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

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. 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. 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. In humans, these receptors are represented by LDLR, LDLR-related protein 1 (LRP1), very low-density lipoprotein receptor (vLDLR), ApoER2, LRP2, LRP1B, and LRP4 (1Dieckmann M. Dietrich M.F. Herz J. Lipoprotein receptors--an evolutionarily ancient multifunctional receptor family.Biol. Chem. 2010; 391: 1341-1363Crossref PubMed Google Scholar). In circulation, LDLR, LRP1, and vLDLR are responsible for endocytosis of various proteins and lipoproteins (2Gent J. Braakman I. Low-density lipoprotein receptor structure and folding.Cell Mol. Life Sci. 2004; 61: 2461-2470Crossref PubMed Scopus (83) Google Scholar), and misfunction of these receptors may result in atherosclerotic disease and other abnormalities. In other tissues, LRP1, vLDLR, and ApoER2 are involved in cell signaling and tissue remodeling, and all are implicated in Alzheimer's disease (3Cooper J.M. Lathuiliere A. Migliorini M. Arai A.L. Wani M.M. Dujardin S. Muratoglu S.C. Hyman B.T. Strickland D.K. Regulation of tau internalization, degradation, and seeding by LRP1 reveals multiple pathways for tau catabolism.J. Biol. Chem. 2021; 296: 100715Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 4Dlugosz P. Nimpf J. The reelin receptors apolipoprotein E receptor 2 (ApoER2) and VLDL receptor.Int. J. Mol. Sci. 2018; 19: 3090Crossref Scopus (19) Google Scholar). Other pathological processes implicating the LDLR family receptors involve cardiovascular diseases, type 2 diabetes, obesity, Parkinson's disease, and others (5Lane-Donovan C. Philips G.T. Herz J. More than cholesterol transporters: Lipoprotein receptors in CNS function and neurodegeneration.Neuron. 2014; 83: 771-787Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Au D.T. Strickland D.K. Muratoglu S.C. The LDL receptor-related protein 1: At the crossroads of lipoprotein metabolism and insulin signaling.J. Diabetes Res. 2017; 2017: 8356537Crossref PubMed Scopus (18) Google Scholar, 7Go G.W. Mani A. Low-density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis.Yale J. Biol. Med. 2012; 85: 19-28PubMed Google Scholar). Better knowledge of respective receptor–ligand interactions is important for understanding these processes and fundamental discoveries in the future. The LDLR family receptors are composed of the same domain types serving specific functional roles. The ligand-binding function is generally served by highly homologous complement-type repeats (CRs) organized in clusters (7Go G.W. Mani A. Low-density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis.Yale J. Biol. Med. 2012; 85: 19-28PubMed Google Scholar, 8Hussain M.M. Strickland D.K. Bakillah A. The mammalian low-density lipoprotein receptor family.Ann. Rev. Nutr. 1999; 19: 141-172Crossref PubMed Scopus (302) Google Scholar). Relatively simple in structure, LDLR, vLDLR, ApoER2, and LRP4 have one cluster formed by seven to eight CRs, whereas other receptors have four clusters of CRs formed by similar or larger numbers of repeats. In a prominent member of the family, LRP1, there are two major ligand-binding clusters termed II and IV, less significant clusters for ligand binding include cluster III and cluster I (Fig. S1), which is known to participate with cluster II in binding of only one ligand, activated forms of alpha-2-macroglobulin (9Actis Dato V. Chiabrando G.A. The role of low-density lipoprotein receptor-related protein 1 in lipid metabolism, glucose homeostasis and inflammation.Int. J. Mol. Sci. 2018; 19: 1780Crossref Scopus (36) Google Scholar, 10Mikhailenko I. Battey F.D. Migliorini M. Ruiz J.F. Argraves K. Moayeri M. Strickland D.K. Recognition of alpha 2-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions.J. Biol. Chem. 2001; 276: 39484-39491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). LRP1 also binds triglyceride-rich particles, fibronectin, matrix proteases, and blood clotting factors with a total number of more than 40 of disparate ligands (9Actis Dato V. Chiabrando G.A. The role of low-density lipoprotein receptor-related protein 1 in lipid metabolism, glucose homeostasis and inflammation.Int. J. Mol. Sci. 2018; 19: 1780Crossref Scopus (36) Google Scholar). Each CR domain is formed from ∼40 amino acids and connected to an adjacent CR domain with a flexible linker that, in the case of LRP1, is composed of three to ten amino acids. Each CR domain's structure is enforced by three internal disulfide bonds formed from six conserved cysteines and by coordination of a Ca2+ ion with four conserved acidic residues (11Fass D. Blacklow S. Kim P.S. Berger J.M. Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module.Nature. 1997; 388: 691-693Crossref PubMed Scopus (297) Google Scholar, 12Guo Y. Yu X. Rihani K. Wang Q.Y. Rong L. The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: Implications in structure and function for the LDL receptor superfamily.J. Biol. Chem. 2004; 279: 16629-16637Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). During ligand binding, 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 (13Verdaguer N. Fita I. Reithmayer M. Moser R. Blaas D. X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein.Nat. Struct. Mol. Biol. 2004; 11: 429-434Crossref PubMed Scopus (123) Google Scholar), ApoER2 with reelin (14Yasui N. Nogi T. Takagi J. Structural basis for specific recognition of reelin by its receptors.Structure. 2010; 18: 320-331Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), LDLR with receptor-associated protein (RAP), and proposed to be common for the ligands' recognition by all LDLR family receptors (15Fisher C. Beglova N. Blacklow S.C. Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors.Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). RAP was described as a molecular chaperone for LRP1 and antagonist for its interactions with ligands (16Bu G. Rennke S. Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein.J. Biol. Chem. 1996; 271: 22218-22224Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 17Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar, 18Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). This indicates that RAP may also serve the chaperone function for other LDLR family receptors, which is supported by several studies (19De Nardis C. Lossl P. van den Biggelaar M. Madoori P.K. Leloup N. Mertens K. Heck A.J. Gros P. Recombinant expression of the full-length ectodomain of LDL receptor-related protein 1 (LRP1) unravels pH-dependent conformational changes and the stoichiometry of binding with receptor-associated protein (RAP).J. Biol. Chem. 2017; 292: 912-924Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 20Sato A. Shimada Y. Herz J. Yamamoto T. Jingami H. 39-kDa receptor-associated protein (RAP) facilitates secretion and ligand binding of extracellular region of very-low-density-lipoprotein receptor: Implications for a distinct pathway from low-density-lipoprotein receptor.Biochem. J. 1999; 341: 377-383Crossref PubMed Scopus (13) Google Scholar, 21Willnow T.E. Armstrong S.A. Hammer R.E. Herz J. Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4537-4541Crossref PubMed Scopus (242) Google Scholar, 22Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.EMBO J. 1996; 15: 2632-2639Crossref PubMed Scopus (234) Google Scholar). In interaction with LRP1, RAP was proposed to bind the CR domains of the newly synthesized receptor to assist their folding, prevent premature binding to other ligands, and deliver the molecule to the cell surface (23Prasad J.M. Young P.A. Strickland D.K. High affinity binding of the receptor-associated protein D1D2 domains with the low density lipoprotein receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysines 60 and 191.J. Biol. Chem. 2016; 291: 18430-18439Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Several studies demonstrated that interaction of RAP and LRP1 (and also LDLR) involves formation of a complex between two "critical" lysines of RAP and a doublet of adjacent CR domains (15Fisher C. Beglova N. Blacklow S.C. Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors.Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Andersen O.M. Christensen L.L. Christensen P.A. Sorensen E.S. Jacobsen C. Moestrup S.K. Etzerodt M. Thogersen H.C. Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor- associated protein (RAP). A conserved acidic residue in the complement- type repeats is important for recognition of RAP.J. Biol. Chem. 2000; 275: 21017-21024Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 25Bajari T.M. Lindstedt K.A. Riepl M. Mirsky V.M. Nimpf J. Wolfbeis O.S. Dresel H.A. Bautz E.K. Schneider W.J. A minimal binding domain of the low density lipoprotein receptor family.Biol. Chem. 1998; 379: 1053-1062Crossref PubMed Scopus (15) Google Scholar, 26Dolmer K. Campos A. Gettins P.G. Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).J. Biol. Chem. 2013; 288: 24081-24090Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), where each lysine docks to a single CR domain; and these binding events provide an additive (avidity) effect enforcing the interaction (15Fisher C. Beglova N. Blacklow S.C. Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors.Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Prasad J.M. Young P.A. Strickland D.K. High affinity binding of the receptor-associated protein D1D2 domains with the low density lipoprotein receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysines 60 and 191.J. Biol. Chem. 2016; 291: 18430-18439Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 26Dolmer K. Campos A. Gettins P.G. Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).J. Biol. Chem. 2013; 288: 24081-24090Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Such a mode of interaction was termed "bivalent" regarding the interaction of LRP1 and an isolated fragment of RAP composed of its D1 and D2 domains (D1/D2) bearing a high-affinity site for binding to LRP1. On D1/D2, the "critical" lysines K60 and K191 are located on D1 and D2, respectively; both these domains are connected with a flexible linker, and therefore both lysines have mutual flexibility (23Prasad J.M. Young P.A. Strickland D.K. High affinity binding of the receptor-associated protein D1D2 domains with the low density lipoprotein receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysines 60 and 191.J. Biol. Chem. 2016; 291: 18430-18439Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In contrast, the two "critical" lysines of the second high-affinity LRP1-binding site of RAP, K256, and K270 are located on the single domain (D3), which has a rigid structure with minimal mutual flexibility of the lysines. The role of these lysines was established by testing interactions of isolated D3 with LRP1 and its CR-doublet 5–6 (26Dolmer K. Campos A. Gettins P.G. Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).J. Biol. Chem. 2013; 288: 24081-24090Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) and also CR-doublet 3–4 of LDLR (15Fisher C. Beglova N. Blacklow S.C. Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors.Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar); therefore the interaction of D3 with both full-size LRP1 and LDLR also corresponds to the bivalent mode. Thus, each of the two sites of RAP can bivalently interact with LRP1; however, it is unclear how these binding events are mutually coordinated during the interaction of both molecules. In LRP1, the binding sites for RAP are located within clusters II, III, and IV. Each of the isolated clusters is able to interact with RAP with affinity comparable to that for the full-length receptor (KD 1–5 nM) (23Prasad J.M. Young P.A. Strickland D.K. High affinity binding of the receptor-associated protein D1D2 domains with the low density lipoprotein receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysines 60 and 191.J. Biol. Chem. 2016; 291: 18430-18439Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 27Jensen G.A. Andersen O.M. Bonvin A.M. Bjerrum-Bohr A.I. Etzerodt M. Thogersen H.C. O'Shea C. Poulsen F.M. Kragelund B.B. Binding site structure of one LRP/RAP complex - implications for a common ligand/receptor binding motif.J. Mol. Biol. 2006; 362: 700-716Crossref PubMed Scopus (55) Google Scholar, 28Kurasawa J.H. Shestopal S.A. Woodle S.A. Ovanesov M.V. Lee T.K. Sarafanov A.G. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.Biochemistry. 2015; 54: 481-489Crossref PubMed Scopus (6) Google Scholar, 29Obermoeller L.M. Warshawsky I. Wardell M.R. Bu G. Differential functions of triplicated repeats suggest two independent roles for the receptor-associated protein as a molecular chaperone.J. Biol. Chem. 1997; 272: 10761-10768Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Within the clusters II and III, the majority of CR doublets were shown to bind RAP and its isolated D1/D2 and D3 fragments with comparable affinities (24Andersen O.M. Christensen L.L. Christensen P.A. Sorensen E.S. Jacobsen C. Moestrup S.K. Etzerodt M. Thogersen H.C. Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor- associated protein (RAP). A conserved acidic residue in the complement- type repeats is important for recognition of RAP.J. Biol. Chem. 2000; 275: 21017-21024Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 27Jensen G.A. Andersen O.M. Bonvin A.M. Bjerrum-Bohr A.I. Etzerodt M. Thogersen H.C. O'Shea C. Poulsen F.M. Kragelund B.B. Binding site structure of one LRP/RAP complex - implications for a common ligand/receptor binding motif.J. Mol. Biol. 2006; 362: 700-716Crossref PubMed Scopus (55) Google Scholar, 28Kurasawa J.H. Shestopal S.A. Woodle S.A. Ovanesov M.V. Lee T.K. Sarafanov A.G. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.Biochemistry. 2015; 54: 481-489Crossref PubMed Scopus (6) Google Scholar, 30Sarafanov A.G. Makogonenko E.M. Andersen O.M. Mikhailenko I.A. Ananyeva N.M. Khrenov A.V. Shima M. Strickland D.K. Saenko E.L. Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.Thromb. Haemost. 2007; 98: 1170-1181Crossref PubMed Scopus (15) Google Scholar). Within cluster IV, the active binding CR doublets have not yet been identified; however, previous studies indicated that the majority of its CRs are capable of binding LRP1 (30Sarafanov A.G. Makogonenko E.M. Andersen O.M. Mikhailenko I.A. Ananyeva N.M. Khrenov A.V. Shima M. Strickland D.K. Saenko E.L. Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.Thromb. Haemost. 2007; 98: 1170-1181Crossref PubMed Scopus (15) Google Scholar, 31Meijer A.B. Rohlena J. van der Z.C. van Zonneveld A.J. Boertjes R.C. Lenting P.J. Mertens K. Functional duplication of ligand-binding domains within low-density lipoprotein receptor-related protein for interaction with receptor associated protein, alpha(2)-macroglobulin, factor IXa and factor VIII.Biochim. Biophys. Acta. 2007; 1774: 714-722Crossref PubMed Scopus (34) Google Scholar). Notably, the data show that the absence of the conserved aromatic residue in any domain of a CR doublet (Fig. S1) correlates with its inability to bind RAP as shown for CRs 1–2 (cluster I), 9–10 (cluster II), and 19–20 (cluster III). Thus, these studies show that numerous LRP1 sites are capable to facilitate bivalent binding combinations with RAP. However, like the sites on RAP, it is unclear how these sites in LRP1 are coordinated during its interaction with RAP. Until now, the chaperone function of RAP has been supported only for LRP1, LRP2, and vLDLR, but not LDLR. Indeed, (i) disruption of the RAP gene in mouse model resulted in impairing the expression of these receptors, except LDLR (21Willnow T.E. Armstrong S.A. Hammer R.E. Herz J. Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4537-4541Crossref PubMed Scopus (242) Google Scholar, 22Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.EMBO J. 1996; 15: 2632-2639Crossref PubMed Scopus (234) Google Scholar); (ii) cotransfection of the RAP gene in cell culture facilitated expression of recombinant vLDLR and LRP1, but not LDLR (20Sato A. Shimada Y. Herz J. Yamamoto T. Jingami H. 39-kDa receptor-associated protein (RAP) facilitates secretion and ligand binding of extracellular region of very-low-density-lipoprotein receptor: Implications for a distinct pathway from low-density-lipoprotein receptor.Biochem. J. 1999; 341: 377-383Crossref PubMed Scopus (13) Google Scholar), and (iii) coexpression of RAP and the LRP1 exodomain in cell culture resulted in increase of the latter's yield (19De Nardis C. Lossl P. van den Biggelaar M. Madoori P.K. Leloup N. Mertens K. Heck A.J. Gros P. Recombinant expression of the full-length ectodomain of LDL receptor-related protein 1 (LRP1) unravels pH-dependent conformational changes and the stoichiometry of binding with receptor-associated protein (RAP).J. Biol. Chem. 2017; 292: 912-924Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). At the same time, expression of recombinant CR fragments of LRP1 and LDLR in cell culture yielded relatively low amounts of correctly folded proteins (28Kurasawa J.H. Shestopal S.A. Woodle S.A. Ovanesov M.V. Lee T.K. Sarafanov A.G. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.Biochemistry. 2015; 54: 481-489Crossref PubMed Scopus (6) Google Scholar, 32Kurasawa J.H. Shestopal S.A. Karnaukhova E. Struble E.B. Lee T.K. Sarafanov A.G. Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.J. Biol. Chem. 2013; 288: 22033-22041Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 33Sarafanov A.G. Makogonenko E.M. Pechik I.V. Radtke K.P. Khrenov A.V. Ananyeva N.M. Strickland D.K. Saenko E.L. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.Biochemistry. 2006; 45: 1829-1840Crossref PubMed Scopus (31) Google Scholar) indicating requirement of a folding factor. Notably, the affinity of RAP for LDLR was found to be similar (32Kurasawa J.H. Shestopal S.A. Karnaukhova E. Struble E.B. Lee T.K. Sarafanov A.G. Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.J. Biol. Chem. 2013; 288: 22033-22041Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) or weaker (34Medh J.D. Fry G.L. Bowen S.L. Pladet M.W. Strickland D.K. Chappell D.A. The 39-kDa receptor-associated protein modulates lipoprotein catabolism by binding to LDL receptors.J. Biol. Chem. 1995; 270: 536-540Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) than that for LRP1. Due to the ability of RAP to interact with all LDLR family receptors, it is used as a model ligand to study their interactions with other ligands based on similarity of the respective mechanisms. In particular, the bivalent binding mode has also been described for the interactions of LRP1 with blood coagulation factor VIII (FVIII) (35Young P.A. Migliorini M. Strickland D.K. Evidence that factor VIII forms a bivalent complex with the low density lipoprotein (LDL) receptor-related protein 1 (LRP1): Identification of cluster IV on LRP1 as the major binding site.J. Biol. Chem. 2016; 291: 26035-26044Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and plasminogen-activator inhibitor 1 (PAI-1) (36Migliorini M. Li S.H. Zhou A. Emal C.D. Lawrence D.A. Strickland D.K. High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor-related protein 1 requires lysines 80, 88, and 207.J. Biol. Chem. 2020; 295: 212-222Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In our study, we aimed to characterize interactions of RAP with selected LDLR family receptors in several model systems on to obtain a deeper insight into these mechanisms. Our basic approach was to test RAP interactions with ligand-binding fragments of LRP1, LDLR, and vLDLR within living cells upon the coexpression of RAP with proteins (Fig. 1A). For this, we used an insect-cell-based platform, which is not capable of providing a relevant folding factor for the receptors as resulted in production of their fragments mostly in misfolded forms (28Kurasawa J.H. Shestopal S.A. Woodle S.A. Ovanesov M.V. Lee T.K. Sarafanov A.G. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.Biochemistry. 2015; 54: 481-489Crossref PubMed Scopus (6) Google Scholar, 32Kurasawa J.H. Shestopal S.A. Karnaukhova E. Struble E.B. Lee T.K. Sarafanov A.G. Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.J. Biol. Chem. 2013; 288: 22033-22041Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 33Sarafanov A.G. Makogonenko E.M. Pechik I.V. Radtke K.P. Khrenov A.V. Ananyeva N.M. Strickland D.K. Saenko E.L. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.Biochemistry. 2006; 45: 1829-1840Crossref PubMed Scopus (31) Google Scholar). To dissect the mechanism of RAP and LRP1 interaction to smaller molecular determinants (Fig. 1B), we tested binding of their fragments, including mutated variants, using a purified system and in silico modeling. The resulting data support the function of RAP as a folding chaperone for the tested receptors and a bivalent mechanism of the interactions, which occur in dynamic mode. First, we tested coexpression of RAP and LRP1 cluster II, based on the previously reported increase of LRP1 exodomain production upon coexpression with RAP in mammalian cell culture (19De Nardis C. Lossl P. van den Biggelaar M. Madoori P.K. Leloup N. Mertens K. Heck A.J. Gros P. Recombinant expression of the full-length ectodomain of LDL receptor-related protein 1 (LRP1) unravels pH-dependent conformational changes and the stoichiometry of binding with receptor-associated protein (RAP).J. Biol. Chem. 2017; 292: 912-924Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The goal was to verify the suitability of an insect-cell-based (baculovirus) system for testing RAP and LDLR family receptor fragments' interactions within living cells. In particular, insect cells are not capable of providing, at least in sufficient amount, a factor to facilitate the folding of the CR domains, which results in protein secretion mostly in misfolded multimeric forms due to mislinking of the conserved cysteines (28Kurasawa J.H. Shestopal S.A. Woodle S.A. Ovanesov M.V. Lee T.K. Sarafanov A.G. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.Biochemistry. 2015; 54: 481-489Crossref PubMed Scopus (6) Google Scholar, 32Kurasawa J.H. Shestopal S.A. Karnaukhova E. Struble E.B. Lee T.K. Sarafanov A.G. Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.J. Biol. Chem. 2013; 288: 22033-22041Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 33Sarafanov A.G. Makogonenko E.M. Pechik I.V. Radtke K.P. Khrenov A.V. Ananyeva N.M. Strickland D.K. Saenko E.L. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.Biochemistry. 2006; 45: 1829-1840Crossref PubMed Scopus (31) Google Scholar). We considered such a background to be favorable f