The Structure of a Functional Unit from the Wall of a Gastropod Hemocyanin Offers a Possible Mechanism for Cooperativity^,

Abstract

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1‐h) reveals disulfide bridges

“…As type 3 copper proteins and their active sites are embedded in a four a-helix bundle with six histidine residues, which coordinate two copper atoms (4)(5)(6)(7)(8). Between them one molecule oxygen is reversibly bound in side-on (l-g 2 :g 2 ) coordination (4,7,9,10).…”

“…Crystal structures of molluscan hemocyanin FUs representing different topological positions in the quaternary structure (wall, inner collar, outer collar) are available (4)(5)(6)(7)(8). The standard molluscan hemocyanin FU (FU-a to FU-f) folds into two domains.…”

“…1). The N-terminal core domain (KLH1-h-D1) contains the type 3 copper active site as in other known FU structures (4,8). The central domain (KLH1-h-D2) is equivalent to the Cterminal domain of other FUs (FU-a to FU-g) and contains a bsandwich as its main structural element.…”

Purification of Hemocyanin from Marine GastropodRapana Thomasianausing Ammonium Sulfate Precipitation Method

“…new hc has been isolated and purified from the hemolymph of this marine gastropod using ultracentrifugation and column chromatography (6). Rapana thomasiana hemocyanin (Rth) has been structurally well characterized (2,3,6,7,8,9,10,12,14,15,16). Recently, we demonstrated the high immunogenicity of Rth as a model antigen and also its properties as a strong protein carrier for viral peptides from Influenza hemagglutinin (17).…”

Small-angle X-ray Scattering-based Three-dimensional Reconstruction of the Immunogen KLH1 Reveals Different Oxygen-dependent Conformations

“…Each FU binds one molecule of oxygen (1,16,17). Based on x-ray structures of FU-g from the octopus O. dofleini (1) and FU-e from Rapana thomasiana (18), a FU contains two subdomains with different folding motifs, a N-terminal ␣-helical part containing the oxygen binding center and a C-terminal ␤-rich part (1,17,18). Depending on the species, covalently bound carbohydrates are located at the interface between the two subdomains (1) or at the N-terminal domain (18).…”