Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na + ,K + -ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five-or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na + ,K + -ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg 2+ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K + in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.Na/K-ATPase | phosphoenzyme | inhibitor | cardiac glycosides | structure C ardiotonic steroids (CTSs) induce diverse physiological effects on, for example, heart muscle and blood pressure regulation, but the underlying mechanisms remain unknown, despite a long history of therapeutic applications and model studies. It is widely recognized that they target Na + ,K + -ATPase, and a direct consequence of their binding is an inhibition of the enzyme. Their positive inotropic effect in cardiomyocytes has been related to coupling between Na + ,K + -ATPase and Na + /Ca 2+ -exchanger through the intracellular Na + concentration, whereas numerous other outcomes observed on the cellular level have led to hypotheses of the existence of signaling cascade mechanisms with Na + ,K + -ATPase acting as a receptor. The minimal functional unit of the enzyme is an αβ-complex, and because there exist four α-and three β-isoforms of the Na + ,K + -ATPase, the variations in the heterodimer composition and a vast number of CTSs differing in apparent isoform specificities (1) add to the complexity and multiplicity of reported physiological responses.The conserved structural core shared by all CTSs includes a cis-trans-cis ring-fused steroid core with two methyl substituents at steroid positions C10 β and C13 β , two hydroxyl groups (OH3 β and OH14 β ), and an unsaturated lactone ring at the C17 β position, among which the lactone and OH14 β are critical for binding to Na + ,K + -ATPase (2, 3). The type of lactone at the C...

The Na + ,K + -ATPase maintains electrochemical gradients for Na + and K + that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na

No abstract

Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.