HIGH-RESOLUTION X-RAY STUDIES ON RABBIT SERUM TRANSFERRIN - PRELIMINARY STRUCTURE-ANALYSIS OF THE N-TERMINAL HALF-MOLECULE AT 2.3-A RESOLUTION

An iron-containing half-molecule fragment (M(r) congruent-to 39 000) of rabbit serum transferrin has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to the space group P3(1)21 with cell parameters a = 67.06 (3), c = 138.33 (11) angstrom. Partial amino-acid sequence analysis has shown that this fragment corresponds to the N-terminal lobe of the intact molecule. Intensity data, to a resolution of 2.3-angstrom, were collected photographically using the oscillation method. The structure was determined by molecular replacement using the coordinates of the N-terminal lobe of the undigested parent molecule as the search model. A preliminary least-squares refinement of the molecular-replacement solution resulted in an R factor of 28% for all data between 7.0 and 2.3-angstrom. The refinement was performed initially with both the iron atom and the carbonate anion excluded from the model. The resulting \F(o)\ - \F(c)\ difference Fourier map was readily interpreted in the vicinity of the iron-binding site in terms of a carbonate anion directly bound to the iron in a bidentate fashion. The current R factor is 22.5% for all data between 7.0 and 2.3-angstrom. The carbonate anion with the four protein ligands (Asp63, Tyr95, Tyr188 and His249) forms a distorted octahedral arrangement around the Fe3+ cation. The anion appears to be 'locked' in position by the formation of hydrogen bonds to residues at the N-terminus of helix 5; these involve the side chains of Thr120 and Arg124 and the main-chain nitrogens of Ala126 and Gly127. The importance of this loop at the N-terminus of helix 5 in stabilizing the Fe3+ transferrin complex is further emphasized by the involvement of Ser125 in hydrogen bonding to the distal oxygen of Asp63; this is also an interdomain hydrogen bond. Water molecules have also been identified, some in close proximity to the iron-binding site, and in particular to the side chain of Arg124.