Asparagine 706 and Glutamate 183 at the Catalytic Site of Sarcoplasmic Reticulum Ca2+-ATPase Play Critical but Distinct Roles in E2 States

Abstract

Mutants with alteration toThe sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (1) is an energytransducing enzyme of the P-type that couples hydrolysis of ATP to translocation of Ca 2ϩ from the cytosol to the endoplasmic reticulum. In this control of cytosolic Ca 2ϩ concentration, the Ca 2ϩ -ATPase plays a vital role in cellular activation events, such as muscle contraction, hormone secretion, immune responses, cell migration, and protein synthesis. Ca 2ϩ transport is coupled to ATP hydrolysis by a reaction cycle (Scheme 1), in which the enzyme is transiently phosphorylated at a conserved aspartic acid residue and undergoes major conformational changes (2, 3).In recent years, several high resolution crystal structures of the Ca 2ϩ -ATPase, each thought to represent a particular intermediate state in the pump cycle, have been solved (4 -9). The Ca 2ϩ -ATPase consists of a membrane-spanning domain of ten helical segments and a large cytoplasmic head piece, comprising three distinct domains, named "N" (nucleotide binding), "P" (phosphorylation), and "A" (actuator). By combining crystallographic data with functional changes in site-specific mutants, an increasingly detailed picture of the mechanisms of energy interconversion and ion translocation in the Ca 2ϩ -ATPase is emerging. Thus, the catalytic function in E1 (autokinase activity) and E2 forms (autophosphatase activity), the movement of Ca 2ϩ ions across the membrane, as well as the major rate-limiting conformational changes of the cycle, i.e. E2 3 E1 and E1P 3 E2P, can all be understood on the basis of the sequential gathering and displacement of certain conserved amino acid motifs in domains N and A relative to the catalytic site in domain P and the coupling of these events to rearrangements of the transmembrane helices containing the high affinity Ca 2ϩ sites. In the present study, we address the role of Asn 706 at the catalytic site of Ca 2ϩ -ATPase and revisit a previously examined mutant, E183A (10). Asn 706 and Glu 183 reside in the conserved 701 TGDGVND 707 (domain P) and 181 TGES 184 (domain A) motifs, respectively, and both residues are found in all known P-type ATPases (11). In fact, Asn 706 is highly conserved even in the superfamily of phosphohydrolases and phosphotransferases (the HAD superfamily), which, given the similarities in reaction mechanism, protein sequence, and structural architecture of the catalytic site, are believed to share a common evolutionary ancestor with the phosphorylation domain of the P-type ATPases (12-14). The side chains of Asn 706 and Glu 183 are both centrally located at the catalytic site in the E2 forms of Ca 2ϩ -ATPase, close to the phosphorylated aspartate, Asp 351 (6, 7, 9). In E1 conformations, Glu 183 has departed the phosphorylation site, whereas Asn 706 retains its close proximity to Asp 351 (4,5,7,8). In our previous study of Glu 183 (10), we demonstrated that substitution of the glutamate with alanine leads to a much reduced rate of both E2P hydrolysis and of the reverse phosphorylation of E2 with P i , suggesti...

Mutational Analysis of the Conserved TGES Loop of Sarcoplasmic Reticulum Ca2+-ATPase

“…binding of the metal fluoride phosphoryl analogs beryllium fluoride, aluminum fluoride, and magnesium fluoride, as well as the binding of vanadate, were determined using the method previously described and validated (20,32). For metal fluoride binding, the enzyme was incubated at 25°C with varying concentrations of AlCl 3 , BeSO 4 , or MgCl 2 with a fixed NaF concentration and without Ca 2ϩ , to stabilize the E2 form of the enzyme.…”

“…We have inquired about the consequences of variation of size, polarity, and charge of the TGES side chains for the overall and partial reactions of the Ca 2ϩ -ATPase studied by steady state and transient kinetic measurements. A newly introduced method (20) was used to examine the effects of the mutations on the binding affinities for the metal fluoride phosphoryl analogs beryllium fluoride, aluminum fluoride, and magnesium fluoride, supposed to mimic the phosphoryl group in the ground state, the transition state, and the E2⅐P i product state, respectively, of E2P hydrolysis.…”

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

“…The rearrangement of the three cytoplasmic domains in the E 1 to E 2 transition and the associated changes in the orientation of the membrane helices implied by the various srCa ATPase crystal structures are believed to be generally conserved (28,29). This assumption has provided the foundation of structure-function analyses for the potassium counter-transport ATPases (12,26,30,31).Our previous homology model of the H,K ATPase (11) was based on the backbone of srCa ATPase in the E 2 state (PDB code 1iwo) (18), the only E 2 conformer available at that time. This form is unphosphorylated, and thus a hypothetical E 2 P model was generated by addition of phosphate at the active site to mimic the HAD protein, phosphoserine phosphatase, to account for binding of K + and the K + competitive inhibitors to the E 2 P conformation in the H,K ATPase.…”

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

“…As seen in Fig. 5, open symbols, S72R, E90R, and 4Gi-46/47 were all able to form a stable Ca 2ϩ -occluded Ca 2 E1⅐AlF 3 Ϫ ⅐ADP complex similar to the wild type (23,24). Then in another set of experiments without AlF 3 Ϫ and ADP, the enzyme was phosphorylated by ATP in the presence of 45 Ca 2ϩ followed by the same EGTA chase and filtration procedure as described above (closed symbols in Fig.…”