Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors
| Autor: | Svetlana A Shestopal, Andrey G Sarafanov, Ekaterina S. Marakasova, James H. Kurasawa, Haarin Chun, Elena Karnaukhova, Dudley K. Strickland, Nancy E. Hernandez, Philip Olivares, Gabriela U. Hassink |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Protein Folding Circular dichroism LRP1 low-density receptor-related protein 1 DNA Mutational Analysis Very Low-Density Lipoprotein Receptor Ligands Biochemistry LDL low-density lipoprotein LDL-Receptor Related Protein-Associated Protein Receptor CD circular dichroism CR complement-type repeat biology Chemistry SE-FPLC size-exclusion fast protein liquid chromatography molecular chaperone ASPP ammonium sulfate/potassium phosphate (buffer) KD dissociation constant LRP1 ApoE3 apolipoprotein E3 protein Cell biology Molecular Docking Simulation LDLR low-density lipoprotein receptor factor VIII (FVIII) Protein folding Low Density Lipoprotein Receptor-Related Protein-1 Protein Binding Research Article Repetitive Sequences Amino Acid In silico SPR surface plasmon resonance LDL-receptor-related protein-associated protein ER endoplasmic reticulum 03 medical and health sciences Humans low-density lipoprotein (LDL) protein expression Molecular Biology RAP LDL-receptor related protein-associated protein EDTA ethylenediaminetetraacetic acid 030102 biochemistry & molecular biology LDL-receptor related protein-associated protein (RAP) fungi Cell Biology 030104 developmental biology Receptors LDL vLDLR very low-density lipoprotein receptor LDL receptor RU response units biology.protein sense organs |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode. |
| Databáze: | OpenAIRE |
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