Limulus polyphemus Hemocyanin: 10 Å 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 (1×6mers) that form the native 8×6mer in a nested hierarchy of 2×6mers and 4×6mers. 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 1×6mer are available, but for the 8×6mer only a 40 Å 3D reconstruction exists. Consequently, the structural parameters of the 8×6mer 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 Å structure (FSC1/2-bit criterion) of the oxygenated 8×6mer 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 Å to the final 10 Å. The 10 Å structure allows firm assessment of various structural parameters of the 8×6mer, the 4×6mer and the 2×6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2×6mer level (II–II, II–IV, V–VI, IV–VI), three form the 4×6mer (V–V, V–VI, VI–IIIB/IV/V), and four are required to assemble the 8×6mer (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.