Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

Ekaterina Marakasova,Philip Olivares,Elena Karnaukhova,Haarin Chun,Nancy E Hernandez,James H Kurasawa,Gabriela U Hassink,Svetlana A Shestopal,Dudley K Strickland,Andrey G Sarafanov

doi:10.1016/j.jbc.2021.100842

Abstract

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.

Highlights

The receptors from the low-density lipoprotein receptor (LDLR) family are expressed in many tissues where they recognize various dissimilar ligands involved in numerous biological processes
Insect cells are not capable of providing, at least in sufficient amount, a factor to facilitate the folding of the complement-type repeat (CR) domains, which results in protein secretion mostly in misfolded multimeric forms due to mislinking of the conserved cysteines [28, 32, 33]
We investigated mechanisms of interaction of receptor-associated protein (RAP) with selected LDLR family receptors mainly focusing on the interaction with LDLR-related protein 1 (LRP1)

Summary

Introduction

The receptors from the low-density lipoprotein receptor (LDLR) family are expressed in many tissues where they recognize various dissimilar ligands involved in numerous biological processes. The conserved acidic residues and an aromatic residue interact with ε-amino group and aliphatic portion of a “critical” lysine of the ligand, respectively, and additional interface residues provide weaker binding energy This mechanism was described for the interactions of vLDLR with a human rhinovirus [13], ApoER2 with reelin [14], LDLR with receptor-associated protein (RAP), and proposed to be common for the ligands’ recognition by all LDLR family receptors [15]. Each of the two sites of RAP can bivalently interact with LRP1; it is unclear how these binding events are mutually coordinated during the interaction of both molecules

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