Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.
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 sixth CR domains 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 noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 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 (K(D) = 1.71 x 10(-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 resonance 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.
Andersen
O M
OM
Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark.
Schwarz
F P
FP
Eisenstein
E
E
Jacobsen
C
C
Moestrup
S K
SK
Etzerodt
M
M
Thøgersen
H C
HC
D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D046911:Macromolecular Substances; D011994:Recombinant Proteins