Loop Electrostatics Modulates the Intersubunit Interactions in Ferritin

Abstract

Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

“…Cells were grown at 37°C, until A 600 nm reached 0.6 -0.8, and subsequently induced with isopropyl 1-thio-␤-D-galactopyranoside (1 mM final concentration) for 4 h. Recombinant ferritins were purified from the harvested cells, as described previously (43). Briefly, cells were sonicated, and the cell-free extract obtained after centrifugation (40 min, 40,000 rpm, 4°C) was incubated for 15 min at 65°C as the first purification step.…”

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ 3 -oxo)Tris[(μ 2 -peroxo)] triiron(III) cluster

“…Several cadmium ions have been observed bound to both ironfree HuLf and HoLf, in the threefold axis channel, on the twofold axis, and on the internal surface of the protein shell, but none on the fourfold axis channel (SI Channels and Metal Ions Binding Sites) (17,18). In the iron-free structures of both L-type ferritins, two Cd 2+ ions are coordinated by Glu57 and Glu60, and by Glu61 and Glu64, respectively.…”

Is His54 a gating residue for the ferritin ferroxidase site?

“…After the catalytic oxidation of the ferrous species, under the effect of new incoming ferrous ions [8,18], the ferric products are released from the ferroxidase site and migrate towards the inner cavity, where they grow to provide a mineral of nanometer dimension [16]. NMR data have suggested that the migration process might involve ferric-oxo cluster of high nuclearity, but confirmatory high-resolution data are still missing [3,19].…”

“…Sequence similarities with bullfrog M ferritin were analyzed to identify the nature of the amino acid present at positions corresponding to His54 using 500 sequences of vertebrate ferritins (identity range 100-55%) [16]. Search for ferritin from vertebrate sequences in UniProtKB database was performed using BLAST and multiple sequence alignment was obtained by ClustalW.…”