Structural role of countertransport revealed in Ca²⁺pump crystal structure in the absence of Ca²⁺

Abstract

Ca 2؉ -ATPase of sarcoplasmic reticulum is an ATP-powered Ca 2؉ pump but also a H ؉ pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-Å-resolution crystal structure of the Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca 2؉ binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca 2؉ -release. They suggest that H ؉ countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca 2؉ -binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a), an integral membrane protein consisting of 994 aa (1), transfers two Ca 2ϩ from the cytoplasm into the lumen of sarcoplasmic reticulum per ATP hydrolyzed and thereby establishes a Ͼ10 4 concentration gradient across the membrane (2). At the same time, Ca 2ϩ -ATPase pumps two or three H ϩ in the opposite direction (3-6) during the reaction cycle. According to the classical E1͞E2 theory (7-9), transmembrane ion-binding sites have high affinity for Ca 2ϩ and face the cytoplasm in E1, whereas they have low affinity and face the lumen of sarcoplasmic reticulum in E2. The opposite applies to H ϩ , which binds to the ATPase in E2 and dissociates in E1, presumably in exchange with Ca 2ϩ . As Na ϩ K ϩ -ATPase and gastric H ϩ K ϩ -ATPase countertransport K ϩ , instead of H ϩ , countertransport of monovalent cations may be a common feature of the P-type ATPase superfamily (2, 10), of which SERCA1a is the best-studied member (11,12). However, the sarcoplasmic reticulum membrane is leaky to monovalent cations including H ϩ (13). As has been pointed out before (14), the membrane potential and pH gradient must be minimized to achieve such a large concentration gradient of Ca 2ϩ . Therefore, the physiological role of H ϩ countertransport has been a puzzle. Although protonation of carboxyls in the Ca 2ϩ -binding site has been suggested from biochemical (15, 16) and mutagenesis studies (17, 18), crystal structures of SERCA1a in various states (19-24) did not clarify the role of countertransport or identify protonation sites.These questions, however, can be addressed computationally by calculating stabilization energy provided by H ϩ binding (25)(26)(27). Protonation probability of a particular residue is related directly to the free energy difference between protonated and unprotonated forms. Such calculations, therefore, should be able to identify residues likely to be protonated in crystal structures and were indeed successful (28) for a Ca 2ϩ -bound form [E1⅐2C...

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

“…In fact, continuum electrostatic calculations predict full protonation of the four acidic residues (Glu309, Glu771, Asp800 and Glu908) within the Ca 2+ binding sites [23,24] in the E 2 (TG + BHQ) state, with pK within or near the pH 6-8 range (see also Hauser and Barth [25] for the E 2 (TG) state, and Fibich et al [26] for the ATPase in the absence of inhibitors). The presence of protons provides important stabilization of the E 2 state by the establishment of hydrogen bonding.…”

“…However, Glu908 may dissociate its H + only as the pH is raised to 8 [24], while Glu58, which does not participate directly in binding, retains a H + by interacting with the Glu309 carbonyl oxygen.…”

“…However, exchange of all four H + for the 2 Ca 2+ transported per each ATPase cycle is not expected since the Glu309 side chain orientation makes unlikely its participation in net Ca 2+ /H + exchange. Furthermore, it is not clear that Glu908 looses stoichiometrically its H + upon Ca 2+ binding [24]. In fact, upon addition of ATP, steady state Ca 2+ /H + exchange has been found to occur with a stoichiometric ratio of two, resulting in electrogenic transport [17].…”

Outside of the box: recent news about phospholipid translocation by P4 ATPases

“…A specific proposal for such a site was suggested by the crystal structure of SERCA in the E2 form (PDB 2AGV) [14]. In this structure, a phospholipid glycerol backbone is tightly bound in a crevasse between the M4 and M2 transmembrane helices.…”

Analysis of the Gastric H,K ATPase for Ion Pathways and Inhibitor Binding Sites

“…This can be shown by comparing PDB code 1wpg to PDB code 2agv (42), a more highly resolved structure nearly superimposable with PDB 1iwo (overall RMS for all backbone atoms of 0.83 Å). The PDB 2agv structure is of sufficient resolution to define it as the likely ion occluded counter-transport form of E 2 in the srCa ATPase (42) and should be more homologous to the K + occluded form of the H,K ATPase.…”