Conformational Transitions and Alternative Access Mechanism in ATP-Driven Calcium Pump SERCA

Abstract

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is an integral membrane protein that uses ATP hydrolysis as a source of free energy to pump two calcium ions per ATP molecule from calcium poor cytoplasm of the muscle cell to the calcium rich lumen of the sarcoplasmic reticulum, thereby maintaining a ten thousand fold concentration gradient. Detailed structural studies of the pump under different conditions provided analogues of various intermediates in the reaction cycle and revealed important changes in the tertiary structure of the protein both in the cytoplasmic and in the transmembrane parts. It is essential to go beyond the static crystal structures and study functionally relevant dynamical motions of the protein to achieve a comprehensive understanding of the mechanism of the pump. Functional transitions in SERCA occur in timescales (milliseconds and longer) far beyond the reach of standard molecular dynamics simulations for molecular systems of this size (a solvated SERCA system comprises ∼291,000 atoms). This problem is overcome by employing the string method in which a transition pathway between two known end-point structures is represented discretely as a chain-of-state. Using this method, we have determined the pathways for all the functional transitions between known crystal structures along the entire transport cycle. Here we present detailed studies on three important steps of the pumping cycle: 1) ion binding from the cytoplasmic side and occlusion; 2) opening of the luminal gate and release of ions to the luminal side; 3) closing of luminal gate and its coupling to dephosphorylation. We clearly demonstrate the tight coupling between the motions of various regions of the protein which explains how information is transmitted between the phosphorylation and transmembrane binding sites that are spatially separated by a large distance. We correlate our observations with biochemical experiments.