title: 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).
abstract: LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteine-rich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system.
authors.affiliation: From the Crystal & Structural Chemistry Group, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht.
authors.forename: Camilla
authors.initials: C
authors.lastname: De Nardis
authors.affiliation: the Biomolecular Mass Spectrometry & Proteomics Group and Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht.
authors.forename: Philip
authors.initials: P
authors.lastname: Lössl
authors.affiliation: the Department of Plasma Proteins, Sanquin Research, 1006 AN Amsterdam, and.
authors.forename: Maartje
authors.initials: M
authors.lastname: van den Biggelaar
authors.affiliation: From the Crystal & Structural Chemistry Group, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht.
authors.forename: Pramod K
authors.initials: PK
authors.lastname: Madoori
authors.affiliation: From the Crystal & Structural Chemistry Group, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht.
authors.forename: Nadia
authors.initials: N
authors.lastname: Leloup
authors.affiliation: the Department of Plasma Proteins, Sanquin Research, 1006 AN Amsterdam, and.|the Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Koen
authors.identifier: 0000-0002-4134-6898
authors.initials: K
authors.lastname: Mertens
authors.affiliation: the Biomolecular Mass Spectrometry & Proteomics Group and Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht.
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJ
authors.lastname: Heck
authors.affiliation: From the Crystal & Structural Chemistry Group, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, p.gros@uu.nl.
authors.forename: Piet
authors.identifier: 0000-0002-7782-2585
authors.initials: P
authors.lastname: Gros
mesh_terms: D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
chemical_list: C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011994:Recombinant Proteins
keywords: LRP1; electron microscopy (EM); mass spectrometry (MS); pH regulation; receptor-associated protein; small-angle X-ray scattering (SAXS); surface plasmon resonance (SPR)