Multiple and Distinct Effects of Mutations of Tyr122, Glu123, Arg324, and Arg334 Involved in Interactions between the Top Part of Second and Fourth Transmembrane Helices in Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

We explored, by mutational substitutions and kinetic analysis, possible roles of the four residues involved in the hydrogen-bonding or ionic interactions found in the Ca 2؉ -bound structure of sarcoplasmic reticulum Ca 2؉ ; it thus contributes to the inclination of the M4/P domain toward the M2/A domain, which is crucial for the appropriate gathering between the P domain and the largely rotated A domain to cause the loss of ADP sensitivity. On the other hand, Tyr 122 most likely functions in the subsequent Ca 2؉ -releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca 2؉ -released form of EP. During the Ca 2؉ -transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 1 is a representative member of P-type ion-transporting ATPases; it catalyzes Ca 2ϩ transport coupled with ATP hydrolysis ( Fig. 1; Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4). In the catalytic cycle, the enzyme is activated by the binding of two Ca 2ϩ ions (E2 to E1Ca 2 , steps 1 and 2) and then autophosphorylated at Asp 351 by MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3). Upon 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) will result in a reduction in affinity and a change in orientation of the Ca 2ϩ -binding sites and, thus, the Ca 2ϩ release into lumen (steps 4 -5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). E2P can also be formed from P i in the presence of Mg 2ϩ and the absence of Ca 2ϩ by reversal of its hydrolysis. The enzyme has three cytoplasmic domains (N, P, and A) which are widely separated in the Ca 2ϩ -bound form (E1Ca 2 ) but are associated in the Ca 2ϩ -unbound and thapsigarginbound form E2(TG) (Refs. 5 and 6; Fig. 2). In E2(TG), the A domain has largely rotated, and the P domain has significantly inclined together with the transmembrane helices M4 and M5 toward A domain to associate with A domain. We showed previously in the proteolysis experiments (7,8) that the large rotation of A domain and its gathering with P and N domains most likely occur during the E1P to E2P transition and Ca 2ϩ release (steps 4 -5) to form the most compactly organized single headpiece in the Ca 2ϩ -released form of E2P; we further suggested that the stabilization energy provided by the intimate contacts between three cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca 2ϩ into lumen. To gain further insight into the energy coupling between cytoplasmic and transmembrane domains, it is crucial to find out the structural elements essential for the changes in the cytoplasmic domain organization and the coordinated movements of transmembrane helices and reveal the actual roles of ...

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

“…The Ca 2ϩ -ATPase activity was nearly completely inhibited in all the mutants in agreement with our previous observation (22,23), and the complete inhibition was found at all the Ca 2ϩ concentrations examined (see supplemental Fig. S1 for the representative mutant Y122A).…”

“…Thus Y122-HC produces the compactly organized structure of E2P. 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).…”

“…In these Y122-HC mutants, the high affinity binding of cytoplasmic Ca 2ϩ , the resulting E1PCa 2 formation, and the loss of the ADP sensitivity were all found to occur normally as in the wild type (22,23). Y122-HC is formed by gathering of the seven residues of the three regions upon their motions; i.e.…”

“…Recently, we found that mutations in a specific hydrophobic interaction network, "Tyr 122 -hydrophobic cluster" (Y122-HC), at the A-P domain interface disrupt markedly the processing of ADP-insensitive EP formed from ATP with Ca 2ϩ and also the hydrolysis of E2P formed from P i without Ca 2ϩ , thus causing nearly complete inhibition of the Ca 2ϩ -ATPase activity (22,23). In these Y122-HC mutants, the high affinity binding of cytoplasmic Ca 2ϩ , the resulting E1PCa 2 formation, and the loss of the ADP sensitivity were all found to occur normally as in the wild type (22,23).…”

Ca2+ Release to Lumen from ADP-sensitive Phosphoenzyme E1PCa2 without Bound K+ of Sarcoplasmic Reticulum Ca2+-ATPase

“…E1PCa 2 to E2P transition. The result agrees with the established mechanism that the two Ca 2ϩ ions are occluded in E1PCa 2 , and Ca 2ϩ release into the lumen occurs very rapidly after the rate-limiting E1PCa 2 to E2PCa 2 transition, E1PCa 2 3 E2PCa 2 3 E2P ϩ 2Ca 2ϩ (11)(12)(13)(14). Thus, the EP transition and Ca 2ϩ release are tightly coupled in the presence of K ϩ .…”