Intermolecular Interactions in the Mechanism of Skeletal Muscle Sarcoplasmic Reticulum Ca2+-ATPase (SERCA1) : Evidence for a Triprotomer

The sarcoplasmic reticulum (SR) Ca2+-ATPase from skeletal muscle (SERCA1) utilizes the energy derived from ATP hydrolysis to transport Ca2+ into the SR against a concentration gradient. Resolution of the crystal structure of SERCA1 in the E1 and E2 conformational states by Toyoshima (1, 2) has facilitated correlation of the biochemical reactions of the Ca2+-ATPase with changes in its atomic and molecular structure associated with unidirectional Ca2+ transport. Despite these recent advances, controversy remains concerning the Ca2+ transport mechanism, and in particular the functional transport unit of the Ca2+ pump. The Ca2+-ATPase is generally thought to operate as a monomer, fully competent and independent of its neighbors. However, this view has been challenged by reports from several laboratories identifying the skeletal muscle SR Ca2+ pump as a Ca2+-ATPase oligomer (3 and references therein). Indeed, transient state kinetic studies have uncovered several features of the enzymatic behavior of native membrane SERCA1 contradictory to the monomer hypothesis. The crystal structure reveals the presence of one nucleotide binding site (1, 4), whereas variation of [ATP] in the micromolar range has been shown to influence the kinetic behavior of several of the enzymatic reactions down-stream from phosphorylation, including the phosphoenzyme transition (5, 6), E2P hydrolysis (7, 8) and the conversion of E2 to E1 (7, 9). ATP has also been shown to modulate the transport stoichiometry of the pump, shifting the ratio from 1 Ca2+ transported per ATP hydrolysed at low (1–10 µM) ATP to 2:1 at high (>100 µM) ATP (10). Although the reaction mechanism contains multiple acid-stable phosphoenzyme intermediates, their consecutive relationship predicts monophasic kinetics of phosphoenzyme formation by ATP and monophasic dephosphorylation kinetics when re-phosphorylation is prevented by Ca2+ chelation with EGTA. In contrast, phosphorylation of Ca2+-equilibrated native SERCA1 by ATP produces biphasic kinetics at 2°C (11–13), while dephosphorylation with EGTA also yields a biphasic pattern of EP decay (14, 15) which becomes monophasic following solubilization with the monomer-forming detergent, C12E8 (3, 16). This behavior is consistent with the presence of parallel catalytic pathways in an oligomer in which out-of-phase coupling of the subunits delays the onset of a reaction in one subunit relative to that in its neighbor (3, 11–13, 15, 16, 17). From the consecutive relationship between E1P and E2P, ADP-sensitive E1P is expected to reach its maximum level in the pre-steady state and then decline while the ADP-insensitive E2P shows the opposite behavior, becoming the predominate phosphorylated intermediate in the steady state. By contrast, when ADP was used to chase the phosphoenzyme at 2°C we found that E2P exceeded E1P in the pre-steady state and then rapidly declined, becoming equimolar with E1P as the reaction entered the steady state (3). In the context of the conventional E1, E2 Ca2+-ATPase linear (monomeric) reaction mechanism this behavior implies that the transformation of E1P to E2P is very fast initially, but then dramatically slows down allowing E1P to accumulate in the steady state. The absence of a unique value for the rate constant controlling the E1P→ E2P transition means that the conventional model fails the test for consistency and that modification of this scheme is necessary to render the transition rate constant time-invariant. Finally, the conventional linear consecutive model failed to reproduce quantitatively the time courses of both phosphoenzyme formation and inorganic phosphate (Pi) release measured at 21°C (3). The data show that the decay of the phosphoenzyme overshoot and the Pi burst occur at the same time; however, the size of the Pi burst is much larger than EP decay. We were unable to resolve this problem by increasing the rate of the back reaction for E2P hydrolysis or by adding additional intermediates to the reaction mechanism (3). In our previous studies (3, 13, 17, 18), we considered various dimer schemes, culminating in the model shown in Fig.1 (3), which consists of two linear pathways coupled together so that the subunits maintain an out-of-phase relationship. This model greatly improved our ability to simulate the kinetic data outlined above, including the biphasic time courses of EP formation and dephosphorylation, and the inverted time dependence of E1P and E2P formation in the pre-steady and steady states. However, as in the monomer simulations, the dimer model failed to reproduce quantitatively the amplitude of the Pi liberation burst while maintaining the observed steady-state EP level. It became clear that a modification of the model was needed that would allow for simultaneous phosphorylation of one subunit and dephosphorylation of another, producing the observed Pi burst while preventing the decay of the EP overshoot. Fig. 1 Dimer model of the skeletal SR Ca2+-ATPase (SERCA1). The model consists of parallel catalytic pathways constrained to out-of-phase (asynchronous) cycling by conformational coupling between the subunits. The subunits oligomerize forming an asymmetric E2/E1 ... In the present study we have addressed the issue of the quantitative discrepancy between the decay of the overshoot and the Pi burst amplitude by considering a model consisting of three interacting SERCA1 subunits. Support for this model was provided by chemical cross-linking studies, which demonstrated the presence of Ca2+-ATPase trimers under cycling and non-cycling conditions, and by AMPPCP titration experiments revealing the presence of high and low affinity nucleotide binding sites in a 2:1 ratio at physiological (0.1 M) KCl. We show here that the SERCA1 trimer model faithfully reproduces of all the kinetic data in our previous studies. By enabling out-of-phase conformational coupling of the subunits in the oligomeric complex, the trimer facilitates inter-subunit free energy transfer (15), accelerating slow steps (E2P hydrolysis) at the expense of faster reactions (E1ATP → E1P; E1P → E2P). This leads to an improvement in the overall catalytic efficiency, increasing the velocity of Ca2+ transport in the presence of an increasing concentration gradient.