Structural Basis for Membrane Binding Specificity of the Bin/Amphiphysin/Rvs (BAR) Domain of Arfaptin-2 Determined by Arl1 GTPase*

Background: Arfaptin-2 is recruited onto Golgi membranes through interacting with Arl1 and induces membrane deformation. Results: A crescent-shaped homodimer of the Arfaptin-2 BAR domain is flanked by two Arl1 molecules, leaving the concave face open for membrane association. Conclusion: Arl1 determines the membrane binding specificity of Arfaptin-2 BAR. Significance: Similar mechanisms underlie targeting of various BAR domain proteins to specific membranes. Membrane-sculpting BAR (Bin/Amphiphysin/Rvs) domains form a crescent-shaped homodimer that can sense and induce membrane curvature through its positively charged concave face. We have recently shown that Arfaptin-2, which was originally identified as a binding partner for the Arf and Rac1 GTPases, binds to Arl1 through its BAR domain and is recruited onto Golgi membranes. There, Arfaptin-2 induces membrane tubules. Here, we report the crystal structure of the Arfaptin-2 BAR homodimer in complex with two Arl1 molecules bound symmetrically to each side, leaving the concave face open for membrane association. The overall structure of the Arl1·Arfaptin-2 BAR complex closely resembles that of the PX-BAR domain of sorting nexin 9, suggesting similar mechanisms underlying BAR domain targeting to specific organellar membranes. The Arl1·Arfaptin-2 BAR structure suggests that one of the two Arl1 molecules competes with Rac1, which binds to the concave face of the Arfaptin-2 BAR homodimer and may hinder its membrane association.

Arfaptin-2 alone can deform liposomes into tubules in vitro (2) and can bind to the Arf and Rac GTPases (10,11), suggesting that membrane remodeling induced by Arfaptin-2 can be modulated by these GTPases. Based on the crystal structure of the Arfaptin-2 BAR domain in complex with Rac1, Rac1 binds to the concave face of the Arfaptin-2 BAR homodimer, likely interfering with membrane association of the BAR domain (12). Moreover, Arf1/Arf6 and Rac1 bind to the Arfaptin-2 BAR domain in a mutually exclusive manner (12). However, the molecular basis for the possible cross-talk between Arf and Rac1 GTPases mediated by Arfaptin-2 remains to be elucidated.
Arfaptins are known to localize to the Golgi region when exogenously expressed in cells (2,13). We have recently shown that both Arfaptin-1 and Arfptin-2 interact not only with Arf GTPases but also with Arf-like 1 (Arl1), yet are recruited to trans-Golgi membranes through interaction with Arl1, rather than with the Arf GTPases (14). Arfaptin-2 colocalizes with Arl1 at dynamic vesicular and tubular structures emanating from the Golgi, suggesting that Arl1 may regulate Arfaptin-mediated membrane deformation at the trans-Golgi (14). Here, we report the crystal structure of the BAR domain  was performed using an A1R-MP confocal microscope (Nikon) as described previously (14). R eq =R max Conc / (Conc + K d ).

Crystal structure of the Arl1-Arfaptin-2 BAR
complex -To elucidate the molecular basis for We previously showed that Arf1, Arf3, Arf5, and Arf6 also bind to Arfaptins (14). The

Interaction involving a charged residue in the Arl1 N-terminal region stabilizes the Arl1-BAR
complex -Although the above pull-down assay ( Fig. 2A) indicated that Glu17 of Arl1 is not critical for its interaction with Arfaptin-2, we were still interested in potential ionic interactions involving the N-terminal charged residues of Arl1, because these residues are conserved between Arl1 and Arf1−Arf6 (Supplemental Fig. S1A). Therefore, we focused on the interaction between Arg19 of Arl1 and Asp220 of Arfaptin-2 suggested by the crystal structure (Fig. 1D, E). As shown in Fig. 5A, introduction of an R19A mutation into Arl1(Q71L) greatly diminished its binding to GST-Arfaptin-2 (compare lanes 11 and 13).

Thus, the interaction between Arg19 of Arl1 and
Asp220 of Arfaptin-2 makes a critical contribution to the Arl1-Arfaptin-2 complex formation, although there remains a possibility that the effect of the D220A mutation could also be due, at least in part, to loss of the hydrophobic interaction of the aliphatic portion of Asp220 with the essential Trp66 residue in the interswitch (Fig. 1E and Fig. 2B, lane 10).
To our knowledge, this is the first demonstration that a residue in the N-terminal region, apart from residues in the switch and interswitch regions, of a small GTPase plays a critical role in effector binding.    (Fig. 7A, B) The concave face of SNX9 PX-BAR engaged in membrane binding is predominantly positively charged (Fig. 7C). SNX9 PX-BAR is known to effectively tubulate phosphoinositide-containing liposomes (7,31).

Structural and functional similarities between the Arl1-BAR complex and the PX-BAR
Similarly, the concave surface of Arl1-Arfaptin-2 BAR is positively charged, though less extensively (Fig. 7D). Thus, like SNX9 PX-BAR, the Arl1-Arfaptin-2 BAR complex appears to be suitable for associating with negatively charged membranes.
As demonstrated by cryo-EM of the