α Helix Content of G Protein α Subunit Is Decreased upon Activation by Receptor Mimetics*

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 α subunit (Gi1α) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPγS binding to soluble Gi1α 2.6-fold with an EC50 of 30 μg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in α helix content of the Gi1α by 7%. Likewise, mastoparan also enhanced the rate of GTPγS binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1α similar to compound 48/80. In corresponding experiments using a K349P-Gi1α, a Gi1α counterpart of the unc mutant in Gsα in which Pro was substituted for Lys349, enhancement of the GTPγS binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1α lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1α. Taken together, our results strongly suggest that the activator-induced unwinding of the α helix of the G protein α subunit is mechanically coupled to the enhanced release of bound GDP from the α subunit.

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