CRYSTALLIZATION AND STRUCTURAL-ANALYSIS OF BULLFROG RED-CELL L-SUBUNIT FERRITINS

Ferritin is a 24 subunit protein that controls biomineralization of ir on in animals, bacteria, and plants. Rates of mineralization vary amon g members of the ferritin family, particularly between L and H type su bunits of animal ferritins which are differentially expressed in vario us cell types. To examine ferritin from a highly differentiated cell t ype and to clarify the relationship between ferritin structure and fun ction, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration co mparable to that of the protein and in the presence of MgCl2 at a conc entration much higher than that of the protein. Under both crystalliza tion conditions, the crystals are tetragonal bipyramids in the space g roup F432 with unit cell dimensions a = b = c = 182 +/- 0.5 Angstrom. Crystals obtained in the presence of manganese and ammonium sulfate di ffract to 1.9 Angstrom, while those obtained in the presence of magnes ium and sodium tartrate diffract to 1.6 Angstrom. Isomorphous crystals have been obtained under similar conditions for a site-directed mutan t with a reduced mineralization rate in which Glu-57, -58, -59, and -6 1 are all replaced by Ala. The structure of wild type L-subunit with m agnesium has been solved by molecular replacement using the calcium sa lt of human liver H subunit (Lawson et al., Nature (London) 349:541-54 4, 1991) as the model, The crystallographic R factor for the 6-2.2 Ang strom shell is 0.21. The overall fold of human H and bullfrog L ferrit ins is similar with an rms difference in backbone atomic positions 0.9 7 Angstrom. The largest structural ifferences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Bec ause red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detaile d comparisons of these structures are likely to shed new light on the relationship between conformation and function. (C) 1994 Wiley-Liss,In c.