Heterotrimeric guanine nucleotide-binding proteins (G-proteins) consist of an alpha subunit (Gα) and the dimeric beta-gamma subunit (Gβγ). The first example of direct cell signaling by Gβγ was the discovery of its role in activating G-protein regulated inwardly rectifying K+ (GIRK) channels which underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate. Published crystal structures have provided important insights into the structures of the G-protein subunits and GIRK channels separately, but co-crystals of the channel and Gβγ together remain elusive and no specific reciprocal residue interactions between the two proteins are currently known. Given the absence of direct structural evidence, we attempted to identify these functionally important channel-Gβγ interactions using a computational approach. We developed a multistage computational docking algorithm that combines several known methods in protein-protein docking. Application of the docking protocol to previously published structures of Gβγ and GIRK1 homomeric channels produced a clear signal of a favored binding mode. Analysis of this binding mode suggested a mechanism by which Gβγ promotes the open state of the channel. The channel-Gβγ interactions predicted by the model in silico could be disrupted in vitro by mutation of one protein and rescued by additional mutation of reciprocal residues in the other protein. These interactions were found to extend to agonist induced activation of the channels as well as to activation of the native heteromeric channels. Currently, the structural mechanism by which Gβγ regulates the functional conformations of GIRK channels or of any of its membrane-associated effector proteins is not known. This work shows the first evidence for specific reciprocal interactions between Gβγ and a GIRK channel and places these interactions in the context of a general model of intracellular regulation of GIRK gating.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4306 |
| Date | 09 October 2012 |
| Creators | Mahajan, Rahul |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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