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Published November 8, 2013 | Published
Journal Article Open

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.

Additional Information

© 2014 American Society for Biochemistry and Molecular Biology, Inc. Received for publication, June 4, 2013, and in revised form, August 31, 2013 Published, JBC Papers in Press, September 12, 2013. We thank Prof. Imke Schroeder (UCLA) and Prof. Wilfred R. Hagen (Delft University of Technology) for the generous gifts of AfFtn, AfFtn-AA, and PfFtn plasmids, respectively. We thank M. Sawaya and the UCLA-DOE X-ray Crystallography Core Facility, which is supported by DOE Grant DE-FC02-02ER63421. We thank M. Capel, K. Rajashankar, N. Sukumar, J. Schuermann, I. Kourinov, and F. Murphy (Northeastern Collaborative Access Team Beamline 24-ID at Advanced Photon Source, which is supported by National Center for Research Resources Grant 5P41RR015301-10 and National Institute of General Medical Sciences Grant 8 P41 GM103403-10 from the National Institutes of Health). Use of the Advanced Photon Source is supported by the Department of Energy under Contract DE-AC02-06CH11357. This work was supported in part by Singapore Ministry of Education Academic Research Fund Tier I Grant RG33/07 and Nanyang Technological University-National Healthcare Group Innovation Seed Grant ISG/11014.

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August 22, 2023
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