Ca
2+
‐ATPase of sarcoplasmic reticulum is known to pump Mn
2+
in addition to Ca
2+
, but whether its transport mechanism is identical to that of Ca
2+
is ambiguous. To clarify, we examined, by ...atomic absorption spectroscopy, competition between Mn
2+
and Ca
2+
in active transport using vesicles of sarcoplasmic reticulum (SR). Here, we demonstrate that Ca
2+
‐ATPase transports Ca
2+
and Mn
2+
concomitantly but has a much lower affinity for Mn
2+
(apparent
K
d
~ 0.5 m
m
). Stoichiometries of transported ions per ATP hydrolysed,
V
max
values and activation energies are very similar. Altogether, Ca
2+
‐ATPase appears to use the same mechanism for transporting Mn
2+
as that for Ca
2+
.
The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis–powered Na+ and K+ electrochemical gradients across the cell membrane. These ion gradients drive co- and ...countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na+ and K+, despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K+ and Na+-bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K+ at the extracellular face (i.e. E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K+ on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na+. Theoretical analysis of the pKa values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na+/K+ selectivities may arise from the protonation or ionization of key residues. The increase in K+ selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp808 protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity.
Under physiological conditions, most Ca
-ATPase (SERCA) molecules bind ATP before binding the Ca
transported. SERCA has a high affinity for ATP even in the absence of Ca
, and ATP accelerates Ca
...binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca
-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca
state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca
-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.
Structural biology of the calcium pump Toyoshima, Chikashi
Proceedings for Annual Meeting of The Japanese Pharmacological Society,
2018, 2018-00-00, Volume:
WCP2018
Journal Article
Open access
Since the publication of the first atomic structure of the Ca2+-pump (SERCA1a) in 2000 (1), more than 10 reaction intermediates that roughly cover the entire reaction cycle have been crystallised, ...allowing us to describe a fairly detailed scenario of ion pumping. We now know how SERCA1a binds two Ca2+ sequentially, utilising more abundant Mg2+ and K+ for acceleration. Furthermore, by developing a technology for visualising lipid bilayers in the crystals, we now begin to understand how P-type ATPases interact with phospholipids as an integral component of the pumping mechanism (2). In this lecture, I would like to overview our current understanding of the SERCA pumps through their atomic structures.1. Toyoshima, et al. Nature 405, 647-655 (2000).2. Norimatsu et al. Nature 545, 193-198 (2017).
The structures of the Ca2+-ATPase (SERCA1a) have been determined for five different states by X-ray crystallography. Detailed comparison of the structures in the Ca2+ bound form and unbound (but ...thapsigargin bound) form reveals that very large rearrangements of the transmembrane helices take place accompanying Ca2+ dissociation and binding and that they are mechanically linked with equally large movements of the cytoplasmic domains. The meanings of the rearrangements of the transmembrane helices and those of the cytoplasmic domains as well as the mechanistic roles of phosphorylation are now becoming clear. Furthermore, the roles of critical amino acid residues identified by extensive mutagenesis studies are becoming evident in terms of atomic structure. PUBLICATION ABSTRACT
Site-directed mutations were produced in the distal segments of the Ca2+-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3–M4 loop), Lys-958, and Thr-960 (M9 – M10 loop) had minor effects ...on ATPase activity and Ca2+ transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90–95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca2+ coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca2+ concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 → Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca2+ was also impaired, thereby explaining the stronger inhibition of Ca2+ transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca2+ following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.
Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca2+ ATPase (SERCA-1). ...Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1·2Ca2+ transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase.
Sarco/endoplasmic reticulum Ca
-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca
from the cytosol into the ER. As its function is associated with various ...biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium
and named iezoside (
). The structure of iezoside (
) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (
7.1 nM). In this article, we report our comprehensive analysis of iezoside (
), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC
6.7 ± 0.4 nM against HeLa cells).
To study domain organization and movements in the reaction cycle of heavy metal ion pumps, CopA, a bacterial Cu+-ATPase from Thermotoga maritima was cloned, overexpressed, and purified, and then ...subjected to limited proteolysis using papain. Stable analogs of intermediate states were generated using AMPPCP as a nonhydrolyzable ATP analog and AlFx as a phosphate analog, following conditions established for Ca2+-ATPase (SERCA1). Characteristic digestion patterns obtained for different analog intermediates show that CopA undergoes domain rearrangements very similar to those of SERCA1. Digestion sites were identified on the loops connecting the A-domain and the transmembrane helices M2 and M3 as well as on that connecting the N-terminal metal binding domain (NMBD) and the first transmembrane helix, Ma. These digestion sites were protected in the E1P·ADP and E2P analogs, whereas the M2–A-domain loop was cleaved specifically in the absence of ions to be transported, just as in SERCA1. ATPase activity was lost when the link between the NMBD and the transmembrane domain was cleaved, indicating that the NMBD plays a critical role in ATP hydrolysis in T. maritima CopA. The change in susceptibility of the loop between the NMBD and Ma helix provides evidence that the NMBD is associated to the A-domain and recruited into domain rearrangements and that the Ma helix is the counterpart of the M1 helix in SERCA1 and Mb and Mc are uniquely inserted before M2.
The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase‐activating proteins (RhoGAPs) and their substrates, ...Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS–/– neurons was higher than that in wild‐type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS–/– mice bore longer neurites than those from wild‐type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.
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