(Received for publication, December 26, 1995; and in revised form, February 13, 1996)
From the
Mutational analysis of several amino acids in the transmembrane
region of the sarcoplasmic reticulum ATPase was performed by expressing
wild type ATPase and 32 site-directed mutants in COS-1 cells followed
by functional characterization of the microsomal fraction. Four
different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type
ATPase; (b) Ca transport inhibited to a
greater extent than ATPase hydrolytic activity; (c) inhibition
of transport and hydrolytic activity in the presence of high levels of
phosphorylated enzyme intermediate; and (d) total inhibition
of ATP utilization by the enzyme while retaining the ability to form
phosphoenzyme by utilization of P
. Analysis of experimental
observations and molecular models revealed short and long range
functions of several amino acids within the transmembrane region. Short
range functions include: (a) direct involvement of five amino
acids in Ca
binding within a channel formed by
clustered transmembrane helices M4, M5, M6, and M8; (b) roles
of several amino acids in structural stabilization of the helical
cluster for optimal channel function; and (c) a specific role
of Lys
in sealing the distal end of the channel,
suggesting that the M4 helix rotates to allow vectorial flux of
Ca
upon enzyme phosphorylation. Long range functions
are related to the influence of several transmembrane amino acids on
phosphorylation reactions with ATP or P
, transmitted to the
extramembranous region of the ATPase in the presence or in the absence
of Ca
.
Understanding the topology of functional domains is required to
clarify the mechanism of cation translocation by membrane-bound
ATPases. With regard to the Ca transport ATPase of
sarcoplasmic reticulum (SR), (
)it is known that the
catalytic and phosphorylation sites reside within the extramembranous
region of the enzyme. On the other hand, the functional relevance of
the transmembrane region was revealed by the mutational work of Clarke et al.(1) , who suggested that six residues
originating from four transmembrane helices (M4, Glu
; M5,
Glu
; M6, Asn
, Thr
, and
Asp
; M8, Glu
) are involved in
Ca
binding (Fig. 1). We have now performed a
mutational analysis of several amino acids in the transmembrane region
to evaluate in detail their roles in Ca
binding and,
more generally, in the catalytic and transport functions of the
Ca
ATPase.
Figure 1:
Diagrammatic representation of
transmembrane helices M4, M5, M6, and M8 of the SR ATPase. The six
putative Ca binding residues identified by Clarke et al.(1) are identified with rectangular boxes. Note that three residues are within M6; only two of these three
residues are confirmed by this paper. Energy transduction occurs
through a long range intramolecular linkage between the phosphorylation
site (Asp
) in the cytosolic extramembranous region and
the Ca
binding domain in the transmembrane
region(2) .
The expressed SERCA-1 ATPase was detected by Western blotting, using monoclonal antibody 9E10 to the c-myc epitope (8) and, in parallel, a monoclonal antibody to the chicken Ca-ATPase, CaF3-5C3(3) . In addition to Western blots, quantitation of SERCA-1 expression was also obtained by enzyme-linked immunosorbent assays in microtiter plates.
ATPase activity was measured by determination of
P (Lanzetta et al.(9) , also as described
by Zhang et al.(5) ). The
Ca
-dependent activity was calculated by subtracting
the Ca
-independent ATPase from the total ATPase. Both
Ca
uptake and Ca
-dependent ATPase
activity were corrected to account for the level of expressed protein
in each microsomal preparation as revealed by immunoreactivity and with
reference to microsomes obtained from COS-1 cells transfected with wild
type cDNA.
The rates of phosphoenzyme decay were determined by first obtaining steady-state levels of phosphoenzyme as explained above. Ten s after the addition of radioactive ATP, 0.5 ml of 1.0 mM nonradioactive ATP was added with rapid mixing, and samples were acid quenched at serial times. A zero time base line was obtained by acid quenching before the chase. The entire procedure was carried out in ice and in the cold room. Washings, electrophoresis, and autoradiography were performed as described above.
The rates of phosphoenzyme decay
were determined by first obtaining equilibrium levels of phosphoenzyme
incubated at 37 °C with 50 µMP
as described above and then adding with rapid mixing 1.0 ml of
1.0 mM (ice-cold) nonradioactive P
. The reaction
was then quenched at serial times by the addition of 0.11 ml of 10.0 M perchloric acid. A zero time sample was obtained by
quenching before the addition of nonradioactive P
. Addition
of carrier protein, centrifugations, washings, electrophoresis, and
autoradiography were carried out as described above.
Figure 2:
Immunologic detection of ATPase
expression. The three rows of bands are examples of
Western blot analysis using microsomal fractions derived from
transfected COS-1 cells. The blots were obtained as described under
``Experimental Procedures,'' using monoclonal antibody 9E10
for detection of the c-myc tag. Note the absence of expression
in pCDL control (cells transfected with plasmid without the cDNA
insert) and K297G (cells transfected with cDNA encoding the Lys
Gly mutant). WT, wild
type.
A special case was the
lack of expression of the Lys
Gly mutant (see Fig. 2), which we failed to recover with the microsomal fraction
of COS-1 cells after repeated transfections with two batches of plasmid
amplified, banded, and sequenced at different times. Northern blot
analysis confirmed the presence of an mRNA transcript for this mutant
consistent with that of wild type (results not shown). It is of
interest that mutation of the same residue to Phe did yield expression,
although at low level, and the Lys
Met,
Lys
Arg, and Lys
Glu mutants
were expressed at normal levels (Fig. 2).
Figure 3:
Examples of ATP-dependent Ca uptake and ATPase activity by microsomal vesicles obtained from
transfected COS-1 cells. The two upper panels show
Ca
uptake; the two lower panels show
Ca
-dependent ATPase activity. Note that in some
mutants (e.g. Val
Ala) Ca
uptake undergoes a relatively greater inhibition than ATPase
activity. The reaction mixture for Ca
uptake
contained 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl
, 0.2 mM CaCl
, 0.26 mM EGTA to yield 1.4 µM free
Ca
(11) , 5-10 µg of microsomal
protein/ml, 5 mM potassium oxalate, and 3 mM ATP. The
reaction was started (30 °C) by the addition of oxalate and ATP and
was terminated at sequential times by vacuum filtration. The reaction
mixture for ATPase activity contained 20 mM MOPS, pH 7.0, 80
mM KCl, 3 mM MgCl
, 0.2 mM EGTA,
0.2 mM CaCl
, 5 mM azide, 30 µg of
microsomal protein/ml, 3 µM ionophore A23187, and 3 mM ATP. Ca
-independent ATPase activity was assayed
in the presence of 2 mM EGTA and no added
Ca
. The reaction was started (37 °C) by the
addition of ATP, and samples were taken at serial times for P
determination. WT, wild
type.
A group of
mutations producing inhibition of ATPase hydrolytic activity is shown
in Table 2. It is of interest that in all of these mutations,
Ca transport was inhibited significantly more than
hydrolytic activity. Since this uncoupling effect is produced by
mutations which do (Table 2) or do not (Table 1) inhibit
hydrolytic activity, it is apparent that a specific structural
perturbation is involved in its onset, independent of other
perturbations that may affect catalytic activity.
Characterization
of the six mutations originally reported by Clarke et
al.(1) , who proposed that the six native residues
participate in Ca binding, is shown in Table 3.
We confirmed that these mutations produce total inhibition of
Ca
transport and hydrolytic activity, and then we
extended the functional characterization as explained below.
It is
noteworthy that the effects of mutations are very specific and
dependent both on the residues that are mutated and the side chains
that are introduced. An example of the high specificity of mutational
effects is found in the total inhibition resulting from the Glu
Ala mutation compared with the full activity retained by
the Glu
Gln mutant (Fig. 3). Of great
interest are also the different and specific effects produced by
mutating Lys
to Gly (no expression), to Phe (low
expression and low activity), to Met (low activity), and to Arg or Glu
(nearly normal ATPase and slight inhibition of Ca
uptake).
Figure 4:
Minimal reaction scheme for the
Ca ATPase. Only the four partial reactions that can
be measured by chemical methods (Ca
binding,
formation of phosphorylated intermediate by utilization of ATP,
vectorial translocation of bound Ca
, and hydrolytic
cleavage of P
) are listed for convenience of easy reference
in the text. As implied under ``Discussion,'' isomeric
transitions (such as, or in addition to the E1 to E2
transition postulated by De Meis and Vianna(12) ) occur in
parallel with the four reactions outlined in the
diagram.
Figure 5:
Examples of phosphoenzyme steady-state
levels formed in the presence of Ca and ATP. The
phosphoenzyme was obtained as described under ``Experimental
Procedures'' and determined by autoradiography. Note the absence
or very low levels of phosphoenzyme when the Glu
Gln, Glu
Gln, Asn
Ala,
Thr
Ala, Asp
Asn, and
Glu
Ala mutants are used. Characterization of
these and all the other mutants is reported in the tables. WT,
wild type.
We then conducted isotopic chase
experiments to evaluate the effect of mutations on the decay of the
phosphorylated enzyme intermediate (reactions 3 and 4 in Fig. 4). We found that mutants sustaining ATPase activity
comparable to that of the wild type enzyme exhibited phosphoenzyme
turnover that was similar to that of the wild type enzyme. On the other
hand, the rate of phosphoenzyme decay was reduced in mutants exhibiting
inhibition of steady-state ATPase activity (e.g. see
Lys
Met, Cys
Ala, and
Ile
Ala in Fig. 6and Table 2).
Figure 6:
Decay of phosphoenzyme obtained by
utilization of ATP in the presence of Ca. Isotopic
chase of phosphoenzyme was performed as described under
``Experimental Procedures.'' Note the delayed decay in the
Lys
Met, Cys
Ala, and
Ile
Ala mutants compared with wild type (WT). Characterization of these and all the other mutants is
reported in the tables.
Observations of mutational effects on phosphoenzyme turnover are very important for two reasons. (a) They confirm the inhibitory effects of mutations under conditions that are not dependent on the enzyme concentration (since phosphoenzyme decay is a first order phenomenon); this dispels doubts on whether observed inhibitions of steady-state ATPase activity (which is dependent on enzyme concentration) may in fact be related to inaccuracies in the immunological determination of expressed ATPase. (b) They indicate that the mutational effect is on ATPase partial reactions that follow formation of the phosphorylated intermediate (step 3 and/or step 4 in Fig. 4), and these reactions are rate-limiting for completion of the catalytic and transport cycle.
When we used samples of wild type enzyme
reacted separately with P under these conditions, we
obtained identical phosphoenzyme levels, indicating that our technique
of detection was highly reproducible (Fig. 7). On the other
hand, the equilibrium levels of phosphoenzyme obtained with various
mutants were quite different. These differences were not related to
whether the mutants exhibited or did not exhibit inhibition of
steady-state ATPase activity and/or enzyme phosphorylation with ATP. In
some cases ( Fig. 7and Table 1Table 2Table 3),
high levels of phosphoenzyme were obtained through the P
reaction with mutants yielding low ATPase activity
(Val
Ala, Cys
Ala,
Ile
Ala), or no ATPase activity and no phosphoenzyme in the presence of ATP and Ca
(Glu
Gln, Glu
Gln). On
the other hand, hardly detectable levels of phosphoenzyme were obtained
through the P
reaction with some mutants (Val
Ala and Leu
Ala, for instance)
retaining relatively high ATPase activity and enzyme phosphorylation
with ATP ( Fig. 7and Table 1).
Figure 7:
Equilibrium levels of phosphoenzyme
obtained by utilization of P in the absence of
Ca
. The upper row of bands was
obtained with wild type (WT) samples that were incubated and
processed separately to demonstrate the accuracy of phosphoenzyme
recovery and autoradiographic detection. The concentrations of mutant
proteins were adjusted (based on immunologic titration) to yield the
same amount of expressed ATPase as in the wild type sample in each of
the two lower rows. Note the very high levels of Val
Ala, Cys
Ala, Ile
Ala, Glu
Gln, and Glu
Gln. Note also the low levels of Leu
Ala and Thr
Ala. See ``Experimental
Procedures'' for experimental
conditions.
As opposed to the
steady-state levels (resulting from all four steps of the diagram in Fig. 4) obtained in the presence of ATP and
Ca, the P
reaction (reverse of step 4 of
the diagram in Fig. 4) may be considered to occur as
where E is the enzyme equilibrated with P. We
then explored the possibility that the observed variations of E-P may be due to altered affinity of the mutants for
P
. We found, however, that the P
concentration
used in our experiments was saturating in all cases (not shown).
Therefore, the observed variations of phosphoenzyme levels were likely
due to mutational effects on the E
P
E-P equilibrium constant, which is dependent on the ratio of
the two bidirectional rate constants. In fact when we followed the
phosphoenzyme decay after isotopic chase, we found that high levels of
phosphoenzyme were in most cases accompanied by slow decays, whereas
low levels of phosphoenzyme were accompanied by fast decays ( Fig. 8and Table 1Table 2Table 3).
Figure 8:
Decay of phosphoenzyme obtained by
utilization of P in the absence of Ca
.
Phosphoenzyme and isotopic chase were obtained as described under
``Experimental Procedures.'' Note the very slow decay of the
Glu
Gln, Glu
Gln, and
Asn
Ala mutants compared with the wild type
sample.
We
extended the functional characterization of these mutants by measuring
the levels of EP formed with P in the presence of
various concentrations of Ca
. We performed these
titrations at mildly acid pH to lower the affinity of the enzyme for
Ca
and to maximize any effect of mutational
perturbations on Ca
binding.
Under these
experimental conditions, half-maximal inhibition of the P reaction with wild type enzyme and several mutants is obtained at
approximately 20 µM Ca
. However, the
same parameter is shifted to the mM range when the Glu
Gln, Glu
Gln, Thr
Ala, Asp
Asn, and Glu
Ala mutants are used (Fig. 9, also (14, 15, 16) ). These experiments are quite
accurate and easy to interpret as they reflect equilibration of
Ca
and P
with the enzyme (as opposed to
studies on the Ca
concentration dependence of ATP
utilization, which depends on several kinetic constants). Our titration
experiments (Fig. 9) show that single mutations do not eliminate
completely the effect of Ca
but rather increase by
2-3 orders of magnitude the effective Ca
concentration (i.e. reduce the affinity of the enzyme
for Ca
). They also demonstrate unambiguously that the
Asn
Ala mutation does not interfere with
Ca
inhibition of enzyme phosphorylation by
P
, even though it does interfere with
Ca
-dependent enzyme phosphorylation with ATP. We
interpret our present findings to indicate that Asn
does
not participate in Ca
binding under our conditions
(as well as those of Clarke et al.(1) ) for enzyme
equilibration with P
. It is possible that Asn
participates in Ca
binding under different
conditions and/or in another specific conformational state of the
enzyme (e.g. Ca
occlusion in the presence of
CrATP(17) ).
Figure 9:
Inhibition of the P reaction
by Ca
. Incubations were carried out as described
under ``Experimental Procedures,'' but various aliquots of
CaCl
were added to yield the free Ca
concentrations (11) given in the
figure.
It should be pointed out that Andersen and
Vilsen (16) reported that the Ca sensitivity
of the Glu
Gln mutant is in the 10 µM range as opposed to the mM range observed in our
experiments (Fig. 9). It is likely that this difference is due
to the higher pH used by Andersen and Vilsen(16) , favoring
ionization of Ca
binding acidic functions with pK near neutrality(18) , thereby increasing the affinity of
the enzyme for Ca
and obscuring the effect of single
mutations.
Finally, we note that mutation of Glu
Ala interferes with inhibition of the P
reaction by Ca
, whereas mutation of Glu
Gln leaves the enzyme perfectly functional (also noted by Clarke et
al.(1) ). This is of specific interest, considering that
mutation Glu
Gln, Glu
Gln, or
Asp
Asn interferes strongly with inhibition of the
P
reaction by Ca
( Table 1and Table 3and Fig. 9). The different effect of the
Glu
mutations to Gln or Ala suggests that although the
Glu
, Glu
, and Asp
contributions to Ca
complexation depend on both
side chain oxygens, Glu
contributes only one side chain
oxygen, which is still present following mutation to Gln, but not to
Ala. As the acidic function is lost by the Gln mutant, we conclude that
the carbonyl oxygen is able to participate in coordination of
Ca
.
An important question is whether it is possible to
have two closely spaced calcium ions bound within the same domain (in
spite of possible charge repulsion). Crystallographic resolution of
several Ca binding structures (for instance, the
duplex Ca
binding site of thermolysin; (20) )
indicates that two Ca
can in fact reside in close
proximity and yields information regarding the appropriate distances
between binding oxygens and Ca
and between the two
bound Ca
.
In previous attempts to model all six
residues suggested by Clarke et al.(1) , we
encountered difficulty in orienting the diverging side chains of the
Asn, Thr
, and Asp
(which
originate from the same helix M6) for simultaneous participation in
Ca
binding. For this reason we proposed exclusion of
Thr
(2) . Our present experimental observations,
however, indicate clearly that Asn
is the residue to be
excluded; and this solves the problem. However, we do not exclude that
Asn
may participate in Ca
binding by
the enzyme in some specific conformational state that we do not see
under our conditions. Andersen and Vilsen suggested (16) that
Asn
may participate in complexation of only the proximal
(closer to the cytosolic side of the membrane) Ca
.
However, modeling shows that Asn
is actually the most
distal of the putative binding residues and is unlikely to interact
with the proximal Ca
.
We considered whether the
two oxygen functions of each acidic residue may converge on the same
Ca or may be shared by the two Ca
.
Indications that the proximal Ca
may be independently
influenced by the Glu
Gln mutation (21) suggest that Glu
contributes both oxygen
functions to the proximal Ca
as shown in model 1 of Fig. 10. Both oxygens of Glu
may then be
approximated to the distal Ca
as proposed by Andersen
and Vilsen (16) to provide adequate distribution of charge.
Approximation of Thr
to the distal Ca
then places Asp
in a position to share the two side
chain oxygens with the proximal and distal Ca
,
thereby contributing binding cooperativity. Finally, we place one of
the Glu
oxygens near the proximal Ca
since the different effects of mutation of this residue to Gln or
Ala indicate that only one oxygen participates in Ca
binding (see ``Results'').
Figure 10:
Two
alternative models of the duplex Ca binding site of
the Ca
ATPase. Model 1 (A and B)
shows coordination of the upper Ca
by
Glu
, Asp
, and Glu
, while the
lower Ca
is coordinated by Glu
,
Thr
, and Asp
. Model 2 (C and D) has coordination of the upper Ca
by
Glu
, Asp
, and Glu
, while the
lower Ca
is coordinated by Glu
,
Thr
, and Asp
. In both models
Glu
, Glu
, and Asp
contribute
two oxygen atoms each, while Thr
and Glu
contribute only one oxygen each. A and C, side
views with the membrane cytosolic side on top. B and D, views from the cytosolic or lumen side, as indicated. The open circles represent two calculated high affinity
Ca
loci in each model, as predicted by analysis of
the model coordinates and valence mapping. Note that no additional high
affinity sites are predicted in the entire model comprising the entire
length of the M4, M5, M6, and M8 cluster. The coordinates of the model
(in PDB format) are available upon request, permitting inspection and
manipulation of the model with the aid of any molecular graphics
program.
Model 1 in Fig. 10is not the only one that is consistent with the
experimental results. For instance, it is possible to reposition the
helices longitudinally so that Glu could bind the distal
Ca
and Glu
the proximal
Ca
, everything else remaining the same (model 2 in Fig. 10).
To accommodate the five residues while excluding
Asn, we found it sterically convenient to alternate the
four transmembrane helices as shown in Fig. 10. In the models
the five binding residues were positioned to approximate their oxygen
ligands within 2.6 Å of one or both calcium ions. Additional
oxygens may be contributed by coordinated water.
Although the
arrangements in Fig. 10are speculative, the important question
that they answer is whether the oxygen functions singled out by
mutational analysis can generate discrete areas of high binding
potential for Ca and if such areas are unique in the
transmembrane helical cluster. To this aim, we subjected the two models
to analysis of structural coordinates to obtain valence maps, using the
algorithm of Nayal and Di Cera(22) , which is a consistent
predictor of Ca
binding sites in proteins of known
structure. Analysis of the entire model generates two distinct areas
with valence points of 1.4 or higher, i.e. a high probability
for binding Ca
. The two areas are shown in Fig. 10, A-D, as cluster of dots. No other site
of high valence is present throughout the four clustered helices,
consistent with the results of extensive mutational work on the four
helices(23, 24) .
Of special interest are the
mutations of Lys, including Lys
Gly,
which interferes with ATPase assembly and expression, Lys
Met and Lys
Phe producing strong
functional inhibition, and Lys
Arg and Lys
Glu producing little or no inhibition ( Table 1and Table 2). In the model, Lys
places its charge at
the distal end of the M4 segment, facing the lumenal end of the
channel. In fact, Lys
provides the only positive charge
within the channel. It is apparent then that the presence of a highly
polar moiety in this position is required to stabilize the channel
structure. The important role of this stabilization is demonstrated by
the interference with ATPase expression produced by replacement of
Lys
with the much less restrictive Gly, which may break
propagation of the M4 helix during protein assembly(27) .
Furthermore, the functional inhibition produced by Lys
replacement with Met or Phe and the lack of inhibition by
Lys
replacement with Arg or even with Glu indicate that
intrusion of a net charge (positive or negative) at the distal end of
the channel seals the lumen and prevents flux of Ca
.
This suggests to us that the M4 helix may rotate (and possibly be
displaced distally so that Lys
can reach the membrane
interface with water) in synchrony with ATPase phosphorylation to allow
exit of transported Ca
into the lumen.
(a)
Mutations of residues that are normally involved in Ca binding affect the catalytic site (i.e. the P
reaction) even in the absence of Ca
. In fact,
mutations Glu
Gln, Glu
Gln,
and Asp
Asn increase the equilibrium level of the
phosphoenzyme obtained with P
(in the absence of
Ca
) through a drastic reduction of its breakdown
kinetics ( Fig. 8and Table 3). An analogous finding was
reported by Andersen (25) regarding mutation of Glu
to Ala or Gly. Therefore, specific mutations in the transmembrane
region produce long range conformational effects favoring
phosphorylation of the catalytic site with P
. This suggests
that the acidic functions of these residues sustain a very important
structural role possibly through hydrogen bonding with neighboring
residues in the absence of Ca
, in addition to
participating in cooperative coordination in the presence of
Ca
. Note that Glu
does not sustain this
function since its carbonyl oxygen, but not its carboxyl function, is
required for Ca
coordination.
(b)
Mutations of neighboring residues that are not involved in
Ca binding interfere with catalytic activity both in
the presence and in the absence of Ca
. A specific
case, in this regard, is Asn
whose mutation to Ala
produced total inhibition of Ca
uptake, ATPase
activity, and phosphoenzyme formation by utilization of ATP in the
presence of Ca
, as well as slow decay of the
phosphoenzyme obtained by utilization of P
in the absence
of Ca
( Table 3and Fig. 8). It is then
apparent that Asn
sustains a very important role in
stabilization of the native enzyme conformation, possibly through
involvement of its side chain oxygen or amino group in hydrogen
bonding.
In several other mutants (Table 2), an inhibitory
effect is primarily manifested with a slow decay of the phosphorylated
intermediate formed by utilization of ATP in the presence of
Ca. Furthermore, in the absence of
Ca
, the inhibition manifests itself with a slow decay
of the phosphoenzyme formed by utilization of P
(e.g. Val
Ala, Cys
Ala,
Ile
Ala; Table 2).
These experimental
observations demonstrate that the long range linkage between the
Ca binding domain and the catalytic site is not
necessarily dependent on Ca
but is rather an
intrinsic feature of the protein structure. It is clear that both the
energy transduction mechanism and kinetic regulation of the
Ca
ATPase are not only related to the organic
chemistry of the catalytic site, but are strongly dependent on extended
features of protein conformation.