10-A cryoEM structure and molecular model of the Myriapod (Scutigera) 6x6mer hemocyanin: understanding a giant oxygen transport protein.

Oxygen transport in Myriapoda is maintained by a unique 6x6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1x6mers). In the sluggish diplopod Spirostreptus, the 1x6mers seem to operate as almost or fully independent allosteric units (h approximately 1.3; P(50) approximately 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h approximately 10; P(50) approximately 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6x6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6x6mer (10.0 A resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6x6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1x6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3x6mers or three oblique 2x6mers. Topologically, the 6x6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3x6mer and three between both 3x6mers. The molecular models of the 6x6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca(2+) or Mg(2+) ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.

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.