(Received for publication, October 5, 1995; and in revised form, January 2, 1996)
From the
Phospholamban is a phosphoprotein regulator of cardiac
sarcoplasmic reticulum which is phosphorylated in response to
-adrenergic stimulation. Previous results have shown that
phospholamban forms Ca
-selective channels in lipid
bilayers. The channel-forming domain has been localized to amino acid
residues 26-52, which form a stable pentameric, helical
structure. The specific residues responsible for stabilizing the
pentameric membrane domain of phospholamban have been identified by
mutational analysis. Residues 26-52 were individually mutated to
Ala or Phe, and the ability of the resulting mutant to form a pentamer
or other oligomer was assessed by SDS-polyacrylamide gel
electrophoresis analysis. Replacement of Leu
,
Ile
, Leu
, Ile
, or Leu
by Ala prevented pentamer formation, indicating their essential
involvement in the oligomeric assembly. The heptad repeats, and
3-4-residue spacing of the essential amino acids suggest that
residues 37-52 adopt a pentameric coiled-coil structure
stabilized by a leucine zipper motif formed by the close packing of
Leu
, Ile
, Leu
, Ile
,
and Leu
. The resulting symmetric structure contains a
central pore defined by the hydrophobic surface of the five stabilizing
leucine zippers, which are oriented to the interior and form the
backbone of the pentamer.
PLB ()is a small oligomeric phosphoprotein of cardiac
SR that regulates Ca
transport across this
intracellular membrane organelle (Tada and Inui, 1983; Sham et
al., 1991). Phosphorylation of PLB activates the Ca
pump of SR and increases Ca
uptake (Lindemann et al., 1983) by a mechanism that remains to be completely
understood (James et al., 1989; Cantilina et al.,
1993; Colyer, 1993; Toyofuku et al., 1994; Voss et
al., 1994). Structural analysis of PLB has been instrumental in
the discovery of its activity as a channel protein (Kovacs et
al., 1988), but the physiological relevance of this activity to
Ca
sequestration is not yet clear (Reddy et
al., 1995).
Following the development of a method to purify PLB
to homogeneity from SR vesicles, analysis by SDS-PAGE suggested that
PLB was a pentamer of identical 5-6-kDa subunits (Wegener and
Jones, 1984; Jones et al., 1985) and further indicated that
each subunit was dually phosphorylated by cAMP- and
Ca/calmodulin-dependent protein kinases (Simmerman et al., 1986; Wegener et al., 1986). Peptide mapping
studies showed that each subunit contained two domains: a cytosolic,
hydrophilic domain incorporating the two phosphorylation sites and a
hydrophobic domain responsible for the oligomeric subunit interactions
(Wegener et al., 1986). Sequence analysis of PLB confirmed
that it is a noncovalent oligomer of identical subunits (Simmerman et al., 1986), each containing 52 amino acids (Fujii et
al., 1987). The amino-terminal hydrophilic domain contains the
residues serine 16 and threonine 17 phosphorylated by cAMP- and
Ca
/calmodulin-dependent kinases, respectively
(Simmerman et al., 1986), whereas the hydrophobic domain is
located within residues 26-52. Empirical analysis of the
carboxyl-terminal residues 26-52 suggested that they could form
an amphipathic helix sufficiently long to traverse the SR membrane and
that five such helices could assemble to a pentameric pore-forming
structure (Simmerman et al., 1986). Single channel recording
experiments have demonstrated that PLB in planar bilayers does in fact
exhibit voltage-regulated Ca
channel activity (Kovacs et al., 1988). Although the primary structure of PLB suggests
that the membrane-embedded region of the protein is localized to
residues 26-52, the role of specific residues in this domain to
the oligomeric stability and channel activity is largely unknown.
Solubilized preparations of residues 26-52 have been shown by
circular dichroism spectroscopy to adopt a predominantly helical
configuration (Simmerman et al., 1989). Residues 26-52
incorporated as pentamers into phospholipid bilayers are also mainly
helical (Tatulian et al., 1995). Further questions remain
regarding the arrangement, orientation, and topography of the native
membrane-spanning domain of PLB in SR vesicles.
To probe the structure and stabilizing interactions of the membrane-spanning channel domain of PLB, mutational analysis has been implemented. Mutagenesis has been used successfully to determine the sequence specificity of interacting helices in glycophorin A (Lemmon et al., 1992a, 1992b), heat shock factor (Rabindran et al., 1993), and the GCN4 DNA-binding domain (van Heeckeren et al., 1992; Harbury et al., 1993). It is assumed that replacement of a residue with Ala, which results in disruption of the PLB pentameric assembly, indicates the essential role of that residue in the stabilization of the quaternary structure. Such has been found the case for a de novo synthetic approach to examining the stability of two-stranded coiled-coils (Zhou et al., 1992). The results of PLB mutagenesis indicate that a leucine zipper stabilizes the pentameric membrane domain of PLB and forms a coiled-coil pore structure. A preliminary account of this work appeared earlier in abstract form (Simmerman et al., 1994).
Figure 1:
In vitro translation of
wild-type PLB and PLB mutated in alanine residues. cRNA encoding
wild-type (WT) PLB and PLB with single alanine mutations
(listed at top of figure) was in vitro translated in
the presence of [S]methionine. After 24 h at
room temperature, SDS sample buffer was added and electrophoresis was
conducted using a 10% polyacrylamide gel (Porzio and Pearson, 1977).
The resulting autoradiograph is shown. Pentamer and Monomer denote the pentameric and monomeric mobility forms of
PLB, respectively. The diffuse area of radioactivity visible above the
PLB monomer band contained PLB dimers, which were poorly resolved in
this gel, but were identified clearly with use of a 7-18%
gradient gel (data not shown). ± Boil indicates whether WT samples were boiled in SDS prior to PAGE. No RNA is a control showing that negligible
S incorporation
occurred when PLB cRNA was omitted from the translation mix. After
autoradiography, the dried gel was placed in a GS-250 Molecular Imager
(Bio-Rad), and the amount of recombinant PLB in each lane was
quantified. Background radioactivity in the No RNA lane was
deducted for each determination. Eight replicate experiments of this
type were performed, and the averaged results for the percentages of
PLB pentamer formation for each mutation are listed in Table 1.
Molecular weight standards (
10
) are shown on
the left.
Figure 2: In vitro translation of phenylalanine mutations. Amino acid residues 26-31, 33-34, and 36-52 of PLB were individually changed to phenylalanine, and the mutant proteins were in vitro translated and analyzed as described in the legend to Fig. 1. In addition, a cysteine 41 to leucine (C41L) mutation was also analyzed. The percentages of pentamer formation for each of the phenylalanine mutations are listed in Table 1.
Changing Cys to Phe was
previously reported to destabilize the pentamer (Fujii et al.,
1989), as is confirmed in Fig. 2and Table 1. We also
tested if substitution with another hydrophobic residue at this
position would disrupt the pentamer. Unexpectedly, changing Cys
to Leu led to a new oligomeric species running with a mobility
expected for a tetramer (Fig. 2). Formation of tetramers by the
C41L mutation is characterized below.
Figure 3: In vitro translation of leucine/isoleucine mutations. Residues 37, 40, 44, 47, and 51 of PLB were mutated to leucine or isoleucine (listed at top of figure) by in vitro translation and analyzed as described in Fig. 1. The percentages of pentamer formation from four separate experiments of this type are listed in Table 2.
Fig. 4demonstrates that wild-type PLB, purified from either
SR vesicles or Sf21 cells, migrated predominately as a pentamer, with
some monomers and dimers also present. Boiling the wild-type proteins
in SDS prior to PAGE dissociated the pentamers and allowed the
visualization of tetramers and trimers, as well as the other two
mobility forms. Most importantly, the C41L mutation migrated
principally as a tetramer, aligning exactly with tetramers formed from
wild-type PLB boiled in SDS. A very weak pentamer band could be
detected with the C41L mutation, which eliminated the trivial
possibility that the tetrameric form of C41L was actually a pentamer
but with an aberrant mobility. As observed by in vitro translation, the L37A mutation migrated principally as a monomer.
Some dimers were also detected, which were also observed with in
vitro translates when gradient gels were used. In other
experiments we were able to detect a minor amount of pentamer formation
by L37A, but only when electrophoresis through the stacking gel was
conducted at a very slow rate with a very high concentration of L37A
(20 µg of protein/gel lane) (data not shown). Thus, unique
association constants for each of the mutant proteins analyzed may
exist, the determination of which is beyond the scope of this study.
Figure 4: SDS-PAGE of purified wild-type (WT) PLB and C41L and L37A mutations. 11 µg of PLB purified from canine cardiac SR vesicles (Cardiac) and insect cells (Sf21 Expressed) were electrophoresed in an 8-17% polyacrylamide gradient gel according to Laemmli(1970), and the gel was stained with Coomassie Blue. WT and mutant proteins are designated at the top of the figure. ± Boil indicates whether samples were boiled in 5% SDS immediately prior to electrophoresis. The numbers 5 to 1 in the left margin indicate the pentameric through monomeric mobility forms of PLB.
In the present site-directed mutagenesis study, residues
responsible for stabilizing the pentameric structure of PLB were
identified by mutation to Ala and were found to be Leu,
Ile
, Leu
, Ile
, and Leu
(Fig. 1). The mechanism by which substitution of Ala for
the critical stabilizing residues prevents the pentameric structure is
not by disrupting the native alpha helical structure, since Ala is a
strong helix-forming residue (Chou and Fasman, 1978). Furthermore,
residues neighboring the stabilizing amino acids may be changed to Ala
with no effect on quaternary structure and, therefore, presumably no
effect on secondary structure either. We conclude that specific
characteristics of the aliphatic side chains of Leu
,
Ile
, Leu
, Ile
, and Leu
stabilize the oligomeric structure of PLB.
The most striking
observation from the results is the spacing between critically
sensitive residues: an alternating series of three leucines and two
isoleucines with each isomeric position separated from the next by
seven residues. The heptad repeat pattern is a diagnostic feature of
the leucine zipper structural motif (Landschulz et al., 1988),
in which the region containing the leucine heptad repeats forms a
helix, and the leucines line up along one face of the helix (at a pitch
of 3.5 residues/turn) to promote oligomerization of the helices in a
parallel orientation. In PLB the repeating isoleucines are offset at
3-4-residue intervals from the leucine heptad repeat (Fig. 5A), conforming to the model of a coiled-coil in
which hydrophobic residues occupy positions a and d of a repeating heptad of a-g residues (O'Shea et al., 1989; Zhou et al., 1992; Zhu et al.,
1993). Thus Leu, Leu
, and Leu
occupy the a position, whereas Ile
and
Ile
occupy the d position in the motif. The
present mutagenesis results are thus consistent with a model of PLB in
which residues 37-52 form a 3.5 residue/turn helix, creating a
leucine zipper of three helical turns and a complimentary pair of
aligned isoleucines with the appropriate axial spacing of two helical
turns to interdigitate with the leucines, forming a symmetric,
coiled-coil pentamer (Fig. 5B). Five identical zippers
formed by interaction between the three leucines of one helix with the
two isoleucines of the adjacent helix thus stabilize the PLB quaternary
structure. Mixed Leu/Ile zippers have been observed previously (Cohen
and Parry, 1990; Atkinson et al., 1991), and the coiled-coil
model for the parallel PLB helices is consistent with the observation
that parallel orientation of helices is very unusual except in a
coiled-coil structure (Oas et al., 1990). The length of the
region containing sites important for PLB pentamer formation further
suggests extensive interhelical interactions that are more consistent
with a coiled-coil model (Zhou et al., 1992) and are not
consistent with a single closest approach crossover point between
adjacent rigid helices.
Figure 5: Heptad repeat cartoon model of PLB monomer and pentamer. A, residues 37-52 of monomeric PLB are configured as a 3.5 residues/turn helix with positions a through g of the heptad repeat circled. Leucine and isoleucine residues constituting the zipper are localized to positions a and d, respectively (filled circles). Changing any amino acid (circled) here to alanine destabilizes the pentamer. B, PLB pentamer. The five monomers are arranged to allow intermolecular contact at positions a and d (shaded), such that the leucine and isoleucine residues of one monomer align with leucine and isoleucine residues of adjacent monomers. Alanine substitutions here disrupt the pentamer. Alanine substitutions at positions e and g do not affect stability, but some phenylalanine mutations here are disruptive.
The proposed model of PLB residues
37-52 (Fig. 5B) suggests that residues occupying
the b, c, and f axial positions are oriented
to the exterior surface of the structure and that the PLB pentamer
would be insensitive to a bulky residue substitution at these sites.
Our observation that all residues predicted to occupy the b, c, and f positions of the heptad repeat, viz. Ile, Leu
, Leu
,
Ile
, Cys
, Val
, and
Leu
, accepted Phe without loss of pentamer formation
supports our model of this domain as a coiled-coil bundle of leucine
zipper helices with these residues oriented to the exterior.
In
contrast to the absence of steric hindrance inherent to the b, c, and f axial positions of the leucine zipper
helical domain model, residues occupying the e and g positions are in closer proximity in the cleft between adjacent
helices (Fig. 5B). Although not specifying the primary
stabilizing interactions between helices, these sites may contribute
secondarily to oligomeric stability through interactions between their
side chains (Hu et al., 1993), and these sites are more likely
to exhibit a restricted range of acceptable substitutions based on the
volume occluded by adjacent structure. Residues Cys and
Ile
in the e position and residues Leu
and Met
in the g position all accepted
replacement with Ala without preventing pentamer formation. However,
replacement with Phe at positions 41 and 50 prevented pentamer
formation, but the bulky side chain did not prohibit pentamer formation
at positions 43 and 48. The results suggest that the cleft positions
within the PLB leucine zipper helical structure are not equivalent,
with positions 41 and 50 more restricted than positions 43 and 48. The
formation of a PLB tetramer upon substitution of Cys
with
Leu ( Fig. 2and 4) further indicates a unique role for this site
in the higher order structure of PLB. A site-directed mutagenesis study
of the role of PLB Cys residues in pentamer stability (Fujii et
al., 1989) has shown previously that PLB quaternary structure is
most intolerant of changes in Cys
. Pentamer formation and
stability decreased as the size of the substituted side chain
increased. Confirmed by the present work, these results are best
interpreted as the effect of steric hindrance on pentamer formation and
the disruption of close packing on stability, consistent with the model
of PLB presented here in which Cys
is confined to an
interfacial cleft between adjacent helices.
It has been proposed
that replacement of the Leu at positions a or d in a
leucine zipper with the -branched Ile may influence the
stoichiometry of the oligomer formed through the specificity of packing
interactions (DeGrado et al., 1989; Zhu et al.,
1993). This has been demonstrated using mutants of the GCN4 leucine
zipper (Harbury et al., 1993). However, that study did not
address structural determinants for oligomers larger than a tetramer.
Our results indicate that the PLB pentamer is primarily stabilized by
interactions between leucines in the heptad position a (residues 37, 44, and 51) with isoleucines in position d (residues 40 and 47) (Fig. 4). According to the algorithm
derived from GCN4 mutants, occupancy of the a position with
Leu and the d position with Ile would be expected to specify a
tetramer. As this is not observed with PLB, other residues in the PLB
heptad pattern must therefore be involved in specifying the number of
subunits in the oligomer. Our observation that substitution of
Cys
for Leu results in a PLB tetramer argues that this
site, in the e position of the heptad, is important in
determining the aggregation number of the PLB oligomer. It thus appears
that whereas occupancy of positions a and d with
either Leu and Ile is necessary for the coiled-coil motif and primarily
responsible for the stability of the resulting oligomer, it is not
sufficient to specify the number of helices in the coiled-coil
quaternary structure.
To probe the role of -branched residues
at positions a and d in determining the oligomeric
state of PLB, we independently mutated Leu
,
Leu
, and Leu
to Ile and changed Ile
and Ile
to Leu. Mutations of Leu
,
Ile
, and Leu
had no effect on the formation
of the native PLB pentamer, whereas mutation of Leu
to Ile
nearly completely eliminated pentameric assembly of PLB and mutation of
Ile
to Leu completely abolished the ability of the PLB
monomers to associate (Fig. 3). Intermediate sized oligomers
were not observed for any of these mutations. These results confirm the
concept that in PLB the a and d heptad positions
stabilize the pentameric assembly but do not specify the number of PLB
helices in the oligomer. The tolerance for substitution of Leu/Ile
isomers at the three C-terminal positions 44, 47, and 51 may indicate a
greater structural flexibility in this region of the oligomer. This
suggests that the contributions of the leucine zipper residues to the
PLB pentamer are not equivalent, with residues Leu
and
Ile
contributing more importantly than residues
Leu
, Ile
, or Leu
. Taken together
with the special sensitivity of Cys
to mutation, the
results suggest that specific contacts in the region of residues
37-41 are primarily responsible for stabilizing the pentameric
PLB assembly, with contacts in other regions of the coiled-coil serving
as secondary sources of stabilizing energy. The location of these
critical residues at the N terminus of the leucine zipper motif
suggests that correct initiation and propagation of the nascent leucine
zipper helix during translation is important for native PLB quaternary
structure.
The prevention of pentamer formation observed by mutation
of Ile to Phe and also by mutations at Gln
,
Phe
, Ile
, and Asn
indicates that
residues 26-36 must contribute in some way to the PLB pentameric
structure, as suggested previously (Simmerman et al., 1986).
Although Ile
fits the PLB pattern of alternating Ile and
Leu residues at 3-4 residue spacing, the zipper must not extend
N-terminally from Leu
to Ile
, as Ala is
accepted at the latter site without loss of pentamer formation.
Therefore, the side chain of Ile
does not contribute to
the essential hydrophobic interactions stabilizing the pentamer. The
aromatic side chain of Phe
appears obligatory at this
position, as its replacement with Ala prevents formation of the PLB
pentamer. The essentiality of a specific, outward facing orientation at
this structural site to enable pentamer formation suggests that
Phe
may play a key function in correct folding of
secondary structural elements during translation, preventing premature
initiation of the zipper-type helix at Ile
. Thus we
propose that although residues 26-36 appear uninvolved in the
close-packed hydrophobic interactions stabilizing the pentamer, they
are critical for appropriate initiation and folding of PLB leucine
zippers and their post-translational orientation and assembly into the
pentamer. Interestingly, the belt of Phe
and Phe
aromatic side chains encircling the pentameric structure defines
the interface between the polar and nonpolar regions of PLB. PLB shares
this structural motif with membrane channel porins (Cowan et
al., 1992; Weiss and Schulz, 1992), in which the band of aromatic
residues may protect polar structural elements at the membrane
interface from conformational fluctuations during vertical
displacements of the protein within the membrane (Kreusch et
al., 1994). It may be that PLB phenylalanines also serve to
correctly position PLB within the bilayer and to protect polar
interfacial residues.
The PLB oligomeric structure is stabilized only by contacts between hydrophobic residues without contribution from side chain electrostatic or hydrogen bonding interactions, commonly observed in other natural coiled-coils (Talbot and Hodges, 1982; Cohen and Parry, 1990). Synthetic two-stranded coiled-coils having only Leu at the a and d heptad positions are much more stable than naturally occurring two-stranded coiled-coils containing a significant proportion of Val and Ala at the critical a and d heptad positions (Hodges et al., 1990). It was thus suggested that natural coiled-coil proteins have not evolved for maximum stability. Considering that the PLB coiled-coil pentamer is stabilized only by Leu and Ile interactions, it appears that the PLB oligomeric structure has uniquely evolved very specifically for great stability.
The PLB membrane domain does not conform to the model of integral membrane proteins as ``inside-out'' with respect to the relative polarity of surface and buried residues (Engelman et al., 1986). Instead, it is a cylinder of close-packed hydrophobic helices (Fig. 5), which may fulfill predictions that the coiled-coil leucine zipper motif may serve as the defining structure for certain channels (Lear et al., 1988; Karle et al., 1988; DeGrado et al., 1989; Popot and Engelman, 1990; Harbury et al., 1993). Modeling suggests the pore formed by a pentameric coiled-coil would have a diameter of 5 Å (Lear et al., 1988), sufficient to accommodate a water molecule (2.8-Å diameter) or a hydrated small ion. The association of channel activity with the pentameric domain of PLB residues 26-52 emphasizes the utility of PLB as a natural model system for elucidating channel structure/function relationships and mechanisms of ion conductance, as suggested previously (Kovacs et al., 1988).
While this work was in progress (Simmerman et al., 1994), a study was published using chimeric protein analysis to examine PLB transmembrane domain structure (Arkin et al., 1994). The two approaches concur that amino acids stabilizing the pentamer line up on faces of a 3.5 residues/turn helix, and the resulting pore structure is defined by hydrophobic residues. However, the wild-type chimeric protein studied by Arkin et al.(1994) did not associate with the same oligomeric distribution as native PLB. The random substitutions created by saturation mutagenesis were confined to residues 35-52, and the enabling role of residues 26-36 in PLB pentameric assembly was not reported. The use of an indirect, qualitative detection method also limited identification of some structurally important residues. For example, the PLB fusion protein containing C41L was expressed as a monomer by Arkin et al. (1994), whereas we clearly show that when native PLB is substituted with C41L, the protein is a tetramer. Our results provide evidence that the leucine zipper helical motif can indeed stabilize an oligomeric membrane domain to form a coiled-coil structure with potential relevance to ion channels.