Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism.
Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe.
Mizutani
K
K
The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 0011, Japan.
Yamashita
H
H
Mikami
B
B
Hirose
M
M
D000818:Animals; D000838:Anions / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D003207:Conalbumin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013431:Sulfates / Q000737:chemistry* / Q000378:metabolism; D013499:Surface Properties
D000838:Anions; D001059:Apoproteins; D010446:Peptide Fragments; D013431:Sulfates; D003207:Conalbumin; D007501:Iron