Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers.

The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1x6mers) that form the native 8x6mer in a nested hierarchy of 2x6mers and 4x6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1x6mer are available, but for the 8x6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8x6mer are not firmly established, and the molecular interfaces between the eight hexamers are still to be defined. This, however, is crucial for understanding how allosteric transitions are mediated between the different levels of hierarchy. Here, we show the 10 A structure (FSC(1/2-bit) criterion) of the oxygenated 8x6mer from cryo-electron microscopy (cryo-EM) and single-particle analysis. Moreover, we show its molecular model as obtained by DNA sequencing of subunits II, IIIA, IV and VI, and molecular modelling and rigid-body fitting of all subunit types. Remarkably, the latter enabled us to improve the resolution of the cryo-EM structure from 11 A to the final 10 A. The 10 A structure allows firm assessment of various structural parameters of the 8x6mer, the 4x6mer and the 2x6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2x6mer level (II-II, II-IV, V-VI, IV-VI), three form the 4x6mer (V-V, V-VI, VI-IIIB/IV/V), and four are required to assemble the 8x6mer (IIIA-IIIA, IIIA-IIIB, II-IV, IV-IV). The molecular model shows the amino acid residues involved, and reveals that several of the interfaces are intriguingly histidine-rich and likely to transfer allosteric signals between the different levels of the nested hierarchy.

Formation, TEM study and 3D reconstruction of the human erythrocyte peroxiredoxin-2 dodecahedral higher-order assembly.

The production of a higher-order assembly of peroxiredoxin-2 (Prx-2) from human erythrocytes has been achieved during specimen preparation on holey carbon support films, in the presence of ammonium molybdate and polyethylene glycol. TEM study suggested that this assembly is a regular dodecahedron, containing 12 Prx-2 decamers (Mr 2.62 MDa, external diameter approximately 20 nm). This interpretation has been supported by production of a approximately 1.6 nm 3D reconstruction from the negative stain TEM data, with automated docking of the available X-ray data of the Prx-2 decamer. Comparison with other known protein dodecahedral and viral icosahedral structures indicates that this arrangement of protein molecules is one of the fundamental macromolecular higher-order assemblies found in biology. Widespread biotechnological interest in macromolecular "cage" structures is relevant to the production of the Prx-2 dodecahedron.

Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy.

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O(2) molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11A (FSC(1/2-bit) criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna.

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data.

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 x 6-mer) in the 75kDa range. A 3D reconstruction of the 1 x 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as alpha-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P.elephas and the American spiny lobster Panulirus interruptus. Comparison of the P.elephas hemocyanin electron microscopy (EM) density map with the known P.interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597-605) and the C-terminal end (654-657), which are absent in the available P.interruptus X-ray data.

3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis.

Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.

Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential.

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units.

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a "pearl-chain" of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O(2) molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 A cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 A resolution (FSC(1/2-bit) criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 A structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.

Minireview: Recent progress in hemocyanin research.

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-Å structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study.

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air. Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry. To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined. 19-Hydroxycholesterol and 7 beta-hydroxycholesterol both induce VCC oligomerization. beta-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate. Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur. Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability. Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside. Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains.