Abstract
Bem1 is a scaffold protein essential for the establishment of cell polarity in Saccharomyces cerevisiae. This work reports the solution structure of a Cdc42 binding module of Bem1 comprising the second SH3 domain (SH3b) and its C-terminal flanking region termed Cdc42 interacting (CI). First, the structure of Bem1 SH3b-CI was determined by NMR spectroscopy, which shows that Bem1 SH3b-CI is a structurally and functionally related domain that binds Cdc42. Next, the solution structure of Bem1 SH3b-CI in complex with the proline-rich region of p21-activated kinase Ste20 (Ste20 PRR) was determined. Finally, the interaction surface of Bem1 SH3b-CI with Cdc42 was identified based on chemical shift perturbation studies which reveals that Bem1 SH3b-CI interacts simultaneously with both Ste20 PRR and Cdc42 using the opposite surfaces. Thus, Bem1 can tether Cdc42 and Ste20 in close proximity so that Cdc42 can efficiently interact with Ste20 Cdc42 and Rac interactive binding (CRIB). Based on the present results together with the previous biochemical studies (Lamson, R. E., Winters, M. J., and Pryciak, P. M. (2002) Mol. Cell. Biol. 22, 2939-2951 and Winters, M. J., and Pryciak, P. M. (2005) Mol. Cell. Biol. 25, 2177-2190), a model was suggested that the autoinhibition of Ste20 kinase activity by CRIB is released through the Cdc42-CRIB interaction, which is mediated by Bem1, and Ste20 is subsequently activated, an initial step for the establishment of the cell polarity.
Highlights
EXPERIMENTAL PROCEDURESProtein Expression and Purification for NMR Samples—The Bem SH3b-Cdc42 interacting (CI)-(156 –260) was cloned into the pGEX-6P-1 vector (GE Healthcare), expressed in Escherichia coli strain BL21 (DE3) as a glutathione S-transferase (GST) fusion protein and purified using glutathione-Sepharose 4B (GE Healthcare)
Cell polarity is essential for the physiological functions of eukaryotic cells
Identification of the SH3b-Cdc42 interacting (CI) Structural Region—For the structure determination, we first expressed a construct of Bem1-(140 – 271) in E. coli strain BL21 (DE3) cells as a glutathione S-transferase (GST) fusion protein, which was originally reported as a Cdc42-binding region and JUNE 18, 2010
Summary
Protein Expression and Purification for NMR Samples—The Bem SH3b-CI-(156 –260) was cloned into the pGEX-6P-1 vector (GE Healthcare), expressed in Escherichia coli strain BL21 (DE3) as a glutathione S-transferase (GST) fusion protein and purified using glutathione-Sepharose 4B (GE Healthcare). The Ste PRR [463– 486] was cloned into the pGEX-6P-1 vector and expressed in E. coli strain BL21 (DE3) as a glutathione S-transferase (GST) fusion protein. Chemical Shift Perturbation Experiments—The Bem SH3bCI and Cdc (1–179, Q61L) were cloned into the pGEX-6P-1 vector and expressed in E. coli strain BL21 (DE3) as GST fusion proteins. The 13C/15N-labeled Bem SH3b-CI and unlabeled Cdc were first purified using glutathione-Sepharose 4B After these GST-fusion proteins were mixed and the GST tag was removed with PreScission protease, Bem SH3b-CI complexed with Cdc was purified by anion exchange chromatography on a Resource Q column (GE Healthcare), and Cdc was activated by loading GTP␥S. In Vitro Pull-down Binding Assays—The Bem SH3b-CI, Ste PRR, and Ste CRIB domain (328 –375) were cloned into the pGEX-6P-1 vector and expressed in E. coli strain BL21 (DE3) as GST fusion proteins. The eluates were subjected to 15% SDS-PAGE and stained by Coomassie Brilliant Blue or immunoblotting using anti-GST and anti-His antibodies
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