Abstract
The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2–4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].
Highlights
The urokinase-type plasminogen activator receptor is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al, 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU)
Zhao et al / Data in Brief 5 (2015) 107–113 methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al, 2015; Gårdsvoll et al, 2006 [5,6]); and (4) solving the threedimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]
Kinetic rate constants for the interaction between immobilized anti-uPAR mAbs and recombinant uPAR mutants were determined by SPR, the structure of the mAb Á uPAR complex was determined by X-ray crystallography Not applicable
Summary
X-ray crystal structures, surface plasmon resonance studies (SPR), and generation of mAbs with defined reactivity X-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility SPR data was recorded on a CM5 chip with a Biacore3000TM (GE Healthcare Life Sciences) Processed Recombinant proteins and monoclonal antibodies were affinity purified to high homogeneity before use. Kinetic rate constants for the interaction between immobilized anti-uPAR mAbs and recombinant uPAR mutants were determined by SPR, the structure of the mAb Á uPAR complex was determined by X-ray crystallography Not applicable. Ncbi.nlm.nih.gov/pubmed/25659907), from the patent (WO 2013/020898 A12013) and the structures deposited in the Protein Data Bank (entries 4QTH and 4QTI). Defines the structure of a closed, active conformation of native uPARwt without covalent modifications; defines a topographic epitope landscape on uPAR for 6 different bins of anti-uPAR mAbs; establish that occupancy of the Vn-binding site by mAbs drives uPAR into to its closed conformation; data defining this interdomain flexibility are important for functional studies on uPAR biology; and for the future design of uPAR-targeted intervention studies in human disease [1,7,8,9]
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