Laure Yatime

Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

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 Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2–MgFx–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 Mg2+. 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+–Mg2+ competition and explain the well-known antagonistic effect of K+ on CTS binding.

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase.

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

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. 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 Mg2+ 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.

Structural basis for activation of the complement system by component C4 cleavage.

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.

Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states.

Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNAiMet. After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNAiMet and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β.

Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3

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. Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site–bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.

Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

Structural insights into the oligomerization mode of the human receptor for advanced glycation end-products.

The receptor for advanced glycation end‐products (RAGE) is a pattern recognition receptor sensing endogenous stress signals associated with the development of various diseases, including diabetes, vascular complications, Alzheimers disease and cancer. RAGE ligands include advanced glycation end‐products, S100 proteins, high mobility group box 1 protein and amyloid β‐peptides/fibrils. Their signalling through RAGE induces a sustained inflammation that accentuates tissue damage, thereby participating in disease progression. Receptor oligomerization appears to be a crucial parameter for the formation of active signalling complexes, although the precise mode of oligomerization remains unclear in the context of these various ligands. In the present study, we report the first crystal structure of the VC1C2 fragment of the RAGE ectodomain. This structure provides the first description of the C2 domain in the context of the entire ectodomain and supports the observation of its conformational freedom relative to the rigid VC1 domain tandem. In addition, we have obtained a new crystal structure of the RAGE VC1 fragment. The packing in both crystal structures reveals an association of the RAGE molecules through contacts between two V domains and the physiological relevance of this homodimerization mode is discussed. Based on homology with single‐pass transmembrane receptors, we also suggest RAGE dimerization through a conserved GxxxG motif within its transmembrane domain. A multimodal homodimerization strategy of RAGE is proposed to form the structural basis for ligand‐specific complex formation and signalling functions, as well as for RAGE‐mediated cell adhesion.

Human C3a and C3a desArg anaphylatoxins have conserved structures, in contrast to C5a and C5a desArg.

Complement is a part of innate immunity that has a critical role in the protection against microbial infections, bridges the innate with the adaptive immunity and initiates inflammation. Activation of the complement, by specific recognition of molecular patterns presented by an activator, for example, a pathogen cell, in the classical and lectin pathways or spontaneously in the alternative pathway, leads to the opsonization of the activator and the production of pro‐inflammatory molecules such as the C3a anaphylatoxin. The biological function of this anaphylatoxin is regulated by carboxypeptidase B, a plasma protease that cleaves off the C‐terminal arginine yielding C3a desArg, an inactive form. While functional assays demonstrate strikingly different physiological effects between C3a and C3a desArg, no structural information is available on the possible conformational differences between the two proteins. Here, we report a novel and simple expression and purification protocol for recombinant human C3a and C3a desArg anaphylatoxins, as well as their crystal structures at 2.3 and 2.6 Å, respectively. Structural analysis revealed no significant conformational differences between the two anaphylatoxins in contrast to what has been reported for C5a and C5a desArg. We compare the structures of different anaphylatoxins and discuss the relevance of their observed conformations to complement activation and binding of the anaphylatoxins to their cognate receptors.

Structural and functional characterization of human and murine C5a anaphylatoxins.

The structure of the human C5aR antagonist, C5a-A8, reveals a three-helix bundle conformation similar to that observed for human C5a-desArg, whereas murine C5a and C5a-desArg both form the canonical four-helix bundle. These conformational differences are discussed in light of the differential C5aR activation properties observed for the human and murine complement anaphylatoxins across species.