Domain organization of the 39-kDa receptor-associated protein

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum re sident protein that binds to the low density lipoprotein receptor-related p rotein (LRP) as well as certain members of the low density lipoprotein rece ptor superfamily and antagonizes ligand binding. In order to identify impor tant functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denatur ation of RAP is highly reversible and occurs in a broad temperature range w ith two well resolved heat absorption peaks. A good fit of the endotherm wa s obtained with four two-state transitions suggesting these many cooperativ e domains in the molecule. A number of recombinant fragments of RAP were ex pressed in bacteria, and their domain composition and stability were charac terized by DSC, circular dichroism, and fluorescence spectroscopy. The resu lts confirmed that RAP is composed of four independently folded domains, D1 , D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-32 3, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent mole cule. Isolated D3 was partially degraded during isolation from bacterial ly sates. The isolated D4 was capable of binding with high affinity to LRP whe reas neither D1 nor D2 bound. At the same time a fragment containing both D 1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containi ng D1 and D2 indicate that these two domains interact with each other and t hat the proper folding of the second domain into a native-like active confo rmation requires presence of the first domain.