Fortuitous structure determination of 'as-isolated' Escherichia coli bacterioferritin in a novel crystal form

Abstract

Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 A resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on 'as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.

Figure 1

(a) A cubic crystal of E. coli bacterioferritin (red) growing between large hexagonal aldolase crystals; (b) silver-stained SDS–PAGE gel of washed crystals. Lane 1, contents of red cubic crystal; lane 2, contents of hexagonal aldolase crystal. Molecular-weight standards (in kDa) are indicated.

Figure 2

Overall structure of E. coli bacterioferritin. (a) Cartoon representation of the eight subunits found in the asymmetric unit. Additional subunits generating the entire protein shell are indicated as traces. (b) The bacterioferritin dimeric building block, consisting of two four-helix bundles. The short capping helices, which are involved in intersubunit contacts, are rendered in a different colour. The haem group is in stick representation and spheres indicate the metal positions of the ferroxidase centre. Figs. 2 ▶ and 3 ▶ were generated with PyMOL (DeLano Scientific; http://pymol.sourceforge.net/).

Figure 3

The ferroxidase centre. (a) Stereo figures of σ_A-weighted F _o − F _c density in the ferroxidase site contoured at 5σ, (b) superposition of the ferroxidase centre of the P2₁3 structure (grey/orange) and the Mn-bound structure (yellow/purple; PDB code 1bcf; Frolow et al., 1994 ▶) of E. coli bacterioferritin and (c) superposition of the FE2 site environment in the P2₁3 E. coli bacterioferritin structure (grey) with the corresponding region in the structure of reduced A. vinelandii bacterioferritin (pale green; PDB code 1fkz; Swartz et al., 2006 ▶). The location of the inner cavity (IC) is indicated. This view is rotated ∼90° with respect to the previous views.

Figure 4

X-ray fluorescence scans around the K edges of Mn, Fe, Cu and Zn, as indicated. Plots are corrected for beam intensity and fluorescence is on an arbitrary scale.