Crystal Structure of D351A and P312A Mutant Forms of the Mammalian Sarcoplasmic Reticulum Ca2+-ATPase Reveals Key Events in Phosphorylation and Ca2+ Release

Abstract

In recent years crystal structures of the sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF 4 ؊ bound (3.5-Å resolution) and D351A with AMPPCP or ATP bound (3.4-and 3.7-Å resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca 2؉ was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca 2؉ -translocating Ca 2 E1P 3 E2P transition, which seems to be due to a stabilization of Ca 2 E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro 312 with alanine in P312A.The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) 5 plays a crucial role in muscle relaxation by re-accumulating Ca 2ϩ into the sarcoplasmic reticulum lumen at the end of the contractile event (1, 2). The fast twitch muscle isoform (SERCA1a) can easily be prepared in large yield from rabbit skeletal muscle, which has facilitated many biochemical and biophysical studies, thus leading to a detailed description of its functional and structural properties (3-7). SERCA belongs to the family of P-type ATPases characterized by the formation during the catalytic cycle of an energy-rich covalent aspartylphosphorylated intermediate. The cycle of phosphorylation and dephosphorylation is coupled with Ca 2ϩ /H ϩ exchange through conformational changes between the so-called "E1" and "E2" forms in phosphorylated and dephosphorylated states. Ca 2ϩ binding from the cytoplasmic side in the E1 form is required for phosphorylation of the enzyme from ATP, whereas the dephosphorylation occurs subsequently to the luminal release of Ca 2ϩ from E2P and leads to proton countertransport (Scheme 1). Key residues involved in the binding of Ca 2ϩ and ATP (8 -11) and in the phosphorylation reaction (12, 13) were initially identified by site-directed mutagenesis, and essential information about residues involved in conformational

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

“…This result is consistent with the earlier findings of MacIntosh et al (24), but inconsistent with the results of Marchand et al (25). It is well known that nonionic detergents like C 12 E 8 and dodecylmaltoside substantially decrease the Ca 2ϩ binding affinity of SERCA pumps (40,41), which may explain the failure of Marchand et al (25) to detect an increase in Ca 2ϩ affinity for D351A. Regarding ATP affinity, we determined a K d value of 9 M for the wild-type enzyme, which is well within the range reported by other investigators for ATP binding at the low-affinity modulatory binding site of E2 measured in the absence of Ca 2ϩ [32][33][34][35].…”

“…7A). This result is consistent with the earlier findings of MacIntosh et al (24), but inconsistent with the results of Marchand et al (25). It is well known that nonionic detergents like C 12 E 8 and dodecylmaltoside substantially decrease the Ca 2ϩ binding affinity of SERCA pumps (40,41), which may explain the failure of Marchand et al (25) to detect an increase in Ca 2ϩ affinity for D351A.…”

“…Regarding ATP affinity, we determined a K d value of 9 M for the wild-type enzyme, which is well within the range reported by other investigators for ATP binding at the low-affinity modulatory binding site of E2 measured in the absence of Ca 2ϩ [32][33][34][35]. For D351A, we noted a modest 2.3-fold increase in ATP affinity relative to wild-type SERCA2a (K d ϭ 4.0 M), in contrast to the two studies above that reported a much higher nucleotide binding affinity for D351A measured under similar conditions (24,25). However, our results appear to be consistent with the recently determined crystal structure of the E2(TG)⅐AMPPCP complex, representing E2 with ATP bound at the modulatory site (34).…”

“…Our results indicate that PLB supershifters act by stabilizing a single conformation of SERCA2a, the nucleotide-bound E2 state, forming a ternary complex (E2⅐ATP⅐PLB) that is highly resistant to traditional Ca 2ϩ pump inhibitors like TG and vanadate, which also act at E2 (18,19). In addition, using these superinhibitory PLB mutants we gained new insights on what effect, if any, PLB has on the V max of enzyme activity (20 -23), and whether the catalytically inactive SERCA mutant, D351A (14), has substantially enhanced Ca 2ϩ binding affinity, as was reported earlier (24), but not subsequently confirmed (25).…”

“…Replacement of aspartic acid at this position renders the enzyme catalytically inactive (14,30). Although inactive at the site of ATP hydrolysis, D351A retains the ability to bind Ca 2ϩ and maintains the thermodynamic equilibrium between E1 and E2 (14,24,25). Therefore, if PLB acts by stabilizing E2 and shifting the E1⅐Ca 2 7 E2⅐PLB equilibrium away from E1, then this effect should be fully reproducible with D351A.…”