The Length of the A-M3 Linker Is a Crucial Determinant of the Rate of the
Ca2+ Transport Cycle of Sarcoplasmic Reticulum
Ca2+-ATPase |
| |
Authors: | Anne Nyholm Holdensen and Jens Peter Andersen |
| |
Institution: | Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Physiology and Biophysics, Aarhus University, DK-8000 Aarhus C, Denmark |
| |
Abstract: | Ion translocation by the sarcoplasmic reticulum Ca2+-ATPase
depends on large movements of the A-domain, but the driving forces have yet to
be defined. The A-domain is connected to the ion-binding membranous part of
the protein through linker regions. We have determined the functional
consequences of changing the length of the linker between the A-domain and
transmembrane helix M3 (“A-M3 linker”) by insertion and deletion
mutagenesis at two sites. It was feasible to insert as many as 41 residues
(polyglycine and glycine-proline loops) in the flexible region of the linker
without loss of the ability to react with Ca2+ and ATP and to form
the phosphorylated Ca2E1P intermediate, but the rate of
the energy-transducing conformational transition to E2P was reduced
by >80%. Insertion of a smaller number of residues gave effects gradually
increasing with the length of the insertion. Deletion of two residues at the
same site, but not replacement with glycine, gave a similar reduction as the
longest insertion. Insertion of one or three residues in another part of the
A-M3 linker that forms an α-helix (“A3 helix”) in
E2/E2P conformations had even more profound effects on the
ability of the enzyme to form E2P. These results demonstrate the
importance of the length of the A-M3 linker and of the position and integrity
of the A3 helix for stabilization of E2P and suggest that, during the
normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize
the Ca2E1P state and thereby to drive the transition to
E2P.The sarco(endo)plasmic reticulum Ca2+-ATPase
(SERCA)2 is a
membrane-bound ion pump that transports Ca2+ against a steep
concentration gradient, utilizing the energy derived from ATP hydrolysis
(1–3).
It belongs to the family of P-type ATPases, in which the γ-phosphoryl
group of ATP is transferred to a conserved aspartic acid residue during the
reaction cycle. Both phospho and dephospho forms of the enzyme undergo
transitions between so-called E1 and E2 conformations
(). The E1 and
E1P states display specificity for reaction with ATP and ADP,
respectively (“kinase activity”), whereas E2P and
E2 react with water and Pi instead of nucleotide
(“phosphatase activity”). The E1 dephosphoenzyme of the
Ca2+-ATPase binds two Ca2+ ions with high affinity from
the cytoplasmic side, thereby triggering the phosphorylation from ATP. In
E1P, the Ca2+ ions are occluded with no access to either
side of the membrane, and Ca2+ is released to the luminal side
after the conformational transition to E2P, likely in exchange for
protons being countertransported. The structural organization and domain
movements leading to Ca2+ translocation have recently been
elucidated by crystallization of SERCA in various conformational states
thought to represent intermediates in the pump cycle
(4–7).
SERCA is made up of 10 membrane-spanning mostly helical segments, M1–M10
(numbered from the N terminus), of which M4–M6 and M8 contribute
liganding groups for Ca2+ binding, and a cytoplasmic headpiece
separated into three distinct domains, named A (“actuator”), P
(“phosphorylation”), and N (“nucleotide binding”). The
A-domain appears to undergo considerable movement during the functional cycle.
In the E1/E1P states, the highly conserved
TGE183S loop of the A-domain is at great distance from the
catalytic center containing nucleotide-binding residues and the phosphorylated
Asp351 of the P-domain, but during the Ca2E1P
→ E2P transition, the A-domain rotates ∼90° around an
axis perpendicular to the membrane, thereby moving the TGE183S loop
into close contact with the catalytic site such that Glu183 can
catalyze dephosphorylation of E2P
(8,
9). During the
dephosphorylation, Glu183 likely coordinates the water molecule
attacking the aspartyl phosphoryl bond and withdraws a hydrogen. Hence, the
movement of the A-domain during the Ca2E1P →
E2P transition is the event that changes the catalytic specificity
from kinase activity to phosphatase activity. During the dephosphorylation of
E2P → E2, there is only a slight change of the position
of the A-domain, and a large back-rotation is needed to reach the E1
form from E2; thus, the A-domain rotation defines the difference
between the E1/E1P class of conformations and the
E2/E2P class. Because the A-domain is physically connected
to transmembrane helices M1–M3 through the linker segments A-M1, A-M2,
and A-M3, the A-domain movement occurring during the
Ca2E1P → E2P transition may be a key event
in the opening of the Ca2+ sites toward the lumen, thus explaining
the coupling of ATP hydrolysis to Ca2+ translocation. An important
unanswered question is, however, how the movement of the A-domain is brought
about. Which are the driving forces that destabilize
Ca2E1P and/or stabilize E2P such that the
energy-transducing Ca2E1P → E2P transition
takes place? To answer this, it seems important to elucidate the exact roles
of the linkers. Intriguing results have been obtained by Suzuki and
co-workers, who demonstrated the importance of the A-M1 linker in connection
with luminal release of Ca2+ from E2P
(10). In this study, we have
addressed the role of the A-M3 linker. An alignment of two crystal structures
thought to resemble the Ca2E1P and
E2·Pi forms
(5), respectively, is shown in
. The A-domain rotation
is associated with formation of a helix (“A3 helix”) in the
N-terminal part of the A-M3 linker, and this helix seems to interact with a
helix bundle consisting of the P5–P7 helices of the P-domain, a feature
exhibited by all published crystal structures of the E2 type
(cf. supplemental Fig. S1 and Ref.
11). Moreover, when structures
of similar crystallographic resolution are compared (as in
), the non-helical part
of the A-M3 linker in E2-type structures has a higher relative
temperature factor (“B-factor”) than the corresponding
segment in Ca2E1P (, thick part colored orange-red for
high temperature factor), thus suggesting a higher degree of freedom of
movement relative to Ca2E1P. Hence, the A-M3 linker
appears more strained in Ca2E1P compared with E2
forms, and the greater flexibility of the linker in E2 forms may
promote the formation of the A3 helix.Open in a separate windowCa2+-ATPase reaction cycle.Open in a separate windowA-M3 linker configuration in E1- and E2-type crystal
structures. Crystal structures with Protein Data Bank codes 2zbd
(Ca2E1P analog) and 1wpg (E2·Pi
analog) are shown aligned. A, overview of structure 2zbd in
bluish colors with green A-M3 linker and structure 1wpg in
reddish colors with wheat A-M3 linker. B,
magnification of the A-M3 linker (corresponding to the red box in
A) with arrows indicating site 1, between Glu243
and Gln244, and site 2, between Gly233 and
Lys234, in both conformations. The green A-M3 linker to
the right is structure 2zbd. The wheat A-M3 linker to the left is
structure 1wpg. Note the kinked A3 helix forming part of the latter structure.
C, same A-M3 linker structures as in B but with the
magnitude of the temperature factor (B-factor) indicated in colors
(red > orange > yellow > green
> blue) and by tube diameter. Because the two crystal structures
selected here as E1- and E2-type representatives have
similar crystallographic resolution (2.40 and 2.30 Å, respectively), the
differences in temperature factor in specific regions provide direct
information about chain flexibility.Here, we have determined the functional consequences of changing the length
(and thereby likely the strain) of the A-M3 linker. Polyglycine and
glycine-proline loops of varying lengths were inserted at two different sites
in the linker (), and
deletions were also studied. Rather unexpectedly, we were able to insert as
many as 41 residues in one of the sites without loss of expression or ability
to react with Ca2+ and ATP, forming Ca2E1P, but
the Ca2E1P → E2P transition was greatly
affected. |
| |
Keywords: | |
|
|