–1200 cm
)1
, and the peptide bands, amide
I (predominantly C¼O stretching) and amide II (predo-
minantly N–H bending). Figures 2 and 5 show spectra for

P4 and P5 absorbance in the spectral range of the amide I
band and from these spectra, the relative levels of peptide
secondary structure were determined as a percentage of
primary structure (Table 2). The major contribution to P4
molecular architecture came from b-sheet structures,
ranging from 63% in the presence of Myr
2
-PEtn to 85%
in the aqueous peptide. Nonetheless, the peptide adopted
significant levels of a-helical structure in the presence of
both Myr
2
-PEtn(37%)andMyr
2
-PGro (20%) although
showing no evidence of such structure either in the presence
of Myr
2
-PCho or in aqueous solution (Table 2). In contrast,
P5 showed high levels of a-helical structure in aqueous
solution (58%), which were generally maintained in the
presence of Myr
2
-PCho, Myr
2
-PEtn and Myr
2
-PGro and
ranged between 43% and 56%.
The effect of proteins on phospholipid phase

transition temperature
Using FTIR spectroscopy, absorbance spectra representing
the effects of either P4 or P5 on the phase transition
temperature and membrane fluidity of membranes formed
from either: Myr
2
-PCho, Myr
2
-PGro or Myr
2
-PEtn, were
derived as a function of temperature (Figs 3 and 6). Control
experiments recorded the T
c
of both Myr
2
-PGro and Myr
2
-
PCho membranes as 25 °C and that of Myr
2
-PEtn mem-
branes as 47 °C (Figs 3A–C and 6A)C). In the presence of
P4, no significant changes in either the membrane fluidity or
the T
c
of Myr
2
-PCho membranes were detected (Fig. 3A).
Similarly, the presence of P4 appeared to have no significant

effect on Myr
2
-PEtn membrane fluidity but did appear to
Fig. 2. FTIR conformational analyses of P5 in the presence of lipid.
(A–D) show FTIR conformational analyses of P5 in the presence of
Myr
2
-PCho, Myr
2
-PEtn and Myr
2
-PGro and in aqueous solution,
respectively. In each case, the major contribution to P5 came from
a-helical structure (1650–1655 cm
)1
) although significant levels of
b-sheet structures (1625–1640 cm
)1
) can also be seen. In all cases,
annotated numbers indicate band peak absorbances.
5824 F. Harris et al. (Eur. J. Biochem. 269) Ó FEBS 2002
have a significant effect on the T
c
of the lipid, with T
c
being
recorded as 42 °C in the presence of the peptide (Fig. 3B).
The presence of P5 had a strong effect on the T
c
and

membrane fluidity of both Myr
2
-PCho and Myr
2
-PEtn
membranes with T
c
being recorded as 13 and 42 °C,
respectively, and in each case, the change was accompanied
by a concomitant increase in membrane fluidity (Fig. 6A,B).
In contrast, in the presence of either P4 or P5, Myr
2
-PGro
membranes showed minor increases in gel phase fluidity,
minor decreases in liquid crystalline phase fluidity with the
gel to fluid phase transition occurring over the interval 20 to
30 °C rather than the 25 °C of the pure lipid (Figs 3C and
6C).
DISCUSSION
Here, we analyzed the C-terminal sequences of PBP4, PBP5,
PBP6 and PBP6b to identify candidates with the potential
to form oblique orientated a-helices [27,30] and based on
their Æl
H
æ ,andÆH
0
æ values, our analyses showed that these
sequence formed two subgroups. The C-terminal regions of
PBP4andPBP6bwerepredictedtobeunlikelytoform
oblique orientated a-helices. However, the C-terminal

regions of PBP5 and PBP6 were predicted to be candidates
for the formation of such a-helices and are similar to the
viral fusion peptide, HA2 (Fig. 1), a peptide shown to
penetrate membranes via an oblique orientated a-helix. The
C-terminal a-helices of PBP5 and PBP6 show many
structural resemblances to the HA2 oblique orientated
a-helix. It can be seen from Fig. 7 that each of these
a-helices possesses a wide hydrophobic face, which includes
bulky tryptophan, phenylaniline and isoleucine amino acid
residues, and a glycine rich hydrophilic face. These struc-
tural features give a-helices an effective wedge shape, which
appears to be utilized by HA2, and a number of other
oblique orientated a-helix forming peptides, to destabilize
membranes, leading to membrane fusion [40,41]. Further-
more, Roberts et al. [21] analyzed the PBP5 and PBP6
C-terminal a-helices according to DWIH methodology and
Fig. 3. FTIR lipid phase transition analysis of P4. (A–C) show the
effect of P4 on the b fi a acyl chain melting behavior of Myr
2
-PCho,
Myr
2
-PEtn and Myr
2
-PGro membranes, respectively, which were
monitored by FTIR spectroscopy as a function of temperature. The T
c
of Myr
2
-PCho membranes alone (j) was recorded as 25 °Candinthe

presence of P4 (d), no significant changes in either the T
c
or the
membrane fluidity of the membrane was detected (A). The T
c
of Myr
2
-
PEtn membranes alone was recorded as 47 °C(j) and although in the
presence of P4 (d), no significant effect on the fluidity of the membrane
was detected, the T
c
of the membrane was recorded as 42 °C(B).The
T
c
of Myr
2
-PGro membranes alone (j) was recorded as 25 °Cwhilst
inthepresenceofP5(d), phase transition occurred over the interval
20 °Cto30°C accompanied by an increase in gel phase fluidity and a
decrease in liquid crystalline phase fluidity (C).
Fig. 4. FTIR overview spectrum of P4 in the presence of Myr
2
-PCho.
The peak absorbances for lipid vibrational bands such as the ester
double bond stretching at 1738 cm
)1
,themethylenechainscissoring
mode at 1464 cm
)1

, and the phosphate antisymmetric stretching at
1240–1200 cm
)1
, and the peptide bands amide I (predominantly C¼O
stretching) and amide II (predominantly N–H bending) are shown.
Ó FEBS 2002 Membrane anchoring by E. coli PBPs 4, 5, 6 and 6b (Eur. J. Biochem. 269) 5825
showed that the nature and order of the amino acid residues
forming these a-helices were highly significant. This is
consistent with the ordered spatial arrangement of amino
acid residues necessary to maintain the hydrophobicity
gradients of oblique orientated a-helices [29]. In contrast to
the PBP5 and PBP6 C-terminal regions, it can be seen from
Fig. 7 that, in an a-helical conformation, the C-terminal
regions of PBP4 and PBP6b show ill-defined hydrophobic
faces and few structural resemblances to the HA2 a-helix.
These observations reinforce the suggestion that there
would be differences between the C-terminal membrane
interactions for PBPs from the two subgroups.
The PBP4 and PBP5 C-terminal anchor sequences were
taken to represent each of these subgroups and the
secondary structural features of these sequences in the
presence of lipid have been investigated. FTIR conforma-
tional analysis showed that both in aqueous solution and
in the presence of each lipid examined, over 60% of
P4 architecture was formed from b-sheet structures
(Fig. 5A–D; Table 2). Nonetheless, in the presence of both
Myr
2
-PEtn and Myr
2

-PGro (Fig. 5B,C; Table 2), the
peptide adopted significant levels of a-helical structure
(37% and 20%, respectively) although showing no evidence
of such structure either in the presence of Myr
2
-PCho
(Fig. 5A; Table 2) or in aqueous solution (Fig. 5D;
Table 2). In contrast, P5 architecture showed high levels
of a-helical structure, of the order of 50%, both in aqueous
solution (Fig. 2D; Table 2) and in the presence of each lipid
examined (Fig. 2A–C; Table 2). Both P4 and P5 were found
to affect the lipid phase transition properties of Myr
2
-PEtn
(Figs 3B and 6B) and Myr
2
-PGro (Figs 3C and 6C).
However, whilst P5 was found to affect the lipid phase
transition properties of Myr
2
-PCho (Fig. 6A) no such effects
were detected in the case of P4 (Fig. 3A). In combination,
these results would support the hypothesis that the ability of
P4 and P5 to interact with lipid membranes is related to the
ability of each peptide to adopt amphiphilic a-helical
structure. Furthermore, these results are consistent with
those of Brandenburg et al. [42] and suggest that the ability of
P4 to adopt such structure may be related to the character-
istics of the interface rather than solely the lipid type.
Our FTIR lipid phase transition analyses showed that the

presence of both P4 and P5 led to a broadening of the T
c
of
Myr
2
-PGro membranes (25 °C) with phase transition
occurring over a temperature range (20–30 °C) accompan-
ied by an increases in gel phase fluidity and decreases in
liquid crystalline phase fluidity (Figs 3C and 6C), This form
of phase transition shows similarities to that of some
cholesterol–lipid systems [43] and implies that the presence
of either P4 or P5 leads to changes in the hydrocarbon chain
packing of Myr
2
-PGro membrane, which result in fluidiza-
tion of the gel phase and rigidification of the liquid
Fig. 5. FTIR conformational analyses of P4 in the presence of lipid.
(A–D) show FTIR conformational analyses of P4 in the presence of
Myr
2
-PCho, Myr
2
-PEtn, Myr
2
-PGro and in aqueous solution,
respectively. In each case, the major contribution to P4 came from
b-sheet structures (1625–1640 cm
)1
). Significant levels of a-helical
structure (1650–1655 cm

)1
) can be seen in the presence of Myr
2
-PEtn
(B) and Myr
2
-PGro (C) but there is no evidence of such structure in the
presence of Myr
2
-PCho (A) or in aqueous solution (D). In all cases,
annotated numbers indicate band peak absorbances.
5826 F. Harris et al. (Eur. J. Biochem. 269) Ó FEBS 2002
crystalline phase. These results do not necessarily mean that
P4 and P5 interact with the Myr
2
-PGro acyl chains region
and in isolation, do not allow a clear interpretation as to the
nature of P4 and P5 interaction with Myr
2
-PGro mem-
branes. Even so, these results clearly show that there is some
level of Myr
2
-PGro membrane penetration by the peptides.
The presence of P4 had no effect on the lipid phase
transition properties of Myr
2
-PCho (Fig. 3A) and no effect
on the membrane fluidity of Myr
2

-PEtn membranes,
although a 5 °C decrease in the T
c
of Myr
2
-PEtn was
observed (Fig. 3B). P5 was found to interact strongly with
Myr
2
-PCho membranes (Fig. 6A) and Myr
2
-PEtn mem-
branes (Fig. 6B) with the presence of the peptide leading to
increased membrane fluidity in both cases, accompanied by
decreases in membrane T
c
of 12 and 5 °C, respectively.
Taken overall, these results clearly show that there are
fundamental differences between the mechanisms of mem-
brane penetration utilized by P4 and P5. P4 shows limited
levels of membrane penetration and would appear to prefer
to interact with the membrane’s surface regions whilst P5
has a preference to interact with the membrane’s lipid acyl
chains.
Taken in combination, our experimental results are
consistent with our suggestion that the PBP5 and PBP6
C-terminal a-helices may be able to penetrate the membrane
lipid core region in an oblique orientation. This form of
membrane penetration would be in accord with the high
levels of interaction shown here by P5 for zwitterionic

membranes. Furthermore, this form of membrane penetra-
tion could explain the high levels of hemolysis shown by
both this peptide and P6, a peptide homolog of the PBP6
C-terminal region [22] for HA2 is hemolytic yet abolition of
the peptide’s hydrophobicity gradient leads to loss of
hemolytic and fusogenic ability [31]. Given the apparent
preference shown by P4 for the membrane’s surface regions,
this suggests that the peptide’s cationic region(s) would
interact with negatively charged moieties within this region.
Nonetheless, taking our results overall, we speculate that
such an interaction would be weak and unlikely to play a
Fig. 6. FTIR lipid phase transition analysis of P5. (A–C) show the
effect of P5 on the b fi a acyl chain melting behavior of Myr
2
-PCho,
Myr
2
-PEtn and Myr
2
-PGro membranes, respectively, which were
monitored by FTIR spectroscopy as a function of temperature. The T
c
of Myr
2
-PCho membranes alone (j) was recorded as 25 °Cwhilstin
the presence of P5 (.)theT
c
of the membrane was recorded as 13 °C,
accompanied by an increase in membrane fluidity (A). The T
c

of Myr
2
-
PEtn membranes alone (j) was recorded as 47 °C whilst in the
presence of P5 (.)theT
c
of the membrane was recorded as 42 °C,
accompanied by an increase in membrane fluidity (B). The T
c
of Myr
2
-
PGro membranes alone (j) was recorded as 25 °C whilst in the
presence of P5 (.), phase transition occurred over the interval 20 to
30 °C accompanied by an increase in gel phase fluidity and a decrease
in liquid crystalline phase fluidity (C).
Fig. 7. Helical wheel representations of protein segments. The C-ter-
minal sequences of PBP4, PBP5, PBP6 and PBP6b, and the primary
sequence of the HA2 fusion peptide (Table 1) modeled as a-helices
according to the methodology of Schiffer and Edmundson [46], assu-
ming an angular periodicity of 100° are shown. The a-helices of HA2,
PBP5 and PBP6 show well defined amphiphilicity and, in common,
possess glycine rich hydrophilic faces with wide hydrophobic faces rich
in bulky amino acid residues. The a-helices of PBP4 and PBP6b show
ill-defined faces and few structural resemblances to that of HA2.
Ó FEBS 2002 Membrane anchoring by E. coli PBPs 4, 5, 6 and 6b (Eur. J. Biochem. 269) 5827
major role in the membrane anchoring mechanism of PBP4.
Indeed, experimental evidence has been presented which
suggests that the membrane interactions of PBP4 may
involve occupation of a specific binding site [12] and

protein–protein interactions [8–11].
In summary, our results show that the PBP4 C-terminal
sequence is able to adopt a-helical and b-sheet structure in
the presence of lipid and may weakly associate with the
membrane lipid headgroup region via predominantly elec-
trostatic interactions. In contrast, our results suggest that
the PBP5 C-terminal region possesses a strong intrinsic
tendency to both adopt a-helical structure and to penetrate
the membrane hydrophobic core region. It appears that this
C-terminal a-helix, and that formed by PBP6, possess
hydrophobicity gradients, which we suggest may facilitate
membrane penetration in an oblique orientation.
REFERENCES
1. Harris, F. (1998) Investigation into the membrane interactive
properties of the Escherichia coli low molecular mass penicillin-
binding proteins. Thesis. University of Central Lancashire, Preston,
Lancashire, UK.
2. Phoenix, D.A. & Harris, F. (1998) Amphiphilic a–helices and lipid
interactions. In Protein Targeting and Translocation (Phoenix,
D.A., ed.), pp. 19–36. Portland Press, London.
3. Baqeuro, M.R., Bouzon, M., Quintela, J.C., Ayala, J.A. & Mor-
eno, F. (1996) dacD,anEscherichia coli gene encoding a novel
penicillin-binding protein (PBP6b) with DD-carboxypeptidase
activity. J. Bacteriol. 178, 7106–7111.
4. Henderson, T.A., Templin, M. & Young, K.D. (1995) Identifi-
cation and cloning of the gene encoding penicillin-binding protein
7ofEscherichia coli. J. Bacteriol. 177, 2074–2079.
5. Henderson, T.A., Dombrosky, P.M. & Young, K.D. (1994)
Artifactual processing of penicillin-binding protein 7 and 1b by the
OmpT protease of Escherichia coli. J. Bacteriol. 176, 256–259.

6. Phoenix, D.A. & Harris, F. (1995) The membrane interactive
properties of the low molecular weight penicillin-binding proteins.
Biochem. Soc. Trans. 23, 976–980.
7. Gittins, R.G., Phoenix, D.A. & Pratt, J.M. (1993) Multiple
mechanisms of membrane anchoring of Escherichia coli penicillin
binding proteins. FEMS Microbiol. Rev. 13, 1–12.
8. Ehlert, K. & Holtje, J V. (1996) Role of precursor translocation in
coordination of murein and phospholipid synthesis in Escherichia
coli. J. Bacteriol. 178, 6766–6771.
9. Holtje, J V. (1996) A hypothetical holoenzyme involved in the
replication of the murein sacculus of Escherichia coli. Microbiology
142, 1911–1918.
10. Holtje, J V. (1996) Molecular interplay of murein synthases and
murein hydrolases in Escherichia coli. Microbial Drug Resistance
Mechanisms Epidemol. Dis. 2, 99–103.
11. Holtje, J V. (1995) From growth to autolysis – the murein
hydrolases in Escherichia coli. Arch. Microbiol. 164, 243–254.
12. Harris, F., Demel, R.A., Phoenix, D.A. & De Kruijff, B. (1997)
Membrane binding of Escherichia coli penicillin-binding protein 4
is predominantly electrostatic in nature and occurs at a specific
binding site. Prot. Peptide, Lett. 5, 63–66.
13. Jackson, M.E. & Pratt, J.M. (1987) An 18 amino acid amphiphilic
helix forms the membrane anchoring domain of the Escherichia
coli penicillin binding protein 5. Mol. Microbiol. 1, 23–28.
14. Pratt, J.M., Jackson, M.E. & Holland, I.B. (1986) The C-terminus
of penicillin-binding protein 5 is essential for localisation to the
Escherichia coli inner membrane. EMBO J. 5, 2399–2405.
15. Van der Linden, M.P.G., de Haan, L., Hoyer, M.A. & Keck, W.
(1992) Possible role of Escherichia coli penicillin-binding protein 6
in stabilisation of stationary-phase peptidoglycan. J. Bacteriol.

174, 7572–7578.
16. Siligardi, G., Harris, F. & Phoenix, D.A. (1997) Alpha-helical
conformation in the C-terminal anchoring domains of E. coli
penicillin-binding proteins 4, 5 and 6. Biochim. Biophys. Acta 1329,
278–284.
17. Jackson, M.E. & Pratt, J.M. (1988) Analysis of the membrane-
binding domain of penicillin-binding protein 5 of Escherichia coli.
Mol. Microbiol. 2, 563–568.
18. Phoenix, D.A. & Pratt, J.M. (1993) Membrane interaction of
Escherichia coli penicillin-binding protein 5 is modulated by the
ectomembranous domain. FEBS Lett. 322, 215–218.
19. Pewsey, A.R., Phoenix, D.A. & Roberts, M.G. (1996) Monte
Carlo analysis of potential C-terminal membrane interactive
a-helices. Prot. Peptide, Lett. 3, 185–192.
20. Phoenix, D.A. (1990) Investigation into structural features of the
Escherichia coli penicillin-binding protein 5 C-terminal anchor.
Biochem. Soc. Trans. 18, 948–949.
21. Roberts, M.G., Phoenix, D.A. & Pewsey, A.R. (1997) An
algorithm for the detection of surface-active a-helices with the
potential to anchor proteins at the membrane interface. CABIOS
13, 99–106.
22. Harris, F. & Phoenix, D.A. (1997) An investigation into the ability
of C-terminal homologues of the Escherichia coli low molecular
mass penicillin-binding proteins 4, 5 and 6 to undergo membrane
interaction. Biochemie 79, 171–174.
23. Harris, F., Demel, R.A., Phoenix, D.A. & De Kruijff, B. (1998)
An investigation into the lipid interactions of peptides corre-
sponding to the C-terminal anchoring domains of Escherichia coli
penicillin-binding proteins 4, 5 and 6. Biochim. Biophys. Acta 1415,
10–22.

24. Phoenix, D.A. & Wallace, J. (2000) Analysis of the membrane
interactive potential of the Escherichia coli PBP6b C-terminus.
Prot. Peptide Lett. 7, 99–104.
25. Bradshaw, J.P., Darkes, M.J.M., Harroun, T.A., Katsaras, J. &
Epand, R.M. (2000) Oblique membrane insertion of viral fusion
peptides probed by neutron diffraction. Biochemistry 39, 6581–
6585.
26. Bradshaw, J.P., Darkes, M.J.M., Katsaras, J. & Epand, R.M.
(2000) Neutron diffraction studies of viral fusion peptides. Physica
B 276, 495–498.
27. Brasseur, R. (2000) Tilted peptides: a motif for membrane desta-
bilisation (hypothesis). Mol. Membr. Biol. 17, 31–40.
28. Harris, F., Wallace, J. & Phoenix, D.A. (2000) Use of the
Hydrophobic moment plot to aid the identification of oblique
orientated a-helices. Mol. Membr. Biol. 17, 201–207.
29. Decout, A., Labeur, C., Vanloo, B., Goethals, M., Vandekerck-
hove, J., Brasseur, R. & Rosseneu, M. (1999) Contribution of the
hydrophobicity gradient to the secondary structure and activity of
fusogenic peptides. Mol. Membr. Biol. 16, 37–246.
30. Brasseur, R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu,
M. (1997) Peptides in membranes: tipping the balance of stability.
TIBS 22, 167–171.
31. Plank, C., Zauner, W. & Wagner, E. (1999) Application of
membrane-active peptides for drug and gene delivery across cel-
lular membranes. Adv. Drug Delivery Rev. 34, 21–35.
32. Martin, I., Ruysschaert, J M. & Epand, R.E. (1999) Role of the
N-terminal peptides of viral envelope proteins in membrane
fusion. Adv. Drug Delivery Rev. 38, 233–255.
33. Han, X., Bushweller, J.H., Cafiso, D.S. & Tamm, L.K. (2001)
Membrane structure and fusion-triggering conformational change

of the fusion domain from influenza hemagluttin. Nat. Struct.
Biol. 8, 715–720.
34. Eisenberg, D., Weiss, R.M., Terwilliger, T.C. & Wilcox, W. (1982)
Hydrophobic moment and protein structure. Faraday Symp.
Chem. Soc. 17, 109–120.
5828 F. Harris et al. (Eur. J. Biochem. 269) Ó FEBS 2002
35. Eisenberg, D., Weiss, R.M. & Terwilliger, T.C. (1982) The helical
hydrophobic moment: a measure of the amphiphilicity of a helix.
Nature 299, 371–374.
36. Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. (1984)
Analysis of membrane and surface protein sequences with the
hydrophobic moment plot. J. Mol. Biol. 179, 125–142.
37. Keller, R.C., Killian, J.A. & De Kruijff, B. (1992) Anionic phos-
pholipids are essential for alpha-helix formation of the signal
peptide of prePhoE upon interaction with phospholipid vesicles.
Biochemistry 31, 1672–1677.
38. Kauppinen, J.K., Moffat, D.J., Mantsch, H.H. & Cameron, D.G.
(1981) Fourier self-deconvolution – a method for resolving
intrinsically overlapped bands. Appl. Spectrosc. 35, 271–276.
39. Brandenburg, K., Kusomoto, S. & Seydel, U. (1997) Conforma-
tional studies of synthetic lipid A analogues and partial structures
by infrared spectroscopy. Biochim. Biophys. Acta 1329, 183–201.
40. White, J.M. (1990) Viral and cellular membrane fusion proteins.
Annu. Rev. Physiol. 52, 675–697.
41. Fujii, G. (1999) To fuse or not to fuse: the effects of electrostatic
interactions, hydrophobic forces and structural amphiphilicity on
protein-mediated membrane destabilisation. Adv. Drug Delivery
Rev. 38, 257–277.
42. Brandenburg, K., Harris, F., Phoenix, D.A. & Seydel, U. (2001)
An FTIR investigation into the lipid interactions of pep-

tides corresponding to the C-terminal anchoring regions of
Escherichia coli penicillin-binding proteins 4 and 5. Biol. Membr.
18, 395–399.
43. Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P. & Slotte, J.P.
(2001) Cholesterol interactions with phospholipids in membranes.
Prog. Lipid Res. 41, 66–97.
44. Mottl, H., Terpstra, P. & Keck, W. (1991) Penicillin-binding
protein 4 of Escherichia coli shows a novel type of primary
structure among penicillin-interacting proteins. FEMS Lett. 78,
213–220.
45. Broome-Smith, J.K., Ionnidis, I., Edelman, A. & Spratt, B.G.
(1988) Nucleotide sequences of the penicillin-binding protein 5 and
6 genes of Escherichia coli. Nucleic Acids Res. 16, 1617.
46. Schiffer, M. & Edmundson, A.B. (1967) Use of helical wheels to
represent the structures of proteins and to identify segments with
helical potential. Biophys. J. 7, 121–135.
Ó FEBS 2002 Membrane anchoring by E. coli PBPs 4, 5, 6 and 6b (Eur. J. Biochem. 269) 5829