文摘

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretorypathway 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 efficientbinding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on theextracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoinedRAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The bindingof U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty(K_D = 2.77 × 10^-6 M and 1.85 × 10^-5 M, respectively), whereas RAP domain 3 binds with a substantiallylower enthalpy, but is favored due to a positive entropic contribution (K_D = 1.71 × 10^-7 M). The heatcapacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K^-1mol^-1. There is an indication of a conformational change in RAP domain 3 upon binding in the surfaceplasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residuesin the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.