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Structural Changes in the Calcium Pump Accompanying the Dissociation of Calcium

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Structural Changes in the Calcium Pump Accompanying the Dissociation of Calcium articles Structural changes in the calcium pump accompanying the dissociation of calcium Chikashi Toyoshima & Hiromi Nomura Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan ........................................................................................................................................................................................................................... In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca21 ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A˚ resolution in a Ca21-free (E2) state, and compare it with that determined previously for the Ca21- bound (E1Ca21) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca21. Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions. P-type ion transporting ATPases, which include NaþKþ-ATPase mined to 3.1 A˚ resolution, is very different from that of E1Ca2þ,yet and gastric HþKþ-ATPase among others, are fundamental in can be compared directly, because no ATP or phosphorylation is establishing ion gradients by pumping ions across biological mem- involved in the transition between them. The movements of branes (reviewed in ref. 1). Of many P-type ATPases known today, cytoplasmic domains are even larger than we described for the Ca2þ-ATPase (SERCA1a) from skeletal muscle sarcoplasmic reti- tubular crystals10. Transmembrane helices undergo drastic culum (SR) is structurally and functionally the best-studied mem- rearrangements that involve shifts normal to the membrane. ber1–3.SRCa2þ-ATPase pumps Ca2þ from the cytoplasm into the These movements have clear mechanistic implication in the release 2þ reticulum, thereby causing the relaxation of muscle cells. Two Ca2þ and binding of Ca . Knowing the second structure in the reaction ions can be transported per ATP hydrolysed and two or three Hþ cycle, we can now begin to understand how ion pumps work. ions are counter-transported4. Active transport of Ca2þ-ATPase is achieved, according to the Structure determination 2þ E1–E2 model5,6, by changing the affinity of Ca -binding sites from The crystals of SR Ca2þ-ATPase were grown by dialysis in the 2þ high (E1) to low (E2)7. The release of Ca ‘occluded’ in the presence of thapsigargin and exogenous lipid. Thapsigargin was transmembrane binding sites takes place during the transition added before the addition of EGTA for removing Ca2þ. Two types of from E1P to E2P (‘P’ indicating that the enzyme is phosphorylated; crystals of different symmetry (P21 and P41) appeared but only ˚ Fig. 1, inset). Autophosphorylation of an aspartyl residue in the those belonging to the space group P41 diffracted to 3.1 A resolution reaction cycle is a characteristic feature of the P-type ATPases. at the BL44XU beam line of SPring-8. The structure was solved by However, the residues constituting the phosphorylation site are molecular replacement and refined to an R free of 26.8%. Inclusion of shared by the members in the haloacid dehalogenase superfamily8 Ca2þ (1 mM) in the dialysis buffer did not affect the crystal quality and by many bacterial response regulators, despite the differences in or bring differences in the final model. The structure of Ca2þ- the folding patterns (reviewed in ref. 9). ATPase in E2(TG) form, in comparison with E1Ca2þ, is shown in We previously determined the structure of SR Ca2þ-ATPase10 Figs 1 and 2. We describe the structural changes as those accom- with two bound Ca2þ in the transmembrane (M) region, which panying the transition from E1Ca2þ to E2(TG), mainly because we þ consists of ten a-helices (Fig. 1; Protein Data Bank, PDB, code prepared the crystals by removing Ca2 . 1EUL). The cytoplasmic part of Ca2þ-ATPase consists of three domains (A, actuator or anchor; N, nucleotide; and P, phosphory- Movements of the cytoplasmic domains 2þ 2þ lation), well separated in this Ca -bound (E1Ca ) form. The To realize the close association of the three cytoplasmic domains phosphorylation residue, Asp 351, is located on the P domain, and widely separated in E1Ca2þ, the N domain inclines nearly 908 with the adenosine moiety of ATP binds to the N domain. Modelling the respect to the membrane (Fig. 2a) and the A domain rotates by structures (PDB codes 1FQU (ref. 10) and 1KJU (ref. 11)) based on about 1108 horizontally (Fig. 2b; Supplementary Information 11,12 low-resolution maps of the tubular crystals , in which the enzyme Animation 3). As a result, the top part of the N domain moves is in a state similar to E2P (ref. 13), showed large movements of the more than 50 A˚ . Also, the trypsin digestion site on the A domain (T2 three cytoplasmic domains to form a compact headpiece. These in Fig. 1), which is well exposed in E1Ca2þ, is partially blocked by rearrangements of the cytoplasmic domains must be associated with the P domain13 (Supplementary Information Animation 3). The the changes in the transmembrane binding sites14, but the molecular cytoplasmic domains move as a whole in an M10-to-M1 direction mechanism was far beyond what we could imagine. (,23 A˚ for the P domain). þ Here we describe the crystal structure of Ca2 -ATPase in the Two components contribute to this large change in inclination of absence of Ca2þ and in the presence of thapsigargin, a potent the N domain: the first is the movement of the P domain (see, for inhibitor that fixes the enzyme in a form analogous to E2 (ref. example, the change in inclination of the P5 helix in Fig. 2a), and the 15), abbreviated as E2(TG). The structure (Figs 1 and 2), deter- other is that of the N domain itself relative to the P domain. An NATURE | VOL 418 | 8 AUGUST 2002 | www.nature.com/nature © 2002 Nature Publishing Group 605 articles analysis by Dyndom16 indicates that the P domain inclines by about both up to about 5 A˚ , close to one turn of an a-helix (5.4 A˚ ; Figs 308 with respect to the membrane, and the N domain by about 508 1 and 2). M1 shows a large lateral movement within the membrane relative to the P domain. This 308 inclination of the P domain is (Fig. 2b), and is unique in this respect. M3 and M5 are strongly directly related to tilting of the transmembrane helices, in particular curved in E2(TG) but in opposite directions (Figs 1 and 2). Our first the M5 helix, and is likely to be a central event in the active transport goal is, therefore, to understand how these complicated movements (see below). are organized and linked with those of the cytoplasmic domains. We Despite the large movements between the two states, the structure can start with the many places where a movement of one helix could of the P and N domains remain virtually the same (r.m.s. deviations cause the movements of others (a ‘domino’ effect). For example, in 0.63 and 0.75 A˚ , respectively; Fig. 3 and Supplementary Information Fig. 4a, inclination of M4 must impinge on M2 at the top, causing Animation 3), except for a few ‘hinge’ residues and those at the A–P M2 itself to tilt. It also helps to note that the movements of helices interface. The hinge region (DPPR starting from Asp 601 and normal to the membrane are in most cases produced by the changes TNQMS starting from Thr 358) is well hydrogen bonded between in their inclinations (for example, M2). However, it seems most the two strands; no hydrogen bonds favouring E2(TG) can be critical to understand the links between the transmembrane helices identified, although it is generally difficult to assign hydrogen and the P domain. ˚ bonds at 3.1 A resolution. This suggests that stabilization of the M5 runs through the centre of the enzyme from the lumenal closed configuration takes place entirely at the A–N and A–P surface to the end of the P domain, where it is integrated as a part of interfaces, where we can identify a few hydrogen bonds (Fig. 1). the Rossmann fold (Fig. 3). Hence, the top part of M5 moves The residues at the A–P interface (Ile 179–Thr 181 in A, and Asp together with the P domain as a single entity. In fact, if the two 703–Asn 706 in P) contain signature sequences of the P-type structures are superimposed with the P domain, the top part of M5 ATPases (reviewed by ref. 1). also superimposes virtually completely (Fig. 3). Here, short anti- parallel b-strands (0 and 7) of the P domain appear to ‘clamp’ the Rearrangement of transmembrane helices M4 and M5 helices (Fig. 3). The middle part of M5 is linked to the The dissociation (or binding) of Ca2þ accompanies dramatic loop (L67) connecting M6 and M7 (through Arg 751; Fig. 1), rearrangements of six (M1–M6) out of ten transmembrane helices perhaps restricting the bowing of M5; this loop is also hydrogen (Figs 2 and 4a). The rearrangements are not limited to those bonded to the P domain (refer to Fig. 8 of ref. 10) and is important constituting the Ca2þ-binding sites (M4–M6, M8) and appear in phosphorylation17. On the opposite side of the P domain to M4, quite complicated: M1 and M2 move upwards (þz direction, M3 is located.
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