Binding Kinetics of LRP Protein with Receptor Related Protein

Division Of Basic Sciences - Nci

Investigators

Abstract

We have begun a new collaboration with Y-X Wang (SBL) to use SPR to study binding kinetics of continuous repeat unit CR567 of Lipoprotein Receptor Related Protein (LRP) with RAP (Receptor Related Protein). LRP is a transmembrane receptor protein that binds with more than 30 ligands in vivo, making it an important receptor in many cellular processes. LRP has many structural domains; domain II contains several continuous repeat (CR) units including CR567. Previous studies have shown that the CR567 units are important for RAP binding, although the role of the specific CR units in RAP binding is still poorly understood. The signaling pathway of LRP is tightly controlled by a number of extracellular and intracellular regulating proteins, including chaperonins and members of the Dickkopf (DKK) family. These proteins down-regulate Wnt-B-catenin pathway as tumor suppressors. LRP6 has been shown to be an oncogenic receptor. This pathway has high potential for molecular targeting. The ligand binding sites of CR567 are cysteine-rich complement type repeats, making expression and purification with proper folding difficult, a major hurdle to biophysical and structural studies. Other laboratories have expressed CR56 as a ubiquitin fusion protein, which is the approach we are using to express CR567. We have cloned the gene encoding CR567 into pET SUMO cloning vector and expressed protein using BL21 (DE3) E. coli strain, and expressed and refolded CR567 protein. We are working on binding assays using SPR, FIA, and intrinsic fluorescence. We are also concurrently cloning and expressing the SUMO fusion protein in Pichia pastoris which can improve expression and folding efficiency of disulfide-rich proteins. We expect to optimize a protocol and express sufficient protein for SPR and other biophysical studies of CR567 binding with RAP, and complete those studies within the coming year. Site directed mutagenesis will be used to define the details of the binding interface of RAP and CR567. This work will complement parallel in vivo and structural work in Dr. Wang's laboratory. The postdoctoral visiting fellow who is working on the project hopes to also produce enough protein for structure determination of the CR567-RAP complex.