Crystallization and preliminary structural analysis of the giant haemoglobin from Glossoscolex paulistus at 3.2 Å

Abstract

Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

Figure 1

Crystals of HbGp obtained under two different conditions. (a) 10% PEG8000, 2.5 mM CaCl₂, 50 mM Tris-HCl pH 7.5, which grew to approximate maximum dimensions of 0.1 mm. These crystals readily deteriorate after about a week (b) and often disappear altogether. Crystals obtained in the presence of 1.2 M sodium citrate, 2.5 mM CaCl₂, 50 mM, Tris-HCl pH 7.5, can grow to a maximum dimension of almost 1 mm (c), but the best diffraction patterns were obtained from well formed crystals with typical dimensions of approximately 0.1 mm in all directions (d).

Figure 2

A typical diffraction image from a cyanomet HbGp crystal. The resolution at the edge of the detector is 3.10 Å and the oscillation angle is 0.5°. The insert refers to the highlighted region.

Figure 3

Electron density maps showing (a) an overview of part of the 2F _o − F _c map of the full particle contoured at 2σ, (b) an F _o − F _c omit map of the region of one of the haem groups contoured at 3σ, and (c) 2F _o − F _c (at 2σ, blue) and F _o − F _c (at 4σ, green) maps of the region of a predicted calcium binding site.

Figure 4

The reconstructed HbGp globin chains based on the molecular replacement solution using a protomer of HbLt as the search model. The structure shows the offset of the vertices of the upper disc with respect to the lower one, characteristic of the type I architecture. This is most evident towards the centre of the structure where subunits from the two layers can be clearly visualized. HbLt is shown for comparison. In both images red refers to subunits of the upper disc whilst green (or blue) refer to those of the lower disc.