title: Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.
abstract: The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments.
authors: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; Danish-Chinese Centre for Proteases and Cancer.
authors: Baoyu
authors: B
authors: Zhao
authors: Cell Matrix Signaling Unit, Institute of Molecular Oncology of the Italian Foundation for Cancer Research, via Adamello 16, 20139 Milan, Italy.
authors: Sonu
authors: S
authors: Gandhi
authors: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; Danish-Chinese Centre for Proteases and Cancer.
authors: Cai
authors: C
authors: Yuan
authors: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; Danish-Chinese Centre for Proteases and Cancer.
authors: Zhipu
authors: Z
authors: Luo
authors: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; Biotech Research and Innovation Centre, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen North, Denmark.
authors: Rui
authors: R
authors: Li
authors: Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen North, Denmark; Biotech Research and Innovation Centre, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen North, Denmark; Danish-Chinese Centre for Proteases and Cancer.
authors: Henrik
authors: H
authors: Gårdsvoll
authors: Cell Matrix Signaling Unit, Institute of Molecular Oncology of the Italian Foundation for Cancer Research, via Adamello 16, 20139 Milan, Italy.
authors: Valentina
authors: V
authors: de Lorenzi
authors: Cell Matrix Signaling Unit, Institute of Molecular Oncology of the Italian Foundation for Cancer Research, via Adamello 16, 20139 Milan, Italy.
authors: Nicolai
authors: N
authors: Sidenius
authors: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China; Danish-Chinese Centre for Proteases and Cancer. Electronic address: mhuang@fjirsm.ac.cn.
authors: Mingdong
authors: M
authors: Huang
authors: Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen North, Denmark; Biotech Research and Innovation Centre, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen North, Denmark; Danish-Chinese Centre for Proteases and Cancer. Electronic address: m-ploug@finsenlab.dk.
authors: Michael
authors: M
authors: Ploug
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013002:Somatomedins / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D055293:Receptors, Urokinase Plasminogen Activator; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D014568:Urokinase-Type Plasminogen Activator
keywords: CD87; LU domains; allosteric regulation; surface plasmon resonance; urokinase