Significance: Na ⁺ ,K ⁺ -ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na ⁺ and K ⁺ across the membrane of animal cells. Cardiotonic steroids constitute a broad class of specific Na ⁺ ,K ⁺ -ATPase inhibitors, including drugs of clinical importance with multiple physiological effects. The existence of several endogenous cardiotonic steroids suggests their involvement in health and disease mediated by various signaling pathways, but the structure–activity relationships are not yet understood. Using X-ray crystallography and analysis of binding kinetics, we characterize Na ⁺ ,K ⁺ -ATPase complexes with a total of five cardiotonic steroids, showing variations in glycosylation, steroid core substituents, and structure of the lactone substituent. This insight is highly relevant for the understanding of physiological effects and future drug development based on cardiotonic steroids.

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 ⁺ ,K ⁺ -ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na ⁺ ,K ⁺ -ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg ²⁺ , and crystallographic studies indicate that Rb ⁺ and Mn ²⁺ , but not Na ⁺ , bind to this site. Comparison with the low-affinity [K ₂ ]E2–MgF _x –ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg ²⁺ . K ⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K ⁺ –Mg ²⁺ competition and explain the well-known antagonistic effect of K ⁺ on CTS binding.

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.

Significance: Fragments of complement component C3 tag surfaces such as those presented by microbial pathogens or dying host cells for recognition by cells from the innate immune system. Complement receptor (CR) 3 enables efficient binding of complement-tagged surfaces by macrophages and dendritic cells, which eventually transport the CR3-bound material into lymph nodes. The study identifies in atomic details the fragments of CR3 and C3 required for such binding. The structural organization permits concomitant recognition by another complement receptor, namely CR2, expressed on cells of the adaptive immune system, suggesting a structural rationale for the exchange of antigens between leukocytes of the innate and adaptive immune systems critical in the formation of humoral immune responses.