The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

The sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal muscles 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 Ϸ11 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-␤-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.crystal ͉ molecular dynamics simulation ͉ neurotransmitter:sodium symport ͉ second substrate (S2)-binding site ͉ scintillation proximity binding assay

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca 2؉ -ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca 2؉ entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990 -1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.Understanding protein-membrane interaction and the activity of lipophilic drugs and specific lipids like sterols attracts increasing attention and represents a challenge to our current models of protein-solvent interactions and protein dynamics. Sarco/endoplasmic Ca 2ϩ -ATPase (SERCA) 6 offers an attractive model for the analysis of such interactions due to favorable biochemical properties with sensitive assays of function, a large resource of inhibitors, and a wealth of crystal structures related to the functional cycle. In conjunction with plasma membrane Ca 2ϩ -ATPases and Ca 2ϩ channel proteins, SERCA orchestrates spatiotemporal changes in the concentration of Ca 2ϩ inside the cell, providing the background essential for the function of Ca 2ϩ as a second messenger in the regulation of numerous cellular processes (1-3). At the same time, high cytosolic (Ͼ1 M) concentrations of Ca 2ϩ , induced by depletion of the endoplasmic Ca 2ϩ stores and uptake of extracellular Ca 2ϩ by the endoplasmic store-regulated mechanism (4), are involved as essential factors in the induction of apoptotic processes, leading to mitochondrial disruption with release of cytochrome c, activation of intracellular caspases, and cell death (5). As a consequence, interference of cellular function by inhibition of SERCA may lead to apoptotic cell death irrespective of the...

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL’s α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL’s catalytic site, including β2, β3–α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3–α3 and progress to β5 and β4–α4, ultimately leading to the irreversible unfolding of regions that form LPL’s catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL’s α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.