Insights into Arf activation from nuclear magnetic resonance (NMR) spectroscopy
Seidel, Ronald Duane
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ADP Ribosylation Factors (Arfs) comprise a family of Ras-related GTPases regulating a wide variety of intracellular signaling pathways, specifically impacting bi-directional membrane traffic within Golgi membranes. Arfs perform these functions through cyclic interactions with guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), membranes, and effectors. Here, novel NMR evidence is presented for a sequence of events within Arf activation leading from membrane association to GTP incorporation. It is clear from previous models that Arfs have the ability to undergo substantial structural changes resulting from activation. This structural switching hinges on exposure of a myristoylated N-terminal ±-helix. A previously reported mechanism whereby this N-terminal domain is exposed is based on a comparison of the basal (Arf1•GDP) and activated ( 17Arf1•GTP, N-terminally truncated) crystal structures. However, it has been assumed that all structural changes leading to activation, observed through these models, are the direct result of GTP incorporation rather than displacement of this N-terminal extension. Separation of conformational changes resulting from nucleotide exchange and N-terminal truncation cannot be addressed using these existing structures because both the bound nucleotide and the N-terminus differ. Results from NMR experiments presented within, including residual dipolar couplings (RDCs), has allowed the generation of an additional model, 17Arf1•GDP, and reveals substantial structural variation resulting solely from N-terminal truncation. Incorporation of these findings within a mechanistic framework centers on displacement of the N-terminal helix upon membrane interaction, previously mimicked through truncation, but what may cause the needed destabilization of the Arf1 N-terminus in full-length forms? It is known that signaling lipids, specifically phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), regulate the activities of a number Arf effectors. The ability of Arf itself to bind these same lipids has also been reported previously, though with minimal structural detail. In what follows, NMR evidence is presented in support of lipid-mediated conformational changes within Arf1 preceding nucleotide release. A patch of positive potential on the protein surface is identified as the lipid-binding site, and structural changes extending to the N-terminal helix are also reported. Taken together, the existing biochemical data and newly presented structural information have allowed the generation of a mechanistic model for Arf1 activation.