Domain V of m-calpain shows the potential to forman oblique-orientated a-helix, which may modulate the enzyme’s activity via interactions with anionic lipid Klaus Brandenburg1, Frederick
Trang 1Domain V of m-calpain shows the potential to form
an oblique-orientated a-helix, which may modulate the enzyme’s activity via interactions with anionic lipid
Klaus Brandenburg1, Frederick Harris2, Sarah Dennison2, Ulrich Seydel1and David Phoenix2
1
Division of Biophysics, Forschunginstitute Borstel, Germany;2Department of Forensic and Investigative Science, University of Central Lancashire, Preston, UK
The activity of m-calpain, a heterodimeric, Ca2+-dependent
cysteine protease appears to be modulated by membrane
interactions involving oblique-orientated a-helix formation
by a segment, GTAMRILGGVI, in the protein’s smaller
subunit Here, graphical and hydrophobic moment-based
analyses predicted that this segment may form an a-helix
with strong structural resemblance to the influenza virus
peptide, HA2, a known oblique-orientated a-helix former
Fourier transform infrared spectroscopy showed that a
peptide homologue of the GTAMRILGGVI segment, VP1,
adopted low levels of a-helical structure ( 20%) in the
presence of zwitterionic lipid and induced a minor decrease
(3C) in the gel to liquid-crystalline phase transition
tem-perature, TC, of the hydrocarbon chains of zwitterionic
membranes, suggesting interaction with the lipid headgroup
region In contrast, VP1 adopted high levels of a-helical
structure (65%) in the presence of anionic lipid, induced a large increase (10C) in the TCof anionic membranes, and showed high levels of anionic lipid monolayer penetration (DSP¼ 5.5 mNÆm)1), suggesting deep levels of membrane penetration VP1 showed strong haemolytic ability (LD50¼ 1.45 mM), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid mono-layers, was greatly reduced In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated a-helix and elec-trostatic interactions We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this a-helix
Keywords: domain V; hydrophobicity gradient; m-calpain; membrane; oblique-orientated a-helix
Calpains are a growing family [1] of structurally related
intracellular Ca2+-dependent cysteine proteases [2,3], with
calpain 10 the most recently characterized member [4] The
physiological functions of calpains are not fully understood
but they are believed to play important roles in such
processes as cytoskeletal remodelling, cell differentiation,
apoptosis and signal transduction [5–7] Calpains are also of
medical importance, having been implicated in a number of
pathological conditions including: cataract formation [8],
type 2 diabetes [9], muscular dystrophy, rheumatoid
arth-ritis, ischaemic tissue damage, and neurodegenerative
con-ditions such as Alzheimer’s and Parkinson’s disease [10,11]
The major calpains are l-calpain (calpain 1) and
m-calpain (calpain 2), which are ubiquitous in mammalian
cells [10] These enzymes are heterodimeric and possess
larger, 80-kDa, subunits, which show high levels of
homo-logy, and smaller, 30-kDa, subunits, which show lower
levels of homology [2,3] Originally based on sequence
comparisons [12], these calpains were assigned a domainal
organization, with the larger subunit divided into domains I
to IV and the smaller subunit divided into domains V and
VI Domain II possesses the active site and is a papain-like cysteine protease domain, and domain IV contains a calmodulin-like Ca2+-binding domain with multiple EF-hand motifs The smaller subunit is divided into domain
VI, which also possesses a calmodulin-like Ca2+-binding domain, and domain V [2,3,13] The recently solved crystal structures of calcium-free rat m-calpain [14] and human m-calpain [15] confirmed that this domain structure is essentially correct
l-Calpain and m-calpain possess similar substrate spe-cificity and show an absolute requirement for Ca2+ for activation, although they differ in the level of this require-ment: 5–50 lMand 250–1000 lM, respectively [2,3] These observations suggested that other factors may be involved in the activation of m-calpain because, clearly, the millimolar levels of Ca2+needed to activate the enzyme in vivo far exceed normal intracellular levels Calpains are known to exist in a membrane-associated form [16,17], and it has been shown that the presence of membrane lipid mixtures can lower the Ca2+ requirement for m-calpain activation to near physiological levels [18] On the basis of these and other results [19–21], it has been suggested that lipid or membrane interaction may modulate the activity of the enzyme [3] Consistent with this suggestion, it has been shown that m-calpain domain III folds into an antiparallel b-sandwich, which is structurally related to C2 domains [14,15,22] These domains bind phospholipid in a Ca2+-dependent manner and are believed to be responsible for orchestrating the
Correspondence to D A Phoenix, Department of Forensic and
Investigative Science, University of Central Lancashire, Preston
PR1 2HE, UK Fax: + 1772 894981, Tel.: + 1772 894381,
E-mail: daphoenix@uclan.ac.uk
Abbreviations: M yr 2 PtdCho, dimyristoylphosphatidylcholine;
Myr 2 PtdEtn, dimyristoylphosphatidylethanolamine; Myr 2 PtdSer,
dimyristoylphosphatidylserine; FTIR, Fourier transform infrared;
SUV, small unilamellar vesicle.
(Received 20 May 2002, accepted 2 September 2002)
Trang 2membrane–Ca2+regulation of enzyme activity of a number
of proteins [23] A number of studies have suggested that
interaction of m-calpain domain V with lipid or membranes
may also be involved in the modulation of the enzyme’s
activity Earlier investigations showed that a C-terminal
segment of domain V, G17TAMRILGG, is required for
lipid interaction of the enzyme [24] More recent
investiga-tions, using peptides homologous to various regions of
m-calpain’s domain V, showed that, although the presence
of the TAMRIL sequence was required for m-calpain–lipid
interaction, the presence of a polyglycine sequence was also
necessary for such interaction [25]
The recently determined crystal structures of m-calpain
did not include domain V [15,16] and no three-dimensional
structure for this domain is currently available
Further-more, the primary structure of domain V shows no apparent
homology with that of any other known protein [22]
However, a recent theoretical analysis of domain V derived
from four different mammalian m-calpains showed that
each possessed a common segment, GTAMRILGGVI,
which was a candidate for formation of a lipid-interactive
oblique-orientated a-helix [26] These a-helices show a
highly specialized structure/function relationship and
pene-trate membranes at a shallow angle because of a
hydro-phobicity gradient along the a-helical long axis [27,28] It
was suggested by Daman et al [26] that the formation of
such an a-helix may feature in the membrane interactions of
m-calpain domain V Here, to investigate this suggestion, we
have undertaken theoretical analysis and studied the lipid–
membrane interactions of a peptide homologue of the
GTAMRILGGVI segment using haemolytic analysis,
monolayer studies, Fourier transform infrared (FTIR)
conformational analysis and FTIR lipid-phase transition
analysis Our results are discussed in relation to the
influenza viral fusion peptide, HA2 [29,30], a known
oblique-orientated a-helix former [31–35]
M A T E R I A L S A N D M E T H O D S
Reagents
The peptide VP1 was supplied by Pepsyn, University of
Liverpool, UK, produced by solid-state synthesis and
purified by HPLC to a purity of greater than 99% The
peptide was stored as a stock solution (10 mM) in 10% (v/v)
ethanol at 4C Packed human red blood cells were
supplied by the Royal Preston Hospital, UK The
phos-pholipids dimyristoylphosphatidylcholine (Myr2PtdCho),
dimyristoylphosphatidylethanolamine (Myr2PtdEtn) and
dimyristoylphosphatidylserine (Myr2PtdSer) and all
sol-vents, which were of spectroscopic grade, were purchased
from Sigma (UK) For FTIR spectroscopy, deuterated Myr2PtdSer, purchased from Avanti, was used
Theoretical analyses of candidate oblique-orientated a-helix-forming segments
The sequences of the influenza viral fusion peptide, HA2, a known oblique-orientated a-helix former [31–35], and that
of the putative oblique-orientated a-helix of m-calpain domain V (Table 1) were analysed by conventional hydro-phobic moment methods [36] The hydrohydro-phobicity of successive amino acids in these sequences are treated as vectors and summed in two dimensions, assuming an amino-acid side chain periodicity of 100 The resultant of this summation, the hydrophobic moment, lH, 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,ÆlHæ, and the corresponding mean hydrophobicity,ÆH0æ (Table 1), were computed using the normalized consensus hydrophobicity scale of Eisenberg et al [37] and plotted on the hydrophobic moment plot diagram of Eisenberg et al [38] as modified by Harris et al [32] (Fig 1)
WINGEN/WINPEG software [39] was used to perform hydropathy plot analysis (Fig 2) of the GTAMRILGGVI sequence using the hydrophobicity scale of Kyte & Dolittle [40] and a seven-residue window The software was also used to represent both this sequence and that of HA2 as two-dimensional axial projections assuming an angular periodicity of 100 (Fig 3)
Haemolytic assay of VP1 Haemolytic assay was conducted as described by Harris & Phoenix [41] Essentially, packed red blood cells were washed three times in Tris-buffered sucrose (0.25M sucrose, 10 mM Tris/HCl, pH 7.5) and resuspended in the same medium to give an initial blood cell concentra-tion of 0.05% For haemolytic assay, this concentration was adjusted such that incubation with 0.1% (v/v) Triton X-100 for 1 h produced a supernatant with A416¼ 1.0, and this was taken as 100% haemolysis Aliquots (1 mL)
of blood cells at assay concentration were then used to solubilize various amounts of stock peptide solution, each of which had been added to a test-tube and dried under nitrogen gas The resulting mixtures were incu-bated at room temperature with gentle shaking After 1 h, the suspensions were centrifuged at low speed (1500 g,
15 min, 25C), and the A416 of the supernatants
Table 1 Hydrophobic moment analysis of protein structure Primary structure of the putative oblique-orientated a-helix-forming segment identified
in m-calpain, domain V [26] and that of the influenza peptide, HA2, a known oblique-orientated a-helix former, obtained from Peuvot et al [47] Values of Æl H æ and ÆH 0 æ for each sequence were determined by the method of Eisenberg et al [36].
+
Trang 3determined Similar experiments were also performed
except that buffered sucrose was replaced with
Tris-buffered saline (100 mMNaCl, 10 mMTris/HCl, pH 7.5)
In all cases, levels of haemolysis were determined as the
percentage haemolysis relative to that of Triton X-100 and
the results recorded (Fig 4) Basal lysis was less than 3%
in all cases
Preparation of phospholipid small unilamellar
vesicles (SUVs)
SUVs were prepared by the method of Keller et al [42]
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 mM The resulting cloudy suspensions were sonicated at 4C 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, 4C)
FTIR conformational analyses of VP1
To give a final peptide concentration of 1 mM, VP1 was solubilized in 50 mM aqueous Hepes (pH 7.5) or suspen-sions of SUVs, which were formed from Myr2PtdSer, Myr2PtdCho or Myr2PtdEtn, prepared as described above Samples of solubilized peptide were spread on a CaF2crystal, and the free excess water was evaporated at room temperature The single-band components of the VP1 amide I vibrational band (predominantly C¼O stretch) was monitored using an FTIR 5-DX spectro-meter (Nicolet Instruments, Madison, WI, USA), and, for each sample, absorbance spectra were produced (Fig 5) These spectra were analysed, and, for those with strong absorption bands, the band parameters (peak position, band width, and intensity) were evaluated 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 [43] were applied after baseline subtraction with the parameters: bandwidth, 22–
28 cm)1; resolution-enhancement factor, 1.2–1.4; Gauss/ Lorentz ratio, 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, Ontario, Canada) An esti-mation of the number of band components was obtained from deconvolution of the spectra; curve fitting was then applied within the original spectra after the subtraction
of baselines resulting from neighbouring bands Similar
to the deconvolution 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 VP1, relative levels of a-helical structure (1650–1655 cm)1) and b-sheet struc-tures (1625–1640 cm)1) were computed and are shown in Table 2
FTIR analysis of phospholipid phase transition properties Using FTIR spectroscopy, the effects of VP1 on the phase-transition properties of phospholipid were investigated To give a final peptide concentration of 1 mM, VP1 was solubilized in suspensions of SUVs formed from Myr2 Ptd-Ser, Myr2PtdCho or Myr2PtdEtn, prepared as described above As controls, SUVs formed from Myr2PtdSer, Myr2PtdCho or Myr2PtdEtn alone were prepared as described above These samples were then subjected to automatic temperature scans with a heating rate of
3CÆ(5 min))1and within the temperature range 0–60C For every 3C interval, 50 interferograms were accumu-lated, apodized, Fourier transformed, and converted into
Fig 1 Hydrophobic moment plot analysis of protein segments A
con-ventional hydrophobic moment plot diagram of Eisenberg et al [38] is
shown with an overlaid grey region delineating candidate
oblique-orientated a-helices [32] 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) The data point representing the m-calpain domain V
seg-ment, GTAMRILGGVI (1), can be seen to lie in the grey region,
proximal to that representing the HA2 peptide (2), indicating that the
segment may be a candidate for oblique-orientated a-helix formation.
Fig 2 Hydropathy plot analysis of protein segments Hydropathy plot
analysis of the m-calpain domain V segment, GTAMRILGGVI, was
performed using a seven-residue window and the software of Hennig
[39] It can be seen that hydrophobicty progressively increases along
the length of the segment with a maximal value centred on the
C-terminal glycine, residue 8.
Trang 4absorbance spectra [44] (Fig 6) These spectra monitored
changes in the bfi a acyl chain melting behaviour of
phospholipids, with these changes determined as shifts in
the peak position of the symmetric stretching vibration of
the methylene groups, ms(CH2), which is known to be a
sensitive marker of lipid order The peak position of ms(CH2)
lies at 2850 cm)1in the gel phase and shifts at a lipid-specific
temperature Tcto 2852.0–2852.5 cm)1in the
liquid-crystal-line state For deuterated Myr2PtdSer, the values for
the peak position of ms(CD2) are at 2089–2093 cm)1,
respectively
Monolayer studies on VP1
All monolayer equipment was supplied by NIMA,
Coven-try, UK Surface tension was monitiored by the Wilhelmy
plate method using a microbalance [45] Studies were
conducted using a 5· 15 cm Teflon trough containing
80 mL buffer subphase (10 mM Tris/HCl, pH 7.5) The trough was equipped with moveable barriers, which responded to the microbalance and could be adjusted to maintain monolayers at either constant surface pressure or constant surface area Monolayers were formed by spread-ing pure phospholipids (10 mM) in chloroform, compressed
to give a surface pressure of 30 mNÆm)1, and then maintained at constant area Stock VP1 was added to the subphase via a reservoir extending into the subphase, which was contiuously stirred by a magnetic bar, and VP1– monolayer interactions were recorded as changes in mono-layer surface pressure (Fig 7)
R E S U L T S Theoretical analyses of candidate oblique-orientated a-helix-forming segments
The sequences of the influenza viral fusion peptide, HA2, and m-calpain domain V were analysed The resulting values of ÆlHæ and ÆH0æ (Table 1) were plotted on the hydrophobic moment plot diagram (Fig 1) The data points representing these sequences lie proximal in the area delineating candidate oblique-orientated a-helix-forming segments, indicating that m-calpain domain V may contain
an oblique-orientated a-helix comparable to that of HA2 Consistent with these results, hydropathy plot analysis of the GTAMRILGGVI sequence showed a progressive increase in hydrophobicity in moving from the N-terminus
to the C-terminus (Fig 2), suggesting the ability to form an a-helix with an asymmetric distribution of hydrophobicty along the a-helical axis Furthermore, when the sequences of HA2 and m-calpain domain V were modelled as a-helices [39], each formed an amphiphilic a-helix with similar structural properties (Fig 3) Each a-helix possesses a glycine-rich hydrophilic face and a wide hydrophobic face, which includes the bulky amino-acid residues tryptophan, phenylalanine, leucine and isoleucine
Fig 3 Two-dimensional axial projections of protein sequences Primary structures of (A) the influenza peptide, HA2, a known oblique-orientated a-helix former and (B) the putative oblique-orientated a-helix-forming segment identified in m-calpain, domain V (Table 1), represented as two-dimensional axial projections using the software of Hennig [39] Annotated numbers represent the relative locations of amino-acid residues within protein primary structure, and hydrophobic residues are circled It can be seen that each a-helix possesses a glycine-rich polar face and a wide hydrophobic face rich in bulky amino-acid residues In the case of the GTAMRILGGVI segment, these residues, isoleucine (6) and leucine (7 and 11), can be seen to be localized in the C-terminal region of the a-helix.
Fig 4 Haemolytic analysis of VP1 Haemolytic curve of VP1,
deter-mined by the method of Harris & Phoenix [41] The peptide was
incubated with either human erythrocytes (r) or these erythrocytes in
the presence of 100 m M NaCl (j) Percentage haemolysis was
deter-mined (n ¼ 3) and plotted as a function of VP1 concentration It can
be seen that at a concentration of 2.4 m M , VP1 showed 100% lysis of
erythrocytes (LD 50 ¼ 1.45 m M ), but, in the presence of 100 m M NaCl,
this ability was reduced by 60%.
Trang 5Haemolytic assay of VP1
It can be seen from Fig 4 that VP1 is strongly haemolytic
with a sigmoidal relationship between VP1 concentration
and percentage haemolysis The peptide achieved 100%
lysis of erthrocyte membranes at 2.4 mM (LD ¼
1.45 mM), but in the presence of 100 mMNaCl, this ability
of VP1 was reduced the order of 60% (LD50¼ 1.85 mM) FTIR conformational analysis of VP1
FTIR spectroscopy was used for conformational analysis of VP1, either in aqueous solution or in the presence of SUVs formed from Myr2PtdSer, Myr2PtdCho or Myr2PtdEtn (Fig 5) In each case, the relative percentages of a-helical secondary structure (1650–1655 cm)1) and b-sheet secon-dary structure (1625–1640 cm)1) were computed and are shown in Table 2 In aqueous solution, VP1 showed no evidence of a-helical structure and was primarily formed from b-sheet structures (> 90%) (data not shown) In the presence of Myr2PtdEtn and Myr2PtdCho, VP1 showed some evidence of a-helical structure ( 20%) but remained predominantly formed from b-sheet structures (48% and 61%, respectively) (Fig 5A,B) In contrast, VP1 showed high levels of a-helical structure (65%) in the presence of Myr2PtdSer and reduced levels of b-sheet structures (32%) (Fig 5C)
FTIR analysis of phospholipid phase-transition properties
Using FTIR spectroscopy, absorbance spectra representing the effects of VP1 on the phase-transition temperature and membrane fluidity of membranes formed from Myr2PtdEtn, Myr2PtdCho or Myr2PtdSer were derived as a function of temperature (Fig 6) Control experiments recorded the lipid phase-transition temperature, Tc, of M yr2PtdCho mem-branes as 27C, Myr2PtdEtn membranes as 55C, and Myr2PtdSer membranes as 37C (Figs 6A-6C) The pres-ence of VP1 had a minor effect on the Tcand membrane fluidity of both Myr2PtdCho and Myr2PtdEtn membranes, with Tcbeing recorded as 24C and 52 C, respectively, and accompanied in each case by a minor increase in membrane fluidity (Fig 6A,B) In contrast, the presence of VP1 led to a large decrease in the fluidity of Myr2PtdSer membrane, accompanied by a large increase in the Tcof the membranes, with Tcbeing recorded as 47C (Fig 6C)
Monolayer studies on VP1 The interactions of VP1 with Myr2PtdSer monolayers were studied as described above MyrPtdSer was found to form
Fig 5 FTIR conformational analysis of VP1 in the presence of lipid.
(A)–(C) Spectra representing FTIR conformational analyses of VP1 in
the presence of lipid with annotated numbers indicating band peak
absorbances For each spectrum, the relative percentages of a-helical
secondary structure and b-sheet secondary structure were computed,
as described in Materials and Methods, and are shown in Table 2 (A)
and (B) show that, in the presence of Myr 2 PtdEtn and Myr 2 PtdCho,
the major secondary structural contribution to VP1 came from b-sheet
structures (1627 cm)1and 1628 cm)1, respectively) In contrast, (C)
shows that, in the presence of Myr 2 PtdSer, a-helical structure made the
major contribution to VP1 secondary structure (1650 cm)1).
Table 2 VP1 secondary-structural contributions Relative levels of a-helical structure and b-sheet structure determined in VP1, a peptide homologue of the putative oblique-orientated a-helix-forming segment identified in m-calpain domain V (Table 1) The peptide was either in aqueous solution (–) or in the presence of lipid Conformational ana-lysis of VP1 was performed using FTIR spectroscopy, and the resulting spectra (Fig 5) were used to determine relative levels of secondary structure as described in Materials and methods.
Trang 6stable monolayers at a surface pressure of 30 mNÆm)1,
which was taken to represent that of naturally occurring
membranes At a final subphase concentration of 20 lM,
VP1 showed maximal levels of Myr2PtdSer monolayer penentration, which led to a change in monolayer surface pressure of 5.5 mNÆm)1(Fig 7) This ability was reduced to negligible levels in the presence of 100 mMNaCl (data not shown)
D I S C U S S I O N There is evidence to suggest that the enzymatic activity of m-calpain is modulated by the membrane interaction of a segment, GTAMRILGGVI, located in domain V of the protein’s smaller subunit [24,25] It has been predicted that this segment may interact with membrane via the formation
of an oblique-orientated a-helix [26], a class of a-helices [29– 35] that penetrate membranes at a shallow angle because of
a hydrophobicity gradient along the a-helical long axis [27,28] Here we have used theoretical techniques to examine the structural characteristics of the putative domain V a-helix In addition, the ability of the GTAMRILGGV segment to adopt a-helical structure and to interact with membranes has been investigated using VP1, a peptide homologue of this segment, in conjunction with haemolytic analysis, monolayer studies, and FTIR spectroscopy Our results are discussed in relation to HA2, a viral fusion peptide known to penetrate membranes via oblique-orien-tated a-helix formation [29–35]
The sequences of the GTAMRILGGVI segment and HA2 were analysed, and data points representing their a-helices were found to lay proximal in the region of the hydrophobic moment plot diagram delineating candidate oblique-orientated a-helices (Fig 1) This observation sug-gests that the GTAMRILGGVI segment may form such an a-helix and therefore may possess a hydrophobicity gradi-ent Consistent with this suggestion, hydropathy plot analysis showed the GTAMRILGGVI segment to become progressively more hydrophobic in moving from the
Fig 6 FTIR phase-transition analysis of lipid in the presence of VP1.
Spectra representing FTIR phase-transition analysis of lipid in the
presence of VP1 In the absence of the peptide, the phase-transition
temperature (T c ) of M yr 2 PtdEtn was recorded as 55 C (j; A), of
Myr 2 PtdCho as 27 C (j; B) and of Myr 2 PtdSer as 37 C (j; C) The
presence of VP1 led to a minor increase in the fluidity of both
Myr 2 PtdEtn and Myr 2 PtdCho membranes, which in each case was
accompanied by a minor decrease in T c , with T c recorded as 52 C for
Myr 2 PtdEtn membranes (h; A) and 24 C for M yr 2 PtdCho
mem-branes (h; B) In contrast, the presence of VP1 led to a large decrease
in the fluidity of Myr 2 PtdSer membranes, accompanied by a large
increase in the T c of the membranes (h; C) with T c being recorded as
47 C.
Fig 7 Monolayer interactions of VP1 Time course for interactions of VP1 with Myr 2 PtdSer monolayers at a surface pressure of 30 mNÆm)1, taken to represent that of naturally occurring membranes VP1 (final concentration 20 l M ) was introduced into the monolayer subphase at time zero and after 100 s showed rapid penetration of Myr 2 PtdSer monolayers Maximal levels of penentration were reached after 600 s, with a concomitant change in monolayer surface pressure of 5.5 mNÆm)1.
Trang 7N-terminus to the C-terminus (Fig 2), and graphical
analysis showed the GTAMRILGGVI a-helix to possess
a number of structural resemblances to the HA2 a-helix
(Fig 3) It can be seen that each a-helix shown in Fig 3
possesses a glycine-rich polar face, which studies on HA2
and a number of other oblique-orientated a-helices have
shown to be critical for maintaining their hydrophobicity
gradients [27,28,46] It can also be seen from Fig 3 that each
a-helix possesses a wide hydrophobic face rich in bulky
amino-acid residues In the case of the GTAMRILGGVI
segment (Fig 3), leucine and isoleucine can be seen to be
preponderant in the C-terminal region of the a-helix This
localization of strongly hydrophobic amino-acid residues is
structurally consistent with the higher levels of
hydropho-bicity predicted for the C-terminal region of the segment
(Fig 2) and possession of a hydrophobicity gradient [47]
Furthermore, when these wide hydrophobic faces, rich in
bulky residues, are combined with narrow polar faces, rich
in glycine residues (Fig 3), a-helices are given an effective
wedge shape, which appears to assist HA2 and other
oblique-orientated a-helix-forming peptides, to destabilize
membranes in the promotion of their biological activity
[46,48]
It is clear from our theoretical analyses that the
GTAMRILGGVI segment has the potential to form an
a-helix with strong structural similarities to the
oblique-orientated a-helix formed by HA2 and other
membrane-interactive peptides Consistent with these observations,
FTIR spectroscopic analysis showed that VP1 was able to
adopt a-helical structure in the presence of lipid membranes
(Fig 5) and to affect the lipid-phase transition properties of
these membranes (Fig 6) In addition, haemolytic analysis
showed that the peptide is able to lyse erythrocyte
membranes (Fig 4), and monolayer studies showed that it
is able to penetrate lipid monolayers mimetic of naturally
occurring membranes (Fig 7) In combination, these results
clearly show that the GTAMRILGGVI segment is able to
form a membrane-interactive a-helix
In aqueous solution, VP1 showed no evidence of a-helical
structure (data not shown) but was found to adopt such
structure in the presence of Myr2PtdCho, Myr2PtdEtn and
Myr2PtdSer (Fig 6) This suggests that VP1 requires the
amphiphilic environment of a lipid interface to adopt
a-helical structure, a requirement also observed for HA2
[49] In the presence of Myr2PtdEtn and Myr2PtdCho
membranes, VP1 adopted relatively low levels of a-helical
structure ( 20%; Fig 5A,B) and induced only minor
decreases in the Tcof these membranes ( 3 C),
accom-panied by minor increases in liquid-crystalline phase fluidity
(Fig 6,AB) Such changes in lipid-phase transition
proper-ties are consistent with VP1 binding to the headgroup
regions of Myr2PtdCho and Myr2PtdEtn membranes and
suggest that the peptide penetrates the surface regions of
these membranes In contrast, VP1 adopted high levels of
a-helical structure in the presence of Myr2PtdSer (65%;
Fig 5C) and induced a 10C rise in the Tc of these
membranes accompanied by a large decrease in its
liquid-crystalline phase fluidity (Fig 6C) Such changes are
consistent with VP1 penetration of the Myr2PtdSer
mem-brane hydrophobic core region and suggest that the peptide
shows high levels of interaction with these membranes
Strongly supporting this suggestion, VP1 was found to show
high levels of MyrPtdSer monolayer penetration (Fig 7),
inducing surface pressure changes of 5.5 mNÆm)1 Com-parable levels of monolayer penetration have been observed for the C-terminal a-helices of Escherichia coli penicillin-binding proteins 5 and 6 [50], which function as membrane anchors for these proteins [51] Interestingly, a recent study has suggested that these anchors may form oblique-orientated a-helices (D Phoenix & F Harris, unpublished work) Taken in combination, these results suggest that VP1 has a low affinity for zwitterionic lipid but a high affinity for Myr2PtdSer and may have a requirement for either this specific lipid, or anionic lipid in general, to achieve higher levels of membrane penetration These results contrast with HA2, which shows an affinity for zwitterionic lipid [52,53] and requires the low pH of the endosomal membrane surface for both oblique-orientated a-helix formation and enhanced levels of membrane penetration via the deproto-nation of its negatively charged residues [35,54]
VP1 was found to be strongly haemolytic, but, in the presence of 100 mMNaCl, this ability was reduced by the order of 60% (Fig 4) and the ability of VP1 to interact with Myr2PtdSer monolayers was reduced to negligible levels (data not shown) In combination, these results suggest a strong electrostatic contribution to VP1–membrane inter-action and further support a VP1 requirement for anionic lipid Indeed, the relatively high LD50(1.45 mM) shown by VP1 for haemolytic action could reflect the fact that erythrocyte membranes possess an asymmetric distribution
of anionic lipids with the extracytoplasmic leaflet depleted of such lipids [55] It is interesting to note that the HA2 a-helix
is also strongly haemolytic and that the mutation of polar face glycine residues, essential for maintaining the HA2 a-helix hydrophobicity gradient, results in the loss of haemolytic and fusogenic ability [49,56]
In conclusion, based on structural similarities to HA2, we have suggested that the segment, GTAMRILGGVI, of m-calpain domain V forms an a-helix, which possesses a hydrophobicity gradient and penetrates membranes in an oblique orientation We speculate that glycine residues in the polar face of this a-helix could play an important role in facilitating this mechanism of membrane penetration This a-helix has a preference for anionic lipid, which leads to higher levels of a-helicity and membrane penetration via electrostatic interactions These results are consistent with those of previous authors, which have shown that m-calpain activity is modulated by the presence of anionic lipid [25]
To satisfy a VP1 requirement for anionic lipid, it seems probable that the peptide’s single positively charged amino-acid residue, arginine (Table 1; Fig 3) would engage in charge–charge interaction with negatively charged mem-brane lipid Furthermore, to achieve the deeper levels of membrane penetration indicated for Myr2PtdSer mem-branes, it is possible that these interactions may involve the snorkelling mechanism that features in the membrane interactions of a number of lipid-interactive a-helices [28,57] According to this mechanism, the VP1 arginine residue would extend or snorkel its long hydrophobic alkyl chain, facilitating penetration of the membrane hydropho-bic core region yet still allowing its positively charged moiety
to interact with negatively charged moieties in the mem-brane lipid headgroup region We speculate that utilization
of this mechanism by the m-calpain domain V a-helix could result in enhanced levels of membrane interaction by domain V, which would support work indicating lipid
Trang 8involvement in the reduction of Ca2+levels necessary for
the efficient activation of m-calpain
R E F E R E N C E S
1 Sorimachi, H & Suzuki, K (2001) The structure of calpain J Biol.
Chem 129, 653–664.
2 Sorimachi, H., Ishiura, S & Suzuki, K (1997) Structure and
physiological function of calpains Biochem J 328, 721–732.
3 Johnson, G.V.W & Guttmann, R.P (1997) Calpains: intact and
active? Bioessays 19, 1011–1018.
4 Ma, H., Fukiage, C., Kim, Y.H., Duncan, M.K., Reed, N.A.,
Shih, M., Azuma, M & Shearer, T.R (2001) Characterization and
expression of calpain 10 A novel ubiquitous calpain with nuclear
localization J Biol Chem 276, 28525–28531.
5 Sato, K & Kawashima, S (2001) Calpain function in the
mod-ulation of signal transduction molecules Biol Chem 382, 743–751.
6 Calafoli, E & Molinari, M (1998) Calpain: a protease in search of
a function? Biochem Biophys Res Commun 247, 193–203.
7 Ono, Y., Sorimachi, H & Suzuki, K (1998) Structure and
phy-siology of calpain, an enigmatic protease Biochem Biophys Res.
Commun 245, 289–294.
8 Azuma, M., Fukiage, C., David, L & Shearer, T.R (1997)
Acti-vation of calpain in lens: a review and proposed mechanism Lens.
Exp Eye Res 64, 529–538.
9 Horikawa, H et al (2000) Genetic variation in the gene encoding
calpain-10 is associated with type 2 diabetes mellitus Nat Genet.
26, 163–175.
10 Huang, Y.H & Wang, K.K.W (2001) The calpain family and
human disease Trends Mol Med 7, 355–362.
11 Wang, K.K.W (2000) Calpain and caspase: Can you tell the
dif-ference Trends Neurosci 23, 20–26.
12 Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M &
Suzuki, K (1984) Evolutionary origin of a calcium-dependent
protease by fusion of genes for a thiol protease and a
calcium-binding protein? Nature (London) 312, 566–570.
13 Croall, D.E & DeMartino, G.N (1991) Calcium-activated
neu-tral protease (calpain) system: structure, function, and regulation.
Physiol Rev 71, 813–847.
14 Hosfield, C.M., Elce, J.S., Davies, P.L & Jia, Z.C (1999) Crystal
structure of calpain reveals the structural basis for Ca 2+
dependent protease activity and a novel mode of enzyme
activa-tion EMBO J 18, 6880–6889.
15 Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R.,
Masu-moto, H., Nakagawa, K., Irie, A., Sorimachi, H., Bourenkow, G.,
Bartunik, H., Suzuki, K & Bode, W (2000) The crystal structure
of calcium-free human m-calpain suggests an electrostatic switch
mechanism for activation by calcium Proc Natl Acad Sci USA
97, 588–592.
16 Molinari, M & Carafoli, E (1997) Calpain: a cytosolic proteinase
active at the membranes J Membr Biol 156, 1–8.
17 Kawasaki, H & Kawashima, S (1996) Regulation of the
calpain-calpastatin system by membranes (review) Mol Membr Biol 13,
217–224.
18 Chakrabarti, A.K., Dasgupta, S., Gadsden, R.H., Hogan, E.L &
Banik, N (1996) Regulation of brain m-calpain Ca 2+ sensitivity
by mixtures of membrane lipids: activation at intracellular Ca 2+
level J Neurosci Res 44, 374–380.
19 Saido, T.C., Shibata, M., Takenawa, T., Murofushi, H & Suzuki,
K (1992) Positive regulation of mu-calpain action by
polypho-sphoinositides J Biol Chem 267, 24585–24590.
20 Melloni, E & Pontremoli, S (1989) The calpains Trends Neurosci.
12, 438–444.
21 Imajoh, S., Kawasaki, H & Suzuki, K (1986) The amino-terminal
hydrophobic region of the small subunit of calcium-activated
neutral protease (CANP) is essential for its activation by
phos-phatidylinositol J Biochem (Tokyo) 99, 1281–1284.
22 Tompa, P., Emori, Y., Sorimachi, H., Suzuki, K & Friedrich, P (2001) Domain III of calpain is a Ca2+-regulated phospholipid-binding domain Biochem Biophys Res Commun 280, 1333– 1339.
23 Rizo, J & Su¨dhof, T.C (1998) C2-domains, structure and func-tion of a universal Ca2+-binding domain J Biol Chem 273, 15879–15882.
24 Crawford, C., Brown, N.R & Willis, A.C (1990) Investigation of the structural basis of interaction of calpain II with phospholipid and with carbohydrate Biochem J 265, 575–579.
25 Arthur, J.S.C & Crawford, C (1996) Investigation of the inter-action of m-calpain with phospholipids: calpain–phospholipid interactions Biochim Biophys Acta 1293, 201–206.
26 Daman, O.A., Biswas, S., Harris, F., Wallace, J & Phoenix, D.A (2001) Theoretical investigation into the lipid interaction of m-calpain Mol Cell Biochem 223, 159–163.
27 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.
28 Phoenix, D.A., Harris, F., Daman, O.A & Wallace, J (2002) The prediction of amphiphilic a-helices Curr Protein Peptide Sci in press.
29 Bentz, J & Mittal, A (2000) Deployment of membrane fusion protein domains during fusion Cell Biol Int 24, 819–838.
30 Luneberg, J., Martin, I., Nussler, F., Ruysschaert, J.-M & Herr-mann, A (1995) Structure and topology of the influenza virus fusion peptide in lipid bilayers J Biol Chem 270, 27606–27614.
31 Brasseur, R (2000) Tilted peptides: a motif for membrane desta-bilisation (Hypothesis) Mol Membr Biol 17, 31–40.
32 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.
33 Chang, D.K., Cheng, S.F., Trivedi, V.D., Yang, S.H (2000) The amino-terminal region of the fusion peptide of influenza virus hemagglutinin, HA2, inserts into sodium dodecyl sulfate micelle with residues 16–18 at the aqueous boundary at acidic pH: oligomerization and the conformational flexibility J Biol Chem.
275, 19150–19158.
34 Macosko, J.C., Kim, C.H & Shin, Y.K (1997) The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR J Mol Biol 267, 1139–1148.
35 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 hemagluttinin Nat Struct Biol 8, 715–720.
36 Eisenberg, D., Weiss, R.M & Terwilliger, T.C (1982) The helical hydrophobic moment: a measure of the amphiphilicity of a-helix Nature (London) 299, 371–374.
37 Eisenberg, D., Weiss, R.M., Terwilliger, T.C & Wilcox, W (1982) Hydrophobic moment and protein structure Faraday Symp Chem Soc 17, 109–120.
38 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.
39 Hennig, L (1999) Wingen/Winpeg: user friendly software for the analysis of amino acid sequences Biotechniques 26, 1170–1172.
40 Kyte, J & Dolittle, R.F (1982) A simple method for displaying the hydropathic character of a protein J Mol Biol 157, 105–132.
41 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.
42 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.
Trang 943 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.
44 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.
45 Demel, R.A (1974) Model membrane monolayers: description of
use and interaction Methods Enzymol 32, 539–545.
46 Fujii, G (1999) To fuse or not to fuse: the effects of electrostatic
interactions, hydrophobic forces and structural amphiphilicity on
protein-mediated membrane destabilisation Advanced Drug
Delivery Review 38, 257–277.
47 Peuvot, J., Schank, A., Lins, L & Brasseur, R (1999) Are the
fusion processes involved in birth, life and death of the cell
depending on tilted insertion of peptides into membranes?
J Theor Biol 198, 173–181.
48 White, J.M (1990) Viral and cellular membrane fusion proteins.
Annu Rev Physiol 52, 675–697.
49 Plank, C., Zauner, W & Wagner, E (1999) Application of
membrane-active peptides for drug and gene delivery across
cel-lular membranes Advanced Drug Delivery Review 34, 21–35.
50 Harris, F., Demel, R.A., Phoenix, D.A & De Kruijff, B (1998) An
investigation into the lipid interactions of peptides corresponding
to the C-terminal anchoring domains of Escherichia coli
penicillin-binding proteins 4, 5 and 6 Biochim Biophys Acta 1415, 10–22.
51 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.
52 Zhou, Z., Macosko, J.C., Hughes, D.W., Sayer, B.G., Hawes, J & Epand, R.M (2000) N-15 NMR study of the ionization properties
of the influenza virus fusion peptide in zwitterionic phospholipid dispersions Biophys J 78, 2418–2425.
53 Duzgunes, N & Shavnin, S.A (1992) Membrane destabilization
by N-terminal peptides of viral envelope proteins J Membr Biol.
128, 71–80.
54 Stegmann, T & Helenius, A (1993) Influenza virus fusion: from models toward a mechanism In Viral Fusion Mechanisms (Bentz, J., ed.) CRC Press, Boca Raton, FL.
55 Op den Kamp, J.A.F (1979) Lipid asymmetry in membranes Annu Rev Biochem 48, 47–71.
56 Tamm, L.K & Han, X (2000) Viral fusion peptides: a tool to disrupt and connect biological membranes Biosci Rep 29, 501– 518.
57 Segrest, J.P., Venkatachalapathi, Y.V., Srinivas, S.K., Gupta, K.B., De Loof, H & Anatharamaiah, G.M (1992) Role of basic amino acid residues in the amphipathic helix: the snorkel hypothesis In Molecular Conformation and Biological Interactions (Balaram, P & Ramaseshan, S., eds), pp 597–635 Indian Academy of Sciences, Bangalore.