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Dominant Thermodynamic Role of the Third Independent Receptor Binding Site in the Receptor-Associated Protein RAP



O M. Andersen, Frederick P. Schwarz, Edward Eisenstein, C Jacobsen, S K. Moestrup, M Etzerodt, H C. Thogersen


The 39 Kda receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP, is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and the sixth CR-domain of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a non-cooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (KD 2.77 x10-6 M, and KD 1.85 x 10-5 M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (KD 1.71 x10-7 M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K-1 mol-1. There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonsnce analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.


binding constant, isothermal titration calorimetry, protein, receptor-associated protein, surface plasmon resonance


Andersen, O. , Schwarz, F. , Eisenstein, E. , Jacobsen, C. , Moestrup, S. , Etzerodt, M. and Thogersen, H. (2001), Dominant Thermodynamic Role of the Third Independent Receptor Binding Site in the Receptor-Associated Protein RAP, Biochemistry (Accessed June 13, 2024)


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Created December 17, 2001, Updated October 12, 2021