Domain 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 Brandenburg
1
, Frederick Harris
2
, Sarah Dennison
2
, Ulrich Seydel
1
and David Phoenix
2
1
Division of Biophysics, Forschunginstitute Borstel, Germany;
2
Department of Forensic and Investigative Science, University of
Central Lancashire, Preston, UK
The activity of m-calpain, a heterodimeric, Ca
2+
-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

Calpains are a growing family [1] of structurally related
intracellular Ca
2+
-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 Ca
2+
-binding domain with multiple
EF-hand motifs. The smaller subunit is divided into domain
VI, which also possesses a calmodulin-like Ca

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 Ca
2+
-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:
Abbreviations:Myr
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)
Eur. J. Biochem. 269, 5414–5422 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03225.x
membrane–Ca
2+
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

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].
MATERIALS AND METHODS
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 m
M
) in 10% (v/v)
ethanol at 4 °C. Packed human red blood cells were
supplied by the Royal Preston Hospital, UK. The phos-
pholipids dimyristoylphosphatidylcholine (Myr
2
PtdCho),
dimyristoylphosphatidylethanolamine (Myr
2
PtdEtn) and
dimyristoylphosphatidylserine (Myr
2
PtdSer) and all sol-
vents, which were of spectroscopic grade, were purchased
from Sigma (UK). For FTIR spectroscopy, deuterated
Myr
2
PtdSer, 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

[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 (0.25
M
sucrose, 10 m
M
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 A
416
¼ 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, 25 °C), and the A
416
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].

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 °Cwitha
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 VP1
To give a final peptide concentration of 1 m
M
,VP1was
solubilized in 50 m
M
aqueous Hepes (pH 7.5) or suspen-
sions of SUVs, which were formed from Myr
2
PtdSer,
Myr
2
PtdCho or Myr
2
PtdEtn, 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
VP1 amide I vibrational band (predominantly C¼O

the relative percentages of primary structure involved in
secondary-structure formation. For VP1, relative levels of
a-helical structure (1650–1655 cm
)1
)andb-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 m
M
,VP1was
solubilized in suspensions of SUVs formed from Myr
2
Ptd-
Ser, Myr
2
PtdCho or Myr
2
PtdEtn, prepared as described
above. As controls, SUVs formed from Myr
2
PtdSer,
Myr
2
PtdCho or Myr
2

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 2852.0–2852.5 cm
)1
in the liquid-crystal-
line state. For deuterated Myr
2
PtdSer, the values for
the peak position of m
s
(CD
2
) are at 2089–2093 cm
)1
,

H
æ and ÆH
0
æ (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

of VP1 was reduced the order of 60% (LD
50
¼ 1.85 m
M
).
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 Myr
2
PtdSer, Myr
2
PtdCho or Myr
2
PtdEtn
(Fig. 5). In each case, the relative percentages of a-helical
secondary structure (1650–1655 cm
)1
)andb-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 Myr
2
PtdEtn and Myr
2
PtdCho, VP1 showed

2
PtdSer membranes as 37 °C (Figs 6A-6C). The pres-
ence of VP1 had a minor effect on the T
c
and membrane
fluidity of both Myr
2
PtdCho and Myr
2
PtdEtn membranes,
with T
c
being recorded as 24 °Cand52°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 Myr
2
PtdSer membrane,
accompanied by a large increase in the T
c
of the membranes,
with T
c
being recorded as 47 °C (Fig. 6C).
Monolayer studies on VP1
The interactions of VP1 with Myr
2
PtdSer monolayers were
studied as described above. Myr
2

spectra (Fig. 5) were used to determine relative levels of secondary
structure as described in Materials and methods.
Lipid % a-helix % b-sheet
––92
Myr
2
PtdSer 65 32
Myr
2
PtdEtn 18 61
Myr
2
PtdCho 19 48
5418 K. Brandenburg et al.(Eur. J. Biochem. 269) Ó FEBS 2002
stable 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 l
M
,
VP1 showed maximal levels of Myr
2
PtdSer 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 m
M

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
)ofMyr
2
PtdEtn was recorded as 55 °C(j;A),of
Myr
2
PtdCho as 27 °C(j; B) and of Myr
2
PtdSeras37°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
,withT
c
recorded as 52 °Cfor
Myr
2
PtdEtn membranes (h;A)and24°CforMyr
2
PtdCho mem-

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

(Fig. 6,AB). Such changes in lipid-phase transition proper-
ties are consistent with VP1 binding to the headgroup
regions of Myr
2
PtdCho and Myr
2
PtdEtn 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 Myr
2
PtdSer (65%;
Fig. 5C) and induced a 10 °CriseintheT
c
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 Myr
2
PtdSer 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 Myr
2
PtdSer monolayer penetration (Fig. 7),
inducing surface pressure changes of 5.5 mNÆm
)1
.Com-
parable levels of monolayer penetration have been observed

(1.45 m
M
) 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 Myr
2

ulation of signal transduction molecules. Biol. Chem. 382, 743–751.
6. Calafoli, E. & Molinari, M. (1998) Calpain: a protease in search of
afunction?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.,

1339.
23. Rizo, J. & Su
¨
dhof, T.C. (1998) C2-domains, structure and func-
tion of a universal Ca
2+
-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-

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.
Ó FEBS 2002 Lipid-interactive a-helical structure in m-calpain (Eur. J. Biochem. 269) 5421
43. 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.