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The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

ABSTRACT: Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to form a tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (?45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues Lys-150 and Arg-151 were replaced by alanine. The data presented here shed light on the role that these residues play in shaping the unique structural features and biophysical properties of the AfFtn. The x-ray crystal structure of the K150A/R151A mutant, solved at 2.1 Å resolution, indicates that replacement of these key residues flips a "symmetry switch." The engineered molecule no longer assembles with tetrahedral symmetry but forms a typical closed octahedral ferritin cage. Small angle x-ray scattering reveals that the overall shape and size of AfFtn and AfFtn-AA in solution are consistent with those observed in their respective crystal structures. Iron binding and release kinetics of the AfFtn and AfFtn-AA were investigated to assess the contribution of cage openings to the kinetics of iron oxidation, mineralization, or reductive iron release. Identical iron binding kinetics for AfFtn and AfFtn-AA suggest that Fe(2+) ions do not utilize the triangular pores for access to the catalytic site. In contrast, relatively slow reductive iron release was observed for the closed AfFtn-AA, demonstrating involvement of the large pores in the pathway for iron release.

SUBMITTER: Sana B

PROVIDER: S-EPMC3820901 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

Sana Barindra B Johnson Eric E Le Magueres Pierre P Criswell Angela A Cascio Duilio D Lim Sierin S

The Journal of biological chemistry 20130912 45

Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to form a tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (∼45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues ...[more]

PMID: 24030827

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Project description:BACKGROUND:Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the asparagine residue in the N-glycosylation sequons. The catalytic subunits of the OST enzyme are STT3 in eukaryotes, AglB in archaea and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three paralogous AglB proteins. We previously solved the crystal structures of the C-terminal globular domains of two paralogs, AglB-Short 1 and AglB-Short 2. RESULTS:We determined the crystal structure of the C-terminal globular domain of the third AglB paralog, AglB-Long, at 1.9 Å resolutions. The crystallization of the fusion protein with maltose binding protein (MBP) afforded high quality protein crystals. Two MBP-AglB-L molecules formed a swapped dimer in the crystal. Since the fusion protein behaved as a monomer upon gel filtration, we reconstituted the monomer structure from the swapped dimer by exchanging the swapped segments. The C-terminal domain of A. fulgidus AglB-L includes a structural unit common to AglB-S1 and AglB-S2. This structural unit contains the evolutionally conserved WWDYG and DK motifs. The present structure revealed that A. fulgidus AglB-L contained a variant type of the DK motif with a short insertion, and confirmed that the second signature residue, Lys, of the DK motif participates in the formation of a pocket that binds to the serine and threonine residues at the +2 position of the N-glycosylation sequon. CONCLUSIONS:The structure of A. fulgidus AglB-L, together with the two previously solved structures of AglB-S1 and AglB-S2, provides a complete overview of the three AglB paralogs encoded in the A. fulgidus genome. All three AglBs contain a variant type of the DK motif. This finding supports a previously proposed rule: The STT3/AglB/PglB paralogs in one organism always contain the same type of Ser/Thr-binding pocket. The present structure will be useful as a search model for molecular replacement in the structural determination of the full-length A. fulgidus AglB-L.

Dataset Information

The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

Publications

The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

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