Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.
Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
M L
ML
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Morgan
W T
WT
D000818:Animals; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006466:Hemopexin / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
D004220:Disulfides; D006418:Heme; D006466:Hemopexin