Abstract
Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein a subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Ga Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gai1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Ga interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Ga. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Ga and possibly at the Ga domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.
| Original language | English |
|---|---|
| Article number | e19238 |
| Journal | eLife |
| Volume | 5 |
| Issue number | DECEMBER2016 |
| DOIs | |
| State | Published - Dec 23 2016 |
Funding
This work was supported by the US National Institutes of health (NIH) (grants R01-GM105993 to S.R. S. The mass spectrometry facility at Montana State University receives funding from the Murdock Charitable Trust and NIH P20RR02437. The CBSD Macromolecular X-ray Diffraction Core (MDXC), whose protein expression facilities were used to conduct this research at the University of Montana receives support from NIH P20-GM103546. We are grateful to Dr. Tung-Chung Mou of the MDXC, and Drs. Charles Heffern and Ali Raja of NanoTember Technologies, Inc. for assistance with differential scanning fluorescence assays, Cindee-Yates Hansen and Marlene Woldsted for excellent technical assistance, Dr. Jonathan Hilmer for assistance with mass spectrometry, Navid Moavhed for helpful discussions on HDX and Dr. Gregory Tall for his mechanistic insights into the function of Ric-8A in cells. We thank Angela Patterson for creating Video 1.
| Funder number |
|---|
| R01GM105993 |
| P20-GM103546, P20RR02437 |