title: Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.
abstract: The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Å resolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Linda J
authors: LJ
authors: Olson
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Davin R
authors: DR
authors: Jensen
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Brian F
authors: BF
authors: Volkman
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Jung-Ja P
authors: JJ
authors: Kim
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Francis C
authors: FC
authors: Peterson
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors: Rebekah L
authors: RL
authors: Gundry
authors: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States. Electronic address: ndahms@mcw.edu.
authors: Nancy M
authors: NM
authors: Dahms
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins
keywords: Crystallization; Escherichia coli expression; Lectin; Mannose 6-phosphate receptor; NMR; Protein purification