From the Public Health Research Institute, New York, New York 10016
Received for publication, December 11, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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The stalk segments of P-type ion-translocating
enzymes are presumed to play important roles in energy coupling. In
this work, stalk segments S4 and S5 of the yeast
H+-ATPase were examined for helical character,
optimal length, and segment orientation by a combination of proline
substitution, insertion/deletion mutagenesis, and second-site
suppressor analyses. The substitution of various residues for
helix-disrupting proline in both S4 (L353P,L353G; A354P; and
G371P) and S5 (D676P and I684P) resulted in highly defective or
inactive enzymes supporting the importance of helical character and/or
the maintenance of essential interactions. The contiguous helical
nature of transmembrane segment M5 and stalk element S5 was explored
and found to be favorable, although not essential. The deletion or
addition of one or more amino acids at positions Ala354 in
S4 and Asp676 in S5, which were intended to either rotate
helical faces or extend/reduce the length of helical segments, resulted
in enzyme destabilization that abolished most enzyme assembly.
Second-site suppressor mutations were obtained to primary site
mutations G371A (S4) and D676G (S5) and were analyzed with a molecular
structure model of the H+-ATPase. Primary site mutations
were predicted to alter the site of phosphorylation either directly or
indirectly. The suppressor mutations either directly changed packing
around the primary site or altered the environment of the site of
phosphorylation. Overall, these data support the view that stalk
segments S4 and S5 of the H+-ATPase are helical elements
that are optimized for length and interactions with other stalk
elements and can influence the phosphorylation domain.
The plasma membrane H+-ATPase of yeast is an essential
enzyme that actively pumps protons across the cellular membrane in
order to maintain intracellular pH and the electrochemical proton
gradient necessary for growth and development. It belongs to the
superfamily of P-type ion-translocating ATPases for which there are
more than 150 members known (1). The fungal H+-ATPase is a
class II, non-heavy metal-transporting enzyme that includes the plant
H+-ATPase and the animal
Na+,K+-ATPase, Ca2+-ATPase, and
H+,K+-ATPase (2). It couples ATP hydrolysis in
the cytoplasmic domain to ion transport in the membrane-embedded domain
forming an acyl-phosphate intermediate during catalysis. Recently, the
molecular structure of the Ca2+-ATPase of skeletal muscle
sarcoplasmic reticulum
(SERCA1a)1 was solved at
2.6-Å resolution (3). As expected, it confirmed a membrane transport
domain with 10 transmembrane segments, a large cytoplasmic ATP
hydrolysis domain, and a narrow stalk domain that links the cytoplasmic
and membrane domains. The cytoplasmic region consists of three well
separated domains, with the phosphorylation site in the central
catalytic domain assuming a fold similar to haloacid dehalogenase and
the adenosine-binding site formed 25 Å away on another domain (3).
These essential features are conserved among P-type enzymes despite
wide ranges in amino acid conservation between family members. A
detailed comparison of 8 Å structures (4) of the H+-ATPase
from Neurospora (5) and the Ca2+-ATPase from
sarcoplasmic reticulum (6) confirmed that important structural
properties are highly conserved.
There is extensive evidence that dynamic changes in protein structure
occur during catalysis (7-9). However, it is not clear how
conformational changes in the catalytic region are transmitted to the
transmembrane domain. It has been suggested that positional changes in
the stalk segments could play a role in mediating the coupling process
(10, 11). In the Ca2+-ATPase, the stalk region is ~24 Å long and is divided into four largely helical, rod-like structures (3,
6, 12). The stalk densities are not as apparent in the 8-Å
Neurospora H+-ATPase structure, which likely
reflects an open conformation being used to derive structural
information (5). Nonetheless, the helical stalk region is expected to
provide a direct physical linkage between functional domains involved
in ATP hydrolysis and ion transport.
The H+-ATPase stalk segments, designated S2
(Gln164-Val189), S3
(Gly270-Asn291), S4
(Val348-Val372), and S5
(Gly670-Tyr689), are the cytoplasmic
extensions from transmembrane segments M2, M3, M4 and M5, respectively.
Segments M4-6 have been implicated in ion binding/release (13). S4 and
S5 are presumed to play a pivotal role in coupling because they are
contiguous with these transmembrane segments and are directly linked to
the nucleotide binding-phosphorylation domain (2, 11). S4 is within 10 residues or 2.5 helix turns from the site of phosphorylation,
Asp378. A mutation in the Ca2+-ATPase, Y763G,
near the membrane interface of S5 uncouples ion transport from ATP
hydrolysis (14). In the Ca2+-ATPase structure, M5-S5 is a
long, centrally oriented, contiguous helical segment that forms a
"mast-like" element extending from the outer membrane surface to
the center of the cytosolic domain (3).
We previously demonstrated that the helical properties of S2 and S3 are
important to enzyme function (15). In this report, we have extended
that analysis to examine the importance of helical character in S4 and
S5, which are more directly linked to coupling of ion transport. In
addition, we have examined spatial interactions of S4 and S5 by
identifying second site suppressor mutations that reverse the defective
phenotypes observed from point mutations within the stalk segments. Our
results strongly support the view that the helical properties of stalk
segments S4 and S5 are important for H+-ATPase function,
and the length of these segments as well as the juxtaposition of
helical faces are optimized to interact with the phosphorylation domain.
Yeast Strains--
Primary site mutations generated in
vitro were transformed into yeast strain SH122 (HO ade6-1
trp5-1 leu2-1 lys1-1 ura3-1 pma1 Site-directed Mutagenesis--
Site-directed mutants were
created in plasmids pGW201 (19), pRS201
pma1 mutants were grown in a 3-ml YPD (1% yeast extract,
2% peptone, 2% dextrose, pH 5.7) culture for 18-20 h, and
chromosomal DNA was isolated using the Wizard Genomic DNA Purification
Kit (Promega). The PMA1 structural gene was amplified as a
3.5-kilobase pair fragment by the polymerase chain reaction using
flanking primers (5'-GCTCCCCTCCATTAGTTTCG and 5'-GCGTGTTGTGAATTGTGC).
PCR products were purified by the Wizard PCR Preps Purification System (Promega). All mutations were verified by DNA sequence analysis of
PCR-amplified products.
Generation of Suppressor Mutations--
Suppressor mutations
were generated by treating vector pRS201 Growth Phenotypes of pma1 Mutants--
Resistance to hygromycin
B was determined on YPD agar plates containing 200 µg/ml hygromycin B
(20). pH-dependent growth measurements were made on YPD
agar at pH 2.5 with 20 mM acetate added or in liquid YPD
with 20 mM acetate and the pH ranging from 2.5 to 8.0, as
described previously (21). Growth rates were evaluated by inoculating a
50-ml culture of YPD (to A590 ~0.2) with
stationary phase cultures of pma1 mutants and growing cells with shaking at 30 or 37 °C. Aliquots (0.3 ml) were removed every hour, and the optical density at 590 nm was determined.
Kinetic Properties of the pma1 Mutants--
Plasma membranes
were isolated from cells grown to mid-log phase at 30 °C, as
described previously (22). Secretory vesicles were isolated as
described Nakamoto et al. (17). ATP hydrolysis assays were
performed in 96-well microplates, as described in Wang et
al. (19). The pH dependence of ATP hydrolysis was determined in a
standard reaction medium with the pH adjusted to 4.5-8.5. Vanadate
sensitivity was measured in a standard reaction containing 0-100
µM sodium vanadate. Kinetic parameters
Km and Vmax were determined
by measuring ATP hydrolysis with equimolar (1:1) complexes of ATP and
MgSO4 from 0 to 10 mM.
Glucose-dependent medium acidification by carbon-starved
cells was examined, as described previously (23).
Other Procedures--
All mutations were identified by DNA
sequence analysis at the New York University Medical Center Sequencing
Facility. Protein concentrations were determined using the Coomassie
Plus Protein Assay Reagent (Pierce). SDS-gel electrophoresis was
performed using pre-cast 10% minigels (NOVEX). Relative protein
abundance measurements were made by gel electrophoresis and Western
blot analysis, as described previously (21).
Targeted Proline Mutagenesis of S4--
Scanning proline
mutagenesis has been used to probe the helical properties of stalk
segments S2 and S3 (15). This approach was extended to assess the
importance of helical backbone structure in S4 and S5 by making
strategic substitutions in largely non-conserved residues near the ends
of the two stalk segments. Table I shows the positions of these mutations and their affects on cell viability, H+-ATPase-dependent growth phenotypes,
hygromycin B resistance, low pH sensitivity (20, 23), enzyme kinetics,
and sensitivity to orthovanadate. The substitution of proline in S4 for
residues Leu353 and Ala354 near the membrane
interface of M4 and at Gly371 resulted in defective enzymes
incapable of supporting growth. A L353G mutation was also highly
disruptive. In each case, the introduction of other amino acid
substitutions produced active enzymes, which demonstrated that the
disruptive propensity of proline, and in some cases glycine, was most
likely due to its affect on backbone structure and not on amino side
group character. An S368P substitution was viable, although the cells
grew slowly and the enzyme displayed less than 50% of normal catalytic
activity (Table I). Several mutations (e.g. S368F) at this
position are known to induce >500-fold decreases in vanadate
sensitivity (16), although the viable S368P mutant was fully
vanadate-sensitive (Table I). However, a G371A mutation did show
~10-fold less vanadate sensitivity consistent with a recent study of
this region (24). The viable mutant enzymes, L353I, A354G, G371A, and
S386P, showed normal Km values and sensitivity to
vanadate and, other than S368P, showed Vmax
levels that were
The prominent growth defects induced by the S368P mutation were most
pronounced when assessing the proton transport properties of this
mutant. Fig. 1 shows a detailed profile
for pH-dependent growth in which the S368P mutant was most
sensitive to acidic medium conditions. This enzyme appeared less
competent in regulating intracellular pH via the H+-ATPase.
The kinetic defect in this enzyme was apparent when examining whole-cell proton efflux mediated by the H+-ATPase from
carbon-starved cells (Fig. 2). In this
case, S368P pumped protons routinely at a rate 55-65%
(n = 3) that of wild type enzyme or other mutants,
which is consistent with its reduced Vmax
in vitro. Mutant enzymes A354G and G371A were somewhat less efficient (15-20%) than wild type (Fig. 2). Interestingly, both of
these enzymes showed rates of ATP hydrolysis that were >92% of wild
type (Table I), suggesting that they may be partially uncoupled. Most
of the other mutants showed wild type-like rates of
H+-ATPase-mediated proton transport.
Targeted Proline Mutagenesis of S5--
In the
Ca2+-ATPase, M5 and S5 form a contiguous, long central
helical element (3) that has been proposed to play a critical role in
the coupling reaction (25). In contrast to the Ca2+-ATPase
and related higher eukaryotic enzymes, the fungal H+-ATPase
has a naturally occurring proline, Pro669, as well as a
Gly670, near the M5 interface, which should disrupt helical
continuity in this region and create hinge-like flexibility. The
conversion of Pro669 to glycine and alanine had modest
effects on enzyme function (Table I), suggesting that backbone
flexibility is not important at this position.
In contrast, the introduction of proline at both Asp676 and
Ile684 at opposite ends of S5 resulted in highly defective
enzymes incapable of supporting growth. As observed with S4 mutations,
the introduction of either glycine or alanine at these positions was
well tolerated by the enzyme (Table I). These results suggest that
helical character of S5 is important for function. Mutations D676R and
D676G, but not D676A, produced enzymes with low rates of ATP
hydrolysis, hygromycin B resistance, and low pH sensitivity indicative
of a highly defective enzyme (Table I; Fig. 1). Whole-cell proton transport assays also confirmed that these mutant enzymes were less
efficient in H+-ATPase-directed proton transport (Fig. 2).
It is likely that side group mass at this position is more important
than charge.
Suppressor Mutations of G371A and D676G--
The prominent growth
characteristics of mutants G371A in S4 and D676G in S5 permitted the
isolation of second-site suppressor mutations to evaluate short and
long range protein-protein interactions. Spontaneous suppressor mutants
were readily isolated but generally resulted in gene replacement events
that restored the wild type PMA1 gene. To promote the
generation of second site suppressor mutations, plasmid
pRS201 Characterization of Deletion and Alanine Insertion
Mutants--
The helical character of the stalk elements was further
probed by single residue deletion and sequential four amino acid
insertions (i + 1 ... i + 4) around
Asp354 in S4 and Asp676 in S5. The deletion and
alanine insertion mutants were expressed in a secretory vesicle system
(17) to better determine the biochemical properties of the mutant
H+-ATPase enzymes. Fig. 3
shows the level of assembled enzyme and rates of ATP hydrolysis for the
mutant enzymes. The data indicate that both S4 and S5 are highly
sensitive to changes in the helical axis. In all cases, except for a
single alanine insertion in S4, the deletion and addition mutations
were highly disruptive resulting in an unstable enzyme that assembled
poorly, even in the Sec vesicles. These mutants were highly reminiscent
of assembly defects encountered with mutations of the phosphorylation
site, Asp378 close to S4 (26). A single alanine addition
was tolerated resulting in a largely assembled enzyme (65% of wild
type) with only 35% of wild type enzyme activity.
The P-type ATPase stalk region is widely viewed as a mediator of
energy coupling between the catalytic ATP binding/hydrolysis and ion
translocation domain. Genetic evidence from several systems support
this view by demonstrating that mutations in several stalk elements
either partially or fully uncouple ion transport from ATP hydrolysis.
These mutations include I183A in S2 (19), G158D near the S2/M2
interface (27), H285Q in S3 (28) of the yeast H+-ATPase,
and Y763G in the S5 (14) of the Ca2+-ATPase. The Y763G
mutation in S5 has been proposed to directly modulate coupling via an
affect on M5/M6, which has been implicated in ion binding (11, 29).
Furthermore, a K758I mutation in the Ca2+-ATPase leads to
changes in the rates of dephosphorylation and Ca2+ binding
(30), and perturbations in S3 produced by either mutation or the
binding of the inhibitor thapsigargin interfere with energy coupling
(31).
In this work, the helical character and length of stalk segments S4 and
S5 of the yeast H+-ATPase were shown to be important for
enzyme function and stability. The substitution of various residues for
helix-disrupting proline (or in some cases glycine) in both S4
(L353P,L353G; A354P; and G371P) and S5 (D676P and I684P)
resulted in highly defective or inactive enzymes (Table I). In each
case, substitutions with residues other than proline restored normal or
near-normal enzymatic function (Table I). Deletion or addition of one
or more amino acids at positions Ala354 in S4 and
Asp676 in S5, intended to either rotate helical faces or
extend/reduce the length of helical segments, were in most cases
deleterious resulting in enzyme destabilization that significantly
decreased enzyme assembly (Fig. 3). The assembly defects observed were
comparable to those observed by mutation of Asp378, the
site of phosphorylation, and neighboring residues (26, 32). These
results indicate that S4 and S5 play more than a simple structural role
bridging the catalytic phosphorylation and nucleotide binding domains
to the membrane-embedded ion transport domain. Rather, the helical
elements are juxtaposed or packed in an optimal manner that is not
highly flexible.
Scanning alanine mutagenesis of S4 by Ambesi et al. (24)
supports the notion that side group interactions are essential in this
segment. A sided preference was observed for inactivating mutations
from Lys362 to Leu375 which may define
potential interaction sites. These authors further demonstrated that
various mutations in consecutive residues Ile359 through
Gly371 produced enzymes with significantly reduced
sensitivity to orthovanadate. Similarly, G371A in this study showed a
10-fold reduced vanadate sensitivity. This behavior is linked to a
shift in E1-E2
equilibrium toward the vanadate-insensitive E1
conformation. It is supported by previous studies on vanadate
insensitivity (16, 33), by related studies on yeast
H+-ATPase M4 (34) and S4 (24), as well as S4 in the
Ca2+-ATPase. In the latter study, mutations in the S4
region altered E1-E2
and/or Ca2+·E1P In the 2.6-Å resolution structure of the Ca2+-ATPase, S5
and M5 form a long continuous The directed proline mutagenesis, helix lengthening, and shortening and
twisting of stalk segment S5 in this work supports the importance of an
optimized helical element. This likely results from the location of
specific residues on one or more faces of the helix to form critical
interactions. In fact, saturation mutagenesis of
S52 revealed two faces of the
helix that tolerate mutation poorly and may be potential regions of
interaction. Assorted S5 mutations in the Ca2+-ATPase alter
the rate of the reaction sequence HnE2 The intramolecular rearrangement of stalk segments during catalysis
remains obscure. The analysis of second site suppressor mutations
(SSSM) enables short and long range interactions within a protein to be
explored. In the current context, the presumptive behavior of primary
and secondary site mutations were analyzed by superposition of the
yeast H+-ATPase primary sequence on conserved portions of
the molecular coordinates of the SERCA Ca2+-ATPase (Fig.
4). Primary site mutation G371A lies on
the edge of stalk 4, seven residues upstream of the site of
phosphorylation Asp378 (Fig. 4). This mutation introduces a
slightly bulkier side group at this position suggesting some steric
crowding. Given its proximity to Asp378 and the finding
that mutations in this region influence the phosphorylation state of
the enzyme (32), it is likely that the G371A mutation alters the
environment around Asp378 via main chain interactions
involving twisting or other distortions. The introduction of proline at
this position was highly destabilizing resulting in a non-viable enzyme
(Table I). Second site mutations appear to have both short and long
range effects that in each case stabilize the main chain containing
Asp378. SSSM T680I lies on an adjacent descending chain and
shows close contact with the primary site mutation G371A. The side
group change may relieve some steric crowding introduced by the primary
site mutation. SSSM P535L lies within the highly conserved sequence D534PPR, which has been linked to coordination of Mg2+ ions
(36). This side group and backbone change may compensate for the
primary site effect by directly impacting the environment around
Asp378, perhaps by making it more favorable for phosphate
transfer through a decreased polarity. SSSM M405I lies 27 residues
downstream of the phosphorylation site. In the Ca2+-ATPase
structure, this residue lies at the end of an elongated chain that
turns abruptly back toward the membrane. It may exert its effects via
main chain twisting, but there is no indication that the chain is
sufficiently structured to facilitate such a molecular distortion.
Rather, in the H+-ATPase, it appears more likely that M405I
actually lies closer to Asp378 because the next 62 of 82 residues, including the immediately following 34 residues of the SERCA
pump, are absent in the yeast enzyme (Fig. 4). This gap is predicted to
bring M405I much closer to Asp378 in which the isoleucine
side group can more directly impact the environment of the site of
phosphorylation. Overall, it is suggested the primary impact of the
G371A mutation is to alter the environment around Asp378,
and the role of the second site suppressor mutations is to restore a
more normal environment either by changing the immediate environment or
altering the main chain distortion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::LEU2/PMA1) (16) or SY4 (MATa
ura3-52 leu2-3 112 his4-619 GAL1::PMA1::URA3
s6-4ts GAL2) (17). Some suppressor mutations
were isolated in the strain YAK2 (MAT
ade2-101
leu2
1 his3-
200, ura3-52
trp1
63 lys2-801 pma1
::HIS3
pma2-
::TRP1) (18), as indicated.
URA3, and
Ycp2HSE-PMA1 (17) using the QuickChange Site-directed Mutagenesis Kit (Stratagene). For chromosomal integration, a
6.1-kilobase pair HindIII fragment containing a desired
pma1 mutation linked to URA3 was transplaced into
yeast strain SH122. Isogenic pma1 mutants were isolated as
described by Harris et al. (16). All transformations were
performed using the Alkali-Cation Yeast Transformation Kit (Bio 101).
URA3 containing
the primary site mutation with 1 M hydroxylamine HCl, 50 mM sodium pyrophosphate, pH 7.0, 100 mM sodium
chloride, 2 mM EDTA for 1-2 h at 75 °C. Hydroxylamine
was removed from the plasmid using the Wizard PCR Preps Purification
System (Promega). Additional suppressor mutations were generated by
passing the plasmid through an XL1-Red mutator Escherichia
coli strain (Stratagene). The mutagenized plasmids were used to
transform the strain YAK2 (18), as described above. The transformed
YAK2 were cured of their primary plasmid by plating on media containing
0.1% 5-fluoroorotic acid, 0.67% (w/v) yeast nitrogen base without
amino acids, 0.2% (w/v) CSM-URA, 2% (w/v) dextrose, 50 µg/ml
uracil, 0.1% 5-fluoroorotic acid, 2% agar). After 5 days of growth,
colonies were isolated and suppressors selected on the basis of full or
partial reversion to wild type growth. Plasmid DNA was isolated using
the Wizard Plasmid Purification Kit (Promega), and suppressor mutations
were located by DNA sequence analysis of the entire gene.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
78% of wild type enzyme.
Growth phenotypes and kinetic properties of glycine/proline pma1
mutants
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Fig. 1.
pH-dependent growth of
pma1 mutants. Mutant cells (103) were
inoculated into rich YPD medium containing 20 mM potassium
acetate and were grown for 24 h at 30 °C. Each point represents
the average of cells grown in triplicate.
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Fig. 2.
H+-ATPase-mediated medium
acidification. Carbon-starved cells were incubated in weakly
buffered medium, and H+-ATPase-mediated proton efflux was
assessed by following medium acidification with the pH indicator dye
bromphenol blue. Changes in the initial linear rate of acid efflux
relative to wild type (GW201) were taken as differences in the rate of
H+-ATPase-mediated proton efflux. Each point is the average
of three measurements.
URA3 containing a primary site mutation was either
chemically mutagenized by treatment with hydroxylamine or passaged
through a bacterial mutator strain (see "Experimental Procedures").
Suppressors to G371A were found at M405I and P535L, both located within
the catalytic domain, and T608I, located in stalk segment S5. One
suppressor mutation of D676G resulted from two second site mutations at
E288K and V562I, which are located in Stalk segment S3 and the
catalytic domain, respectively. The two other D676G suppressors were
found at V748I in the cytoplasmic turn between membrane segments M6 and
M7 and G888S in transmembrane segment M9. All suppressor mutations
restored phenotypic wild type growth and yielded enzymes that had rates
of ATP hydrolysis that were 100% of wild type for G371A and 81% for
D676G. Whole-cell proton transport assays also indicated that the
suppressor mutations largely restored wild type proton transport
function (not shown).
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Fig. 3.
Effect of addition/deletion mutations on
enzyme assembly and activity. Addition and deletion mutants were
evaluated in enzymes expressed in secretory vesicles. The membrane
vesicles were purified, the proteins separated by SDS gel
electrophoresis, and the level of H+-ATPase evaluated with
a whole anti-H+-ATPase antibody. Densitometric scans were
used to visualize the bands (A). ATP hydrolysis was
evaluated in quadruplicate in purified vesicles (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ca2+·E2-P transitions suggesting
that this segment links phosphorylation and Ca2+ binding
(35). Collectively, these results suggest that perturbations in S4 are
linked directly to changes in the disposition of the site of
phosphorylation Asp378.
-helical structure of ~60 Å that
spans the membrane domain and a significant portion of the cytoplasmic domain. It is worth noting that a naturally occurring proline, Pro669, occurs at the interface of S5 and M5 in the yeast
H+-ATPase. This proline could disrupt the continuity of the
extended M5-S5 helical segment observed in the Ca2+-ATPase.
However, it is likely that the distortion imposed by the naturally
occurring proline is minimal since Pro669 can be converted
to alanine thereby restoring helical integrity with little affect on
enzyme behavior (Table I). This behavior may reflect the fact that
Pro669 lies at the interface of helical elements
constrained by different dielectric environments and/or through close
interactions with other stalk and membrane elements.
Ca2E1 associated with
Ca2+ binding on sites at the cytoplasmic face sites and on
the rate of the dephosphorylation of the ADP-insensitive phosphoenzyme, HnE2P
HnE2 (25). S5 is likely to play a pivotal role in mediating communication between the
Ca2+-binding pocket and the catalytic domain. In addition,
a critically conserved residue, Arg751, appears important
for both structural and functional integrity of the enzyme (25). S5 is
linked directly to the transmembrane ion binding domain composed of
M4-M6, and M8, through its connection with M5.
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Fig. 4.
Putative locations for primary and
secondary site mutations. The primary amino acid sequence of yeast
PMA1p was overlaid on the molecular structure coordinates
for the SERCA Ca2+-ATPase (A) by an iterative
single residue matching process and visualized with the InsightII
(Molecular Simulations, Inc.) structure modeling package. The backbone
trace of the SERCA pump is shown with conserved PMA1
residues (26.4% identity; 37.3% similarity) shown in green
(identical) and orange (similar), non-conserved residues
shown in black, and residues absent in the PMA1p
enzyme shown in dark gray. B and C
represent a topside view of the cytoplasmic portion of the enzyme
without the membrane portion displayed and show the backbone position
of second site suppressor mutations (green dots) obtained
for primary site mutations G371A and D676G (red dots),
respectively. The highly conserved 11-amino acid stretch
(Ile374-Thr384) main chain, containing the
site of phosphorylation, Asp378 (blue dot), is
designated by the solid arrow (blue-gray). The
structure was displayed using Swiss-PDBViewer version 3.61 (Glaxo
Wellcome).
Primary site mutation D676G on stalk 5 is complemented by SSSM V748I, G888S, and E288K,V562I. Asp676 is upstream of the highly conserved Asp634 and Asp638, which are presumed to help coordinate nucleotide-bound divalent cation (37, 38). One likely effect is that the D676G mutation alters this coordination. SSSM E288K maps to stalk segment S3 that lies in the same horizontal plane as D676G, suggesting a close interaction during catalysis. SSSM V748I maps to the cytoplasmic region between M6 and M7 further suggesting another potential interaction with S5. Finally, V562I lies just outside the conserved motif LTGD560. In the haloacid dehalogenase family, the highly conserved residue, Ser118, which is equivalent to PMA1 Thr558, is believed to form a hydrogen bond with substrate carboxylate (37, 38). The V562I mutation may help increase this interaction, which was diminished by the primary site mutation D676G. In this scenario, changes in S5 are strongly linked to changes in nucleotide binding at the catalytic site.
The wide distribution of second site suppressor mutations clustering on protein structure elements that directly or indirectly impinge upon the catalytic Asp378 suggests that the conformational dynamics of the enzyme are highly dependent on the phosphorylation state of this residue, as expected. More generally, this work suggests that second site suppressor studies, when coupled with a working molecular structure model, can yield valuable information about local protein structure interactions and dynamics. Such studies can be especially useful when evaluating interactions occurring as a result of a primary site mutation that alters a short lived conformational state.
Overall, the results in this study provide evidence that stalk segments
S4 and S5 of the yeast H+-ATPase are optimized helical
elements that are closely linked to phosphorylation events occurring
within the catalytic site.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 38225 (to D. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Public Health Research
Institute, 455 First Ave., New York, NY 10016. Tel.: 212-578-0820; Fax:
212-578-0804; E-mail: perlin@phri.nyu.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M011115200
2 M. Miranda and C. W. Slayman, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SERCA, skeletal muscle sarcoplasmic reticulum; PCR, polymerase chain reaction; SSSM, second site suppressor mutations.
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REFERENCES |
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