Conformational Fluctuations of the Ca²⁺-ATPase in the Native Membrane Environment

Abstract

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca²⁺-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1∼P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg²⁺, conversion of E2 to E1 pattern occurs (even when Ca²⁺ is absent) as H⁺ dissociates from acidic residues. Mutational analysis demonstrates that the Glu³⁰⁹ and Glu⁷⁷¹ acidic residues (empty Ca²⁺-binding sites I and II) are required for stabilization of E2. Glu³⁰⁹ ionization is most important to yield E1. However, a further transition produced by Ca²⁺ binding to E1 (i.e. E1·2Ca²⁺) is still needed for catalytic activation. Following ATP utilization, H⁺/Ca²⁺ exchange is involved in the transition from the E1∼P·2Ca²⁺ to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1∼P·2Ca²⁺ to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca²⁺. The E2 state and luminal path closure follow removal of conformational constraint by phosphate.