The sarcoplasmic (SERCA 1a) Ca²⁺-ATPase is a membrane protein abundantly present in skeletal mucles where it functions as an indispensable component of the excitation–contraction coupling, being at the expense of ATP hydrolysis involved in Ca²⁺/H⁺exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca²⁺transporting proteins count Na⁺, K⁺-ATPase and H⁺, K⁺-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3–3 Å) resolution 3D-crystals of Ca²⁺-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca²⁺-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca²⁺and H⁺, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca²⁺across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na ⁺ ; later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule ≈11 Å above the bound leucine and 2 Na ⁺ . We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na ⁺ -coupled substrate symport. Here, we show a profound inhibitory effect of n -octyl-β- d -glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 Å the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na ⁺ -coupled symport.

We present crystal structures of the calcium‐free E2 state of the sarcoplasmic reticulum Ca2+‐ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg2+ ion in accordance with previous Fe2+‐cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca2+ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca2+ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca2+ activation of phosphorylation leading directly from the compact E2‐ATP form to the Ca2E1‐ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.