title: Crystallization and X-ray diffraction of LGN in complex with the actin-binding protein afadin.
abstract: Asymmetric stem-cell divisions are fundamental for morphogenesis and tissue homeostasis. They rely on the coordination between cortical polarity and the orientation of the mitotic spindle, which is orchestrated by microtubule pulling motors recruited at the cortex by NuMA-LGN-Gαi complexes. LGN has emerged as a central component of the spindle-orientation pathway that is conserved throughout species. Its domain structure consists of an N-terminal TPR domain associating with NuMA, followed by four GoLoco motifs binding to Gαi subunits. The LGN(TPR) region is also involved in interactions with other membrane-associated proteins ensuring the correct cortical localization of microtubule motors, among which is the junctional protein afadin. To investigate the architecture of LGN(TPR) in complex with afadin, a chimeric fusion protein with a native linker derived from the region of afadin upstream of the LGN-binding domain was generated. The fusion protein behaves as a globular monomer in solution and readily crystallizes in the presence of sulfate-containing reservoirs. The crystals diffracted to 3.0 Å resolution and belonged to the cubic space group P213, with unit-cell parameter a = 170.3 Å. The structure of the engineered protein revealed that the crystal packing is promoted by the coordination of sulfate ions by residues of the afadin linker region and LGN(TPR).
authors: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.
authors: Manuel
authors: M
authors: Carminati
authors: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.
authors: Valentina
authors: V
authors: Cecatiello
authors: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy.
authors: Marina
authors: M
authors: Mapelli
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: D000199:Actins; C102754:GPSM2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; C108835:afadin
keywords: LGN; fusion proteins; spindle orientation