Hephaestin architecture and construct design.

<p><b>A</b>, Schematic illustration of HEPH function. Ferroportin (FPN) exports divalent iron into plasma, where HEPH catalyses the conversion to trivalent metal. <b>B</b>, The structure of ceruloplasmin shown in cartoon representation, with the three copper binding domains coloured in different shades of grey. Copper ions are shown as orange spheres. In ceruloplasmin, 3 peripheral Type I copper sites shuttle electrons from the Fe²⁺—Fe³⁺ oxidation, with the electrons being transferred to the trinuclear copper cluster. <b>C</b>, The overall construct design used in this study.</p

Oxidase activity of mouse HEPH.

<p>In all assays WT mouse serum and serum from a mouse CpKO were used as control. Details can be found in the main text. <b>A</b>, An oxidase assay monitoring the oxidation of <i>p</i>PD (increase of A₅₃₀). The rate of HEPH catalysed oxidation reaction increased with an increasing protein concentration. <b>B</b>, <i>p</i>PD oxidation by HEPH was inhibited by the copper-specific chelator D-penicillamine (D-P). <b>C</b>, A ferrozine-based assay was also used to follow the oxidation of Fe²⁺ to Fe³⁺ by HEPH (monitored as a decrease of A₅₇₀). Similarly to the <i>p</i>PD assay, an increased catalytic activity can be observed with an increasing amount of HEPH present. <b>D</b>, The catalytic activity was, as in the pPD assay, inhibited by addition of D-P. <b>E</b>, A velocity versus substrate concentration curve used to obtain <i>K</i>_m and <i>V</i>_max for HEPH. Error bars represents 1 S.D, N = 3.</p

The distinctive flattened shape of trimeric Hfx_cass1

<p><b>(PDB 3FUY). </b><b>A.</b> Dimensions highlight distinctive flattened form. Topology map indicates (dashed line) subfold segment found in zinc transporter domain CzrB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Cherezov1" target="_blank">[50]</a>, to which <b>Hfx_cass1</b> is not functionally related. <b>B.</b> Electrostatic surface potential of the trimer surface highlights polar cavities (arrowed) and exposed acidic clusters on external loops. <b>C.</b> Residues from β-strands 3 and 4 form a polar crevice (blue) surrounded by surface loops containing charged residues (red). Solvent molecules trapped within the crevice are shown (spheres).</p

Crystallographic data collection and refinement statistics for structure determination.

a<p>Chains per asymmetric unit (a.s.u).</p>b<p>∑∑<i>_i</i>|I<i>_hi −</i> I<i>_hi</i>|/∑∑<i>_i</i>I<i>_h</i>, where I<i>_h</i> is the mean intensity of reflection <i>h.</i></p>c<p>From MolProbity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Davis1" target="_blank">[36]</a>.</p

Hfx_cass5 contains domain-swapped dimers (PDB 3IF4).

<p><b>A.</b> Ribbon depiction of two domain-swapped chains, each coloured from N-terminus (blue) to C-terminus (red). <b>B.</b> Tetrameric organisation depicted in ribbon form (left panel), showing engagement between two dimers (green and blue), each comprising two chains. Side chain stacking of key contacting residues (Tyr28, Tyr30, Arg31 and Glu35) is depicted. Corresponding view of electrostatic surface is also shown (right), with deep cleft centrally positioned. <b>C.</b> Rotated view of electrostatic surface, positioned to emphasise narrow dimensions of this basic slot along one tetramer surface.</p

The Vpc_cass2 dimer (PDB 3JRT).

<p><b>A.</b> Ribbon of monomeric unit with colour spectrum (blue to red) across six helical components (α1, α2, α3, α′, α″, α4), named as indicated. A loop of weak density connecting helices 2 and 3 is represented by dotted line. <b>B.</b> Contrasting shapes of dimers of <b>Vpc_cass2</b> (left panel) and structural relative HI0074 from <i>Haemophilus influenza </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Lehmann1" target="_blank">[55]</a> (right panel). <b>C.</b> View (left panel) across <b>Vpc_cass2</b> dimer interface shown as electrostatic surface (in blue to red from +5 to −5 kbT/e <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Baker1" target="_blank">[57]</a>), highlighting the basic cluster unique to this protein. Right panel provides segmental view of dimer ribbon (green) and side chains of putative active site residues of <b>Vpc_cass2</b> straddling the dimer interface. Residues conserved in <b>Vpc_cass2</b> and its sequence homologs from <i>Shewanella</i> and <i>Moritella sp</i> are coloured orange.</p

Structural elements and topology of novel gene cassette proteins.

<p>Sequences of gene cassette ORFs aligned with secondary structural features observed within crystal structures (arrows, β-strands; blocks, α-helices; dashed lines, undefined flexible regions). Sequences do not include additional affinity tags used for recombinant production. Schematic diagrams show structures of monomer forms for proteins <b>A. Hfx_cass2, B. Vpc_cass2, C. Hfx_cass1, D. Hfx_cass5, E. Vch_cass3</b> and <b>F. Vch_cass14</b>.</p

Vch_cass14 dimer with sequestered ligand (PDB 3IMO).

<p><b>A.</b> Ribbon depiction of dimer coloured in spectrum from N-terminus (blue) to C-terminus (red) for each chain. <b>B.</b> Pronounced basic feature (in blue) dominates electrostatic surface of <b>Vch_cass14</b> (left); corresponding orientation of protein chain (right) outlines location of contributing Arg and Lys residues. <b>C.</b> Surface with ribbon representation (left) of the <b>Vch_cass14</b> binding pocket. Regions with ≥80% sequence conservation across the small family of homologs are coloured pink. Side chains contributing to the hydrophobic pocket are depicted. The 1.8 Å 2<i>F_o-F_c</i> map (contoured at 1σ level, mid-panel) shows side chains that line the extended cavity, as well as an area of electron density attributed to a sequestered small ligand. Residues lining this hydrophobic binding pocket are detailed (right panel). The single water molecule (red sphere) and unidentifiable ligand (grey) are also shown. The hydrogen-bonding network engaging Arg21, Tyr14, acetate and water is dashed in red.</p

Helical packing in the Hfx_cass2 dimer (PDB 3FXH).

<p><b>A</b>. Ribbon depiction with colour spectrum from N-terminus (blue) to C-terminus (red) for each chain. <b>B.</b> Dimer interface engages hydrophobic residues from Chain A (tan) and Chain B (green). Acidic groups located either side of a small hydrophobic pocket on the external face are indicated (red). <b>C.</b> Electrostatic surface potential of the dimer surface. Key acidic features (Glu47, Asp60 and helix 2 side chains) are labelled.</p

Titration of Cass2 with cationic ligand.

<p>Fluorescence quenching is plotted during tetraphenylphosphonium chloride (TPP) binding to <b>Cass2</b> (▴) and free tryptophan (▪) for A) wild-type and B) E134Q mutant forms of the protein. Relative fluorescence quenching (ΔF) values were calculated by non-linear regression plot for C) wild-type and D) mutant, leading to determination of K_D values (0.2 µM). Fluorescence emission was monitored at 350 nm following excitation at 295 nm (slit widths 10 and 5 nm, respectively).</p