Dissection of RAP-LRP interactions: binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP.
The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7-CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7-CR8, with the higher affinity site (K(d) approximately 1microM) located in the C-terminal two-thirds and the weaker site (K(d) approximately 5microM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5-CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding.
Lazic
Ana
A
Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60607, USA.
Dolmer
Klavs
K
Strickland
Dudley K
DK
Gettins
Peter G W
PG
D001665:Binding Sites; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D002352:Carrier Proteins; D026502:LDL-Receptor Related Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D010750:Phosphoproteins; C046282:RNA polymerase II-binding proteins; D011994:Recombinant Proteins