BAG1 as a Link between Cell Growth and the Stress Response: An NMR Study
EMSL Project ID
2296
Abstract
BAG1 is a multifunctional protein that modulates responses to cell death and cell stress. The 'BAG domain', located in the C-terminal region of BAG1 is necessary for interactions with two interacting proteins: the molecular chaperone Hsc70 and the serine-threonine kinase RAf-1. BAG1 acts as a nucleotide exchange factor for the Hsc70 chaperone and also binds and activates the signaling molecule Raf-1. It has been proposed that Raf-1 and Hsc70 may compete for binding to BAG1. BAG1 may be the link between cell growth and the signal to halt proliferation. We determined the solution structure of BAG1 by NMR methods, using data collected at the EMSL HFMRF facility at PNNL. The BAG1 molecule consists of an antiparallel three-helix bundle in which alpha2-alpha3 correspond to the most conserved region. Then NMR-monitored titrations of BAG1 with synthetic peptides from the ATPase domain of Hsc70, and mutagenesis, were used to define the contact surfaces between BAG1 and the ATPase domain. Now we will extend the studies to BAG1 interactions with Raf-1. BAG1 NMR assignments, BAG1 mutants and a construct corresponding to the kinase domain of Raf-1 are already in hand for this project. For BAG1/Hsc70 binding, the interactions involve residues in BAG helices alpha2-alpha3, while the contacts for Raf-1 are expected to be located predominately in helices alpha1-alpha2. The interface between BAG1 and the kinase domain of Raf-1 will be probed by NMR. In order to identify chemical shifts of BAG1 bound to Raf-1, TROSY or CRINEPT will be applied to the Raf-1/2H,15N-labeled BAG1 complex. The recently developed TROSY-baseed 3D 15N-editied NOESY will be implemented as well as cross-saturation methods to identify amides located at binding interfaces in large complexes. The experiments will require high fields because the complex approximates 52 kDa.
Project Details
Project type
Capability Research
Start Date
2002-02-06
End Date
2002-08-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
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