The major overall aim of the research described in this MIRA proposal is to elucidate the structural and dynamic characteristics of large macromolecules and their complexes, which generally contain both well- structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems have been under study in our lab for several years. Members of the NF?B family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (I?Bs) until the cell receives an external signal. NF?B is activated by phosphorylation and ubiquitination of the I?B?, which is then targeted for proteasomal degradation, releasing the NF?B to translocate into the nucleus. In a classical negative feedback mechanism, NF?B upregulates transcription of I?B? in addition to signal-specific stress-response genes: newly-synthesized I?B? kinetically enhances NF?B dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process and in an exciting new observation, it was recently shown that the stability of the resting NF?B-I?B? complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NF?B-I?B?-DNA complex formed in the nucleus during molecular stripping. We propose the structural characterization of this NF?B-I?B?-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between the binary and ternary complexes of NF?B, I?B? and DNA, and the ternary NF?B-I?B?-RNA complex using specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts containing the over-expressed, folded co-chaperones required according to literature reports for the formation of a stable complex with Hsp90. The advantage of this cell-free system is that it can be tuned to the optimization of complex formation by varying the relative amounts of the component cell extracts.

Public Health Relevance

Two multi-component macromolecular complexes will be characterized using a biophysical approach combining a range of different techniques. The nuclear factor ?B (NF?B) signaling system regulates cell growth, immune responses, inflammatory viral responses, and apoptotic death and is often misregulated in cancer, arthritis, asthma, diabetes, AIDS, and viral infections. The chaperone Hsp90 is involved in many critically important cellular functions, including stabilizing the structured of client proteins such as transcription factors and kinases, and assisting in the movement of proteins across membranes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM131693-01
Application #
9697170
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcguirl, Michele
Project Start
2019-06-01
Project End
2024-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037