(Received for publication, August 29, 1994; and in revised form, November 28, 1994)
From the
Studies on the membrane binding of cytochrome c revealed liposome-associated and soluble cytochrome c not to be in rapid equilibrium. In brief, cytochrome c attached to pyrene phospholipid-labeled, fluorescent liposomes containing either 17.6 mol % cardiolipin (CL) or 30 mol % egg phosphatidylglycerol (PG) is practically not at all or very slowly, respectively, detached by a subsequently added excess (up to 20-fold) of nonlabeled liposomes containing these acidic lipids. Cytochrome c was fully dissociated from PG-containing liposomes by increasing the ionic strength by NaCl, whereas dissociation from CL-containing membranes was less complete, presumably because of the scavenging of the protein within inverted intramembrane micelles. Importantly, the apparent irreversibility of the binding of cytochrome c to liposomes is strongly dependent on the structure of the acidic phospholipid. Cytochrome c bound to lyso-PG/PC liposomes could be dissociated with an excess of nonlabeled PG-containing liposomes. Cytochrome c was also efficiently bound to membranes containing the negatively charged dicetylphosphate yet could be readily dissociated by nonlabeled PG-containing liposomes. We conclude both proper geometry of the phosphate group and the presence of two acyl chains to be required for the tight binding of cytochrome c to acidic phospholipids. These data provide evidence for the membrane association of cytochrome c by an acidic phospholipid in the extended conformation (Kinnunen, P. K. J., Kõiv, A., Lehtonen, J. Y. A., Rytömaa, M., and Mustonen, P. (1994) Chem. Phys. Lipids 73, 181-207) in which one of the acyl chains of the lipid becomes accommodated within a hydrophobic cavity of the protein. Based on the crystal structure of cytochrome c we putatively assign the invariant Asn-52 (horse heart cytochrome c) as the site liganding the protonated phosphate of the lipid, whereas Lys-72 and -73 should bind the deprotonated form.
Peripheral membrane interactions have been widely studied and include the lipid association of proteins such as cytochrome c(1, 2, 3, 4, 5, 6, 7, 8, 9) and protein kinase C (10) as well as nucleic acids (11, 12) and drugs such as adriamycin(13, 14) . The lipid requirements for the membrane association of cytochrome c and protein kinase C are fairly well understood. Accordingly, cytochrome c binds to membranes containing acidic phospholipids and cardiolipin either as such or complexed with cytochrome c oxidase is likely to provide its binding site at the inner mitochondrial membrane(15, 16) . For the activation of protein kinase C, diacylglycerol is needed in addition to phosphatidylserine(17) .
Cytochrome c has been
considered as a paradigm for electrostatically interacting peripheral
membrane proteins since it is dissociated from lipid membranes by
increasing ionic strength(18, 19, 20) .
However, also hydrophobic interactions have been reported to be
involved in the membrane association of cytochrome c(21) . The binding of cytochrome c to
liposomes has been shown to induce conformational alterations in the
protein as well as in the
phospholipids(22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
We have previously suggested that there are two distinct acidic
phospholipid binding sites in cytochrome c and have
tentatively nominated these as A- and C-site, for anion and cardiolipin
binding, respectively(20, 32) . These putative sites
are characterized as follows. The A-site interacts with anions such as
deprotonated acidic phospholipids, Cl, and
nucleotides. Accordingly, the binding of cytochrome c to
liposomes by the A-site is likely to result from electrostatic
interactions between the protein and acidic phospholipids and can be
reversed with increasing ionic strength and anions. In contrast the
C-site presumably involves hydrogen bonding between cytochrome c and protonated acidic phospholipids, and the protein is not
dissociated with nucleotides or increasing ionic strength. However,
sufficiently high ionic strength results in the deprotonation of acidic
phospholipids and thus causes the dissociation of cytochrome c. The nature of the membrane association of cytochrome c can be controlled by factors affecting the protonation of the
acidic phospholipids, i.e. pH(20) , ionic strength, or
surface charge density, that is the amount of acidic phospholipids, in
liposomes(32) .
In the course of our studies we observed
that once cytochrome c became bound to pyrene-labeled,
fluorescent liposomes via either its A-site or C-site it was no longer
dissociated by nonfluorescent liposomes added afterwards. This
phenomenon and its dependence on the acidic phospholipid structure are
reported here. Formation of the hexagonal H phase as a
consequence of the association of cytochrome c to
cardiolipin-containing membranes has been demonstrated(33) .
Therefore, although CL (
)is likely to constitute the
physiological binding site for cytochrome c we also studied
the reversibility of the association of cytochrome c with
PG-containing liposomes in order to avoid ambiguities in the
interpretation of the results arising because of the formation of
H
phase. The experiments were conducted using large
unilamellar liposomes containing either 17.6 mol % CL, or 30 mol % PG,
lyso-PG, or dicetylphosphate. Under these conditions the A-site is
functional, and cytochrome c is detached from the liposomes by
ATP.
Figure 1:
A, binding of cytochrome c to
PC (10.6 µM total phospholipid) liposomes containing 1 mol
% PPDPC and 17.6 mol % CL (,
,
, +). The open symbols show the binding of cytochrome c to
PPDPC/CL/PC liposomes mixed with nonfluorescent liposomes (17.6 mol %
CL in PC) prior to the addition of the protein. The molar ratios of
pyrene-labeled/nonlabeled liposomes were 1/1 (
), 1/5 (
),
1/10 (
), 1/20 (
). B, effect caused by the addition
of nonlabeled liposomes containing 17.6 (
, +), 33.3
(
), or 100 (
) mol % CL subsequent to the binding of
cytochrome c to labeled, PPDPC-containing liposomes. The upward arrow indicates the dissociation of cytochrome c by 150 mM NaCl. The lipid concentration indicated on the x-axis in panel B represents the concentration of
phosphate groups. For the graph labeled by (+), cytochrome c was added only up to a concentration of 123 nM (panel
A, downward arrow), after which nonlabeled PC liposomes
containing 17.6 mol % CL were added. Cyt,
cytochrome.
Figure 4:
A, binding of cytochrome c to PC
liposomes (12.5 µM total phospholipid) containing 1 mol %
PPDPC and 30 mol % PG (,
,
, +). The open
symbols show the binding of cytochrome c to PPDPC/PG/PC
liposomes mixed with nonlabeled (30 mol % PG in PC) prior to the
addition of the protein. The molar ratios of pyrene-labeled/nonlabeled
liposomes were 1/1 (
), 1/5 (
), 1/10 (
), and 1/20
(
). B, the addition of nonlabeled liposomes containing
30 (
, +), 50 (
), or 100 (
) mol % PG subsequent
to the addition of cytochrome c. The upward arrow indicates the dissociation of cytochrome c by 150 mM NaCl. Similarly to Fig. 1for the graph labeled (+),
cytochrome c was added only up to a concentration of 123
nM (panel A, downward arrow), after which
nonlabeled liposomes (30 mol % PG in PC) were added. Cyt,
cytochrome.
Figure 2:
Lack of reversal of the membrane
association of cytochrome c (244 nM) caused by
subsequently added nonlabeled liposomes containing 17.6 (), 33.3
(
), or 100 mol % (
) CL. The open symbols show the
effect of the same concentrations of nonlabeled liposomes added prior
to the addition of cytochrome c. The data were taken from Fig. 1and other similar measurements. Lipid concentration in
fluorescent liposomes was 10.6
µM.
Figure 3:
Time course of the detachment of
cytochrome c from pyrene-labeled liposomes by subsequently
added nonlabeled liposomes. Cytochrome c (244 nM) was
allowed to bind to liposomes (12.5 µM phosphate)
containing in addition to PPDPC and PC either 30 mol % PG (,
) or 17.6 mol % CL (
), after which a 20-fold excess (i.e. 250 µM phosphate) of nonlabeled PG/PC (30
mol % PG) (
), PC (
), or CL/PC (17.6 mol % CL) (
)
liposomes was included. Arrows indicate the dissociation of
cytochrome c by 150 mM NaCl.
Figure 5:
The reversal of the membrane association
of cytochrome c (244 nM) caused by nonlabeled
liposomes containing 30 (,
), 50 (
,
), or 100
mol % (
,
) PG. The closed symbols show the effect
of nonlabeled liposomes added subsequent to the addition of cytochrome c, and the open symbols show the effect of nonlabeled
liposomes added prior to the addition of cytochrome c. The
data were taken from Fig. 4and other similar measurements. The
lipid concentration of fluorescent liposomes was 12.5
µM.
The above experiments utilized liposomes in the fluid, liquid crystalline state. The tight binding of cytochrome c to liposomes containing 30 mol % egg PG was not dependent on the membrane phase state, however. Accordingly, nonlabeled liposomes did not dissociate cytochrome c from PPDPC/DPPG/DPPC liposomes below their main phase transition temperature at 41 °C. Likewise, the presence of up to 50 mol % (from the final lipid concentration) cholesterol did not affect the apparently irreversible binding of cytochrome c described above (data not shown).
Figure 6:
A, binding of cytochrome c to
liposomes containing in addition to 1 mol % of PPDPC 30 mol % lyso-PG
(); 20 mol % lyso-PG, 10 mol % PG, 10 mol % lyso-PC (
); 10
mol % lyso-PG, 20 mol % PG, 20 mol % lyso-PC (
); or 30 mol % PG,
30 mol % lyso-PC (+), the balance in each case being PC. B, the dissociation of cytochrome c by nonlabeled
liposomes containing 30 mol % PG in PC. The arrow indicates
the dissociation of cytochrome c by 150 mM NaCl. Cyt, cytochrome.
Figure 7:
A, binding of cytochrome c to
labeled (1 mol % of PPDPC) PC liposomes containing 30 mol % lyso-PG
(). B, the dissociation of cytochrome c by
nonlabeled PC liposomes. Cyt,
cytochrome.
To exclude
possible interference caused by transfer of lyso-PG between liposomes
we mixed lyso-PG/PPDPC/PC (30/5/65 molar ratio) with a 5-fold molar
excess of PG/PC (30/70 molar ratio) liposomes. If lyso-PG would
equilibrate between the two liposome populations this would result in
an enrichment of the fluorescent lipid PPDPC. This in turn would cause
an increase in I/I
, i.e. the ratio of excimer/monomer fluorescence emission
intensities(38) . However, practically no changes in I
/I
were observed within a
30-min incubation, indicating that the spontaneous transfer of lyso-PG
between liposomes under these conditions is slow. Notably, transfer of
lyso-PG in the above system would also involve curvature changes, which
are counteracted by the bending rigidities of the membranes. We also
investigated the possibility that the above results (Fig. 6)
could be explained by a rapid equilibration of the lyso-PG-cytochrome c complex between labeled and nonlabeled liposomes. If this
was the case the extent of fluorescence quenching caused by the binding
of cytochrome c to a 1/1 (molar ratio) mixture of two liposome
populations of either (a) labeled lyso-PG/PC liposomes and nonlabeled
PC liposomes or (b) labeled PC liposomes and nonlabeled lyso-PG/PC
liposomes should be identical. However, compared with cytochrome c bound to labeled lyso-PG/PC liposomes the quenching of pyrene
fluorescence decreases only slightly (from 49 to 41%) when cytochrome c is bound to a mixture of labeled lyso-PG/PC and nonlabeled
PC liposomes, whereas little binding to labeled PC vesicles is observed
in the presence of nonlabeled lyso-PG/PC liposomes (19% quenching, data
not shown).
Figure 8:
A, binding of cytochrome c to PC
liposomes containing 1 mol % PPDPC and 30 mol % DCP (three identical
measurements are shown). B, the addition of nonlabeled
liposomes containing in addition to PC, 30 mol % dicetylphosphate
(), 30 mol % PG (
), or 100 mol % PG (
). The arrow
indicates the dissociation of cytochrome c by 150 mM NaCl. Cyt, cytochrome.
We have recently shown that the peripheral binding of
cytochrome c to membranes also involves nonelectrostatic
interactions (20) dependent on the protonation of acidic
phospholipids(32) . In brief, we proposed two distinct acidic
phospholipid binding sites in cytochrome c, the C-site
recognizing protonated and the A-site recognizing deprotonated acidic
phospholipids. Dissociation of cytochrome c attached to
membrane via its A-site, i.e. from liposomes containing 30 mol
% PG can be induced with increasing ionic strength as well as by the
addition of ATP. The electrostatically membrane-associated cytochrome c should be in equilibrium with the protein in solution, thus
resulting in the reversal of fluorescence quenching caused by the
addition of an excess of nonlabeled liposomes. However, as shown in the
present work, cytochrome c bound via its A-site to CL- or
PG-containing liposomes is poorly detached by a 20-fold excess of
subsequently added nonlabeled liposomes. We have previously noted the
biphasic association of cytochrome c with acidic phospholipids
containing liposomes and have suggested saturation of the high affinity
binding sites to be followed by a loose, less specific association of
cytochrome c to the surface of liposomes(20) . It
appears that only the latter, less tightly bound cytochrome c appears to be readily removable by subsequently added
nonfluorescent liposomes. Dissociation of cytochrome c from
membranes containing PG is immediately (within a few seconds) induced
by increasing ionic strength. Accordingly, the apparent irreversibility
of cytochrome c binding to liposomes is unlikely to involve
penetration of the protein into the lipid bilayer. In this context it
is worth noticing that highly analogous behavior has been described for
the membrane-bound protein kinase C, which does not equilibrate with
subsequently added liposomes while rapid dissociation of protein kinase
C is induced by chelating Ca with EGTA(41) .
The inability of excess nonlabeled liposomes to dissociate membrane
bound cytochrome c could result from either a highly
cooperative membrane association or very slow dissociation kinetics of
the cytochrome c-lipid complex, thus favoring the
membrane-bound form of the protein. However, it is difficult to
reconcile how the latter could be compatible with an electrostatic
membrane association of cytochrome c.
The binding of cytochrome c to DCP- or lyso-PG-containing liposomes is almost identical, in keeping with the electrostatic interaction of cytochrome c with these lipids. Likewise, cytochrome c is effectively displaced from these lipids by ATP, in keeping with their acidic headgroup interacting with the A-site of the protein (data not shown). In DCP the two alkyl chains are attached directly to the phosphate group. The rapid dissociation of cytochrome c from DCP-containing liposomes by PG/PC liposomes reveals the importance of the correct glycerophospholipid structure for the tight and apparent nonequilibrium membrane association of the protein. Another structural alteration in the acidic lipid resulting in the rapid detachment of bound cytochrome c by PG/PC liposomes is substitution of lyso-PG for PG in the fluorescent vesicles. Because of the negative charge of lyso-PG simultaneous substitution of lyso-PG for lyso-PC is somewhat inadequate in maintaining the curvature of the liposomes. However, since it was desirable to maintain a constant negative surface charge density lyso-PC provides the best available control for the curvature changes caused by lyso-PG. In addition, the dissociation of cytochrome c from lyso-PG-containing liposomes by nonlabeled liposomes is unlikely to be caused by increased membrane curvature by this lyso-component since the efficiency of cytochrome c induced fluorescence quenching actually slightly increases when lyso-PC is present in PG-containing liposomes. Accordingly, if an increase in membrane curvature increases the binding of cytochrome c to liposomes the increase should be even greater when liposomes contain lyso-PG. However, upon gradually substituting lyso-PG for PG the extent of fluorescence quenching caused by the membrane association of cytochrome c decreases, and the detachment of cytochrome c from labeled liposomes by nonlabeled liposomes becomes progressively increased. Taken together, two requirements for the tight, slowly equilibrating membrane association of cytochrome c emerge. These are (a) the presence of two acyl chains in the acidic diacylphospholipid and (b) the correct structure of its phosphate group.
We have recently proposed a novel mechanism for the peripheral attachment of proteins to lipid membrane surfaces(42) , based on the so-called extended lipid conformation (43) in which the two acyl chains of a lipid are pointed to opposite directions from the headgroup. This requires the torsion angle between the glycerol backbone carbon atoms C-2 and C-3 to be in the antiperiplanar range, as seen with NMR(44) . The extended lipid anchorage of peripheral proteins to membranes involves the accommodation of one of the lipid acyl chains within a hydrophobic cavity in the protein while the other chain remains in the membrane. This configuration thus results in a hydrophobic interaction between the protein and the lipid surface without penetration of the protein into the membrane hydrocarbon region(42) . Interestingly, Kahana et al.(45) reported that spectrin and some other red cell membrane cytoskeletal proteins have fatty acid binding sites, and they suggested this property to be a general feature of peripheral membrane proteins. Importantly, there is in cytochrome c a channel with an unknown function, lined by hydrophobic amino acid residues and leading from the surface of the protein into the heme crevice(46) . One side of the opening of this channel on the protein surface is constituted by a highly conserved cluster of positively charged lysines. This cluster is opposed on the other side by the invariant asparagine (Asn-52 in horse cytochrome c)(46) . In light of the structural studies on cytochrome c and the features of the lipid association of this protein we are suggesting the putative mechanism illustrated in Fig. 9for the binding of deprotonated and protonated acidic phospholipids to the A- and C-site, respectively. In both mechanisms one acyl chain would be accommodated within the hydrophobic channel. The A-site would involve electrostatic interactions of the deprotonated phosphate with the conserved lysines at positions 72 and 73. The C-site in turn would involve the invariant Asn-52 forming two hydrogen bonds with the protonated phosphate (Fig. 10). For CL with two phosphates, both polar interactions, i.e. docking of one protonated phosphate moiety to Asn together with a simultaneous electrostatic attachment of the other, deprotonated phosphate to the lysines, are possible. Hydrogen bonding by Asn is greatly enhanced in a hydrophobic environment, i.e. in a low dielectric medium(47) . The enhanced ability of this Asn to form hydrogen bonds is also evident from studies on the structural water molecules in cytochrome c(48) . Accordingly, one of the water molecules (Wat-1) is hydrogen bonded to Asn-52, Tyr-67, and the carbonyl of Ile-75(48) . This structure of the C-site would also be compatible with the close vicinity of one of the CL phosphates to the heme moiety(29, 30) . In this context it is of interest that Asn and Gln are abundant in the interfacial regions of transmembrane proteins, adjacent and in close contact with the headgroups of membrane lipids(49) . Examples of such proteins are Escherichia coli lactose permease (50) and the platelet-activating factor receptor(51) . Accordingly, it is tempting to suggest that Asn and Gln would be generally involved in liganding protonated acidic phospholipids to the boundary of transmembrane proteins.
Figure 9: Model for the binding of cytochrome c to acidic phospholipids. Top, the A-site binding of cytochrome c to PG. Bottom, the C-site binding of cytochrome c to PG. See ``Discussion'' for details.
Figure 10:
Hydrogen bonding between Asn and
protonated phosphate of an acidic phospholipid. Rrepresents the glycerol backbone
containing two acyl chains. R
represents
the polar head group glycerol in PG.
The depicted mechanism is compatible with the available data on cytochrome c-lipid association, such as the involvement of hydrophobic interactions in the absence of penetration of cytochrome c into lipid monolayers(52) , as well as the depletion of acyl chains from the lipid monolayer in contact with cytochrome c demonstrated by x-ray studies(53) . Catalytic hydrogenation of the unsaturated lipids of mitochondria reveals that in the presence of membrane proteins the acyl chain double bonds of CL are less accessible to reduction than those of other mitochondrial lipids (54) . However, differences between the various lipids were abolished in the absence of membrane proteins. This could easily be understood if some of the acyl chains in CL are accommodated within the hydrophobic channel of cytochrome c and perhaps also other mitochondrial proteins.
For the A- and C-site interactions the rate-limiting
contribution would be ionic and hydrogen bonding, respectively.
Accordingly, the former interaction would be competitively blocked by,
for instance, ATP(20) , whereas the latter would be efficiently
controlled by the protonation of the phosphate head group of the acidic
lipids, further influenced by factors such as pH, surface charge
density, and membrane potential(32, 55) . The rate
constant for the association of cytochrome c to either PG- or
CL-containing surfaces (k) should greatly exceed
the value for the rate constant for detachment (k
). This difference between the rate constants
should arise because of the involvement of hydrophobic interactions. It
is possible that an electrostatic interaction of cytochrome c with liposomes is initially required for the binding of the
protein to the liposome surface in the right orientation and perhaps
also for the opening of the cavity of the protein. Conformational
changes and destabilization of protein structure (27) have been
reported as a consequence of cytochrome c-lipid
interactions(25, 26, 27, 28, 29, 30, 31) .
The mechanism for cytochrome c lipid interactions illustrated
here could also be involved in the membrane association of other
proteins such as protein kinase C, CTP:phosphocholine
cytidyltransferase, and annexins(42) . For example, in the case
of annexin V an interaction between the phosphate group and the protein
and a hydrophobic interaction between the sn-2 acyl chain and
the protein seem to be involved(56) .