Dynamic structure of membrane-anchored Arf*GTP

Abstract

ADP ribosylation factors (Arfs) are N-myristoylated GTP/GDP switch proteins that have key regulatory roles in vesicle transport in eukaryotic cells. ARFs execute their roles by anchoring to membrane surfaces, where they interact with other proteins to initiate budding and maturation of transport vesicles. However, existing structures of Arf*GTP are limited to nonmyristoylated and truncated forms with impaired membrane binding. We report a high-resolution NMR structure for full-length myristoylated yeast (Saccharomyces cerevisiae) Arf1 in complex with a membrane mimic. The two-domain structure, in which the myristoylated N-terminal helix is separated from the C-terminal domain by a flexible linker, suggests a level of adaptability in binding modes for the myriad of proteins with which Arf interacts and allows predictions of specific lipid binding sites on some of these proteins.

Figure 1

Structure of myr-yArf1•GTP in a bicelle solution. (a) Twenty lowest-energy structures of myr- yArf1•GTP out of 60 calculations are superimposed through the C terminus (Glu17 to Leu177). The primary sequence of yeast Arf1 is displayed on top with residues corresponding to the important structural landmarks such as N terminus, linker, switches, and secondary structural elements colored in the same scheme as in the superimposed structures. (b) Structural overlay of the C terminus (residues 18–181) of myr-yArf1•GTP (red), Arf1•GTP-Δ17-Q71L (blue, PDB: 1o3y), and myr-yArf1•GDP (yellow, PDB: 2k5u), highlighting the conformational differences of Switch I, Switch II, and the inter-switch loopλ3. (c) Solvent accessible surface representations of myr-yArf1•GDP (left) and myr-yArf1•GTP (right). The residues lining the myristoyl binding pocket of myr-yArf1•GDP are colored yellow. (d) Left: N-terminal structures of myr-yArf1•GTP (red) and yArf2•GDP (blue, PDB: 1mr3). The helix of myr-yArf1•GTP is significantly longer than that of yArf2•GDP, despite the small difference in primary sequence, i.e. Phe4 of yArf1 is replaced by a tyrosine in yArf2. The myristoyl group is shown in purple and phenylalanine side chains in yellow. Middle and right: Surface representations of the amphipathic N terminus of myr-yArf1•GTP highlighting the hydrophobic (yellow) and hydrophilic (blue) surfaces.

Figure 2

Lipid interaction of myr-yArf1 studied through rotational correction times (τ_c). The residual specific correlation times myr-yArf1•GTP with bicelles of [DMPC]:[DHPC] (q) = 0.25, 0.5 and 1.0 are plotted with open circles, solid triangles, and open squares. The total DMPC/DHPC content is fixed at 10% (w/v) in all assays to minimize viscosity differences.

Figure 3

Ensemble structural fitting to RDCs and PREs. (a) Agreement between experimental and back- calculated RDCs for a 1-state ensemble (left) and a 3-state ensemble (right). Data include NH, NC′, and phenyl CH (the latter two are normalized to NH). RDCs are collected in positive and negative gels (b) Agreement between experimental and back-calculated PREs for a 1-state ensemble (left) and a 3-state ensemble (right). Data include PREs from T55C, K59C, R83C, R117C, and S176C.

Figure 4

Reweighted atomic density maps of the C-terminal domain (Glu17 to Leu177) showing the space on the membrane surface that is sampled by the 3 dynamic states. The maps are created from the 44 lowest-energy N=3 structures from 200 calculations. Only backbone atoms are used for density calculation. The all-backbone-atom maps for the 3 states are colored in light blue, light green, and pink. The switch region densities for the corresponding states are colored in darker blue, green, and red. The right and left views are related by a 90° rotation around the bicelle norm.

Figure 5

Modeling of Arf complexes on the membrane. (a) Arf/N-GAT complex modeled by superimposing the structure of myr-yArf1•GTP with that of mouse Arf1•GTP-Δ17-Q71L in the crystal structure of the Arf1•GTP-Δ17-Q71L/N-GAT complex (PDB: 1j2j). The structure of mouse Arf1•GTP-Δ17-Q71L is not displayed. (b) Arf/ArfBD-ARHGAP21 complex modeled by superimposing the structure of myr-yArf1•GTP with that of mouse Arf1•GTP-Δ17 in the crystal structure of the Arf1•GTP-Δ17/ArfBD-ARHGAP21 complex (PDB: 2j59). Segments of ArfBD-ARHGAP21 separated by loops of missing electron densities are connected by red lines. The structure of mouse Arf1•GTP is not displayed. In (a) and (b), Arf is shown in blue and the binding partners are shown in yellow.