Importance of ligand-induced conformational changes in hemopexin for receptor-mediated heme transport.
Hemopexin alters conformation upon binding heme as shown by circular dichroism (CD), but hemopexin binds the heme analog, iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS), without undergoing concomitant changes in its CD spectrum. Moreover, FeTPPS, unlike heme, does not increase the compactness of the heme-binding domain (I) of hemopexin shown by an increased sedimentation rate in sucrose gradients. On the other hand, like heme, FeTPPS forms a bishistidyl coordination complex with hemopexin and upon binding protects hemopexin from cleavage by plasmin. Competitive inhibition and saturation studies demonstrate that FeTPPS-hemopexin binds to the hemopexin receptor on mouse hepatoma cells but with a lower affinity (Kd 125 nM) more characteristic of apo-hemopexin than heme-hemopexin (Kd 65 nM). This provides evidence that conformational changes produced in hemopexin upon binding heme, but not upon binding FeTPPS, are important for increasing the affinity of hemopexin for its receptor. The amount of cell-associated radiolabel from 55FeTPPS-hemopexin increases linearly for up to 90 min but at a rate only about a third of that of the mesoheme-complex. As expected from the recycling of hemopexin, more iron-tetrapyrrole than protein is associated with the Hepa cells, but the ratio of 55Fe-ligand to 125I-hemopexin is only 2:1 for FeTPPS-hemopexin compared to 4:1 for mesoheme complexes. [55Fe]Mesoheme was associated at 5 min with lower density fractions containing plasma membranes and at 30 min with fractions containing higher density intracellular compartments. In contrast, 55FeTPPS was found associated with plasma membrane fractions at both times and was not transported into the cell. Although FeTPPS-hemopexin binds to the receptor, subsequent events of heme transport are impaired. The results indicate that upon binding heme at least three types of conformational changes occur in hemopexin which have important roles in receptor recognition and that the nature of the ligand influences subsequent heme transport.
Smith
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Tatum
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Muster
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Burch
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Morgan
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D000818:Animals; D001693:Biological Transport, Active; D002451:Cell Compartmentation; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D006418:Heme / Q000378:metabolism*; D006466:Hemopexin / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008665:Metalloporphyrins / Q000378:metabolism; D051379:Mice; D011166:Porphyrins / Q000378:metabolism; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide; D013056:Spectrophotometry, Ultraviolet
D008024:Ligands; D008665:Metalloporphyrins; D011166:Porphyrins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C047453:hemopexin receptor; C016136:tetraphenylporphine sulfonate; D006418:Heme; D006466:Hemopexin