The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4

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Abstract
The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 angstrom resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6 JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6- mediated motor switch regulatory function. The EMBO Journal (2009) 28, 2835-2845. doi: 10.1038/emboj.2009.209; Published online 30 July 2009