Arf Functional Landscapes

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Summary Membrane organization in eukaryotic cells is controlled by ADP ribosylation factors (Arfs), small GTPases that function as molecular switches to activate signaling cascades. Arfs regulate vesicular transport of lipids and proteins between the ER and the Golgi (Class I-Arf1) and endosome-plasma membrane trafficking (Class III-Arf6), implicating Arf function in cytokinesis, cell shape, organelle transport, mitochondrial and lipid droplet function and pH-dependent regulation of cell size. Mutations in Arfs or their partners have been linked to genetic neurological diseases causing severe malformation of the cerebral cortex or mental retardation. Moreover, many pathogenic bacteria and viruses commandeer Arfs as they invade cells, thereby promoting infection. Our overall goal is to understand the nucleotide exchange transitions of Arf GTPases, the mechanisms of which cannot be deduced from their static structures. We hypothesize that the Arf conformations specifically recognized by their cognate exchange factors correspond to significantly disrupted excited states that are populated at very low levels under standard conditions. Specifically, we aim to map the GDP/GTP switches of Arf1 and Arf6 (Aims 1 and 2), and using mutational analysis, establish the underlying molecular mechanisms of their functional specificity (Aim 3). Our approach combines experimental biophysical tools (multi- dimensional NMR, SAXS and fluorescence) with pressure perturbation and coarse-grained molecular dynamics simulations constrained by our data, to provide structural ensembles and pseudo-free energy landscapes that will reveal functionally relevant excited states implicated in Arf function and specificity. These excited state structures will provide novel target sites for inhibiting Arf signaling pathways, offering new avenues for developing approaches to mitigate the invasive capacity of bacteria and viruses. More generally, the pressure-based mapping approach proposed here represents a powerful means to characterize elusive states of proteins implicated in their functions.