Val200 Residue in Lys189–Lys205 Outermost Loop on the A Domain of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Rapid Processing of Phosphoenzyme Intermediate after Loss of ADP Sensitivity

Abstract

Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 1 is a 110-kDa membrane protein and a representative member of P-type ion-transporting ATPases. SERCA1a catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4). According to the E1/E2 transport mechanism ( Fig. 1) (3-7), the enzyme is activated by the binding of two Ca 2ϩ ions (E1⅐Ca 2 , steps 1-2) and then autophosphorylated by MgATP to form an ADP-sensitive phosphoenzyme (E1P, step 3). On formation of this EP, the bound Ca 2ϩ ions are occluded in the transport sites. The subsequent isomeric transition to the ADP-insensitive form (E2P, step 4) will result in a reduction in affinity and a change in orientation of the Ca 2ϩ binding sites and thus a Ca 2ϩ release into lumen (step 5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). The main kinetic limitation in this cycle is associated with the mechanism of Ca 2ϩ release before the hydrolysis of E2P (8, 9). E2P can also be formed from P i in the presence of Mg 2ϩ and absence of Ca 2ϩ by reversal of the hydrolysis of E2P. The three-dimensional structure of Ca 2ϩ -ATPase with bound Ca 2ϩ (E1⅐Ca 2 ) and, very recently, the structure without bound Ca 2ϩ and with bound thapsigargin (E2(TG)), were solved by x-ray crystallography at the atomic level (10, 11). The enzyme has three cytoplasmic domains (A, N, and P) that are widely separated in E1⅐Ca 2 and associated in E2(TG). The modeling with a low resolution map of tubular crystals formed with decavanadate (E2V) revealed (10) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V (see Fig. 7). Our previous limited and systematic proteolysis experiments showed (12, 13) that E2V is very similar to the Ca 2ϩ -released form of E2P in the domain organization and that this EP is the intermediate with the most compactly organized headpiece in the catalytic cycle. The results further indicated that a large motion of the A domain (i.e. rotation by ϳ90° (10)) and the strong association of the A domain with the P and N domains most likely occur during the isomerization of EP and Ca 2ϩ release and suggested that the stabilization energy provided by intimate contacts between all three cytoplasmic domains in E2P provides energy for moving transmembrane helices and releases the bound Ca 2ϩ ions. To substantiate such changes in cytoplasmic domain organization and their roles in the Ca 2ϩ transport, it is essential to find the regions and residues involved in the domain-domain interactions and reveal their actual roles in the catalytic steps. The conserved outermost TGES loop (Thr 181 -Ser 184 ) on the A domain is situated at the interface of the A and P domains in E2V. This loop was previously found to be essential for the isomerization of EP (14) and predicted by iron-catalyzed cleavage with Na ϩ /K ϩ -ATPase to participate in Mg 2ϩ binding with specific residues in the conserved TGDGVND loop (starting

Roles of Tyr122-hydrophobic Cluster and K+ Binding in Ca2+-releasing Process of ADP-insensitive Phosphoenzyme of Sarcoplasmic Reticulum Ca2+-ATPase

“…Our previous analyses indicate that, in the Y122-HC mutants, there is a kinetic limit after the loss of ADP sensitivity and before the hydrolysis of the Ca 2ϩ -free E2P, therefore the Ca 2ϩ release from E2PCa 2 is likely retarded (22,23). Almost the same kinetic results were found with the mutations in another A-P domain interaction network at the Val 200 loop of the A domain (24). Notably, E2PCa 2 , the ADP-insensitive EP with two Ca 2ϩ ions occluded at the transport sites was recently identified and trapped successfully by the elongation of the A/M1-linker with two or more amino acid insertions (9).…”

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

“…The intimate contact of the domains also produces the hydrophobic atmosphere (and the fine-tuning) around the phosphorylation site, so that the specific water molecule can attack the acylphosphate bond and the essential residues can participate in the hydrolysis. Upon the E2P hydrolysis in step 6, the domain organization changes to the more relaxed E2 state in which the interactions of the Lys 189 -Lys 205 loop with P domain are likely lost (9), but A domain has not yet rotated back to the state found in E1Ca 2 and still associated with P and N domains (6,8) by the possible interactions between A and N domains and between P domain and the loops connecting A domain to M2 and M3 as found in E2(TG) (6).…”

“…9) besides Arg 560 at the three-domain interface.) In the subsequent step 5, the final process of gathering of A and P domains is accomplished by the formation of strong interaction between the polar residues surrounding Val 200 Lys 189 -Lys 205 loop (another outermost loop of A domain besides the TGES 184 loop) and those on P domain (Arg 678 and Glu 680 on the basis of E2V) to produce the most compactly organized single headpiece (9). This intimate contact of A and P domains will likely provide the conformational energy enough to further distort P domain and rearrange more the transmembrane helices and thus to open the luminal gate and release the bound Ca 2ϩ .…”

“…We have recently identified the Lys 189 -Lys 205 outermost loop of A domain as to make intimate contact with P domain for formation of the proper structure of Ca 2ϩ -released form of E2P in step 5 (9). P domain was actually documented with the crystal structures E1Ca 2 and E2(TG) to function as a coordinator for transmitting the movements of the transmembrane helices to the cytoplasmic domains (6).…”

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

“…In addition, the point mutations R678Q, S186A, S186P, and Q202A as well as the double mutation Q202A/D203A were included in the study, the latter two motivated by previous reports that replacement of Gln 202 with alanine causes reduced Ca 2ϩ transport (32) and ATPase activity (33). The wild type and mutant Ca 2ϩ -ATPase constructs were expressed in COS-1 cells to similar high levels, allowing the study of the kinetics of the partial reactions of the pump cycle as well as direct measurements of nucleotide binding.…”