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

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 A 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.

Figure 1

The overall structure of the ARF6–JIP4-LZII complex. ARF6–GTP is drawn in blue with the switch regions in grey. The two monomers of JIP4-LZII are drawn in yellow and orange. Two orthogonal views of the complex are shown.

Figure 2

The second leucine zipper structure of JIP4. (A) Schematic representation of the JIP4 protein. (B) Sequence alignement of JIP3 and JIP4. The heptad repeats, the characteristic leucine residues at position ‘d' in each heptad, and their respective sequence numbers are indicated. On the JIP4 sequence, the ARF6-binding site (residues A412–D432) is highlighted in grey. (C) The JIP4-LZII structure is shown (with only one ARF6 molecule bound) aligned along the JIP3/JIP4 sequence alignment. It should be noted that the C-terminal part of the linker between the histidine tag and JIP4 has defined density at the N-terminus of the chain C and thus has been modelled (residues A386–F391), whereas the ninth heptad repeats of both monomers are not seen in the density and are not modelled.

Figure 3

Stoichiometry of the ARF6–JIP4-LZII complexes in solution determined by analytical ultracentrifugation. Continuous sedimentation coefficient distribution analysis of ARF6 (100 μM; dashed blue line open triangles), JIP4 (100 μM; dashed red line filled triangles) and ARF6–JIP4 mixtures (12.5 μM ARF6, 50 μM JIP4; black line open squares and 50 μM ARF6, 12.5 μM JIP4; green line filled squares). The total integrated signal of each c(S) distribution was normalized. Sedimentation coefficients are expressed in Svedberg: 1 S=10⁻¹³ s.

Figure 4

The ARF6–JIP4-LZII interaction surface. (A) An overall view of the ARF6–JIP4-LZII interface. (B) An ‘open book' representation of the ARF6–JIP4 interface. Residues involved in the complex interface are labelled and shown as sticks. For ARF6, the residues that interact with chains C and D of JIP4 are shown, respectively, in orange and yellow; residues that interact with both chains are shown in red. For JIP4-LZII, residues that interact with the switch II and interswitch regions are shown, respectively, in light and dark grey; residues that interact with both are shown in mid grey. (C) Cross-eye stereo view of the interface. All the residues from ARF6 and JIP4-LZII that are involved in the interface are labelled and shown as sticks.

Figure 5

Binding affinities of Arfs–JIP4-LZII complexes studied by surface plasmon resonance (SPR). (A) Real-time association and dissociation SPR profiles corresponding to the injection of ARF6 (3.9 μM) over immobilized wild-type and variant JIP4-LZII and (B) ARF6/(JIP4)₂ molar ratio as a function of ARF6 concentration and corresponding fitted K_d values. (C) Real-time association and dissociation SPR profiles corresponding to the injection of the different Arf variants (1.8 μM) over immobilized JIP4-LZII_WT and (D) ARF/(JIP4)₂ molar ratio as a function of Arf concentration and corresponding fitted K_d values.

Figure 6

The structural basis of JIP4 specificity. (A) A sequence alignment of the ARF6 and ARF1 N-termini is shown (left). Sequence differences at the switch regions are indicated on the ARF6–JIP4-LZII structure by spheres (right). Residues in contact with JIP4-LZII or likely to affect the JIP4-LZII binding are indicated in red. (B–D) Detailed views of ARF6–JIP4-LZII interactions at positions not conserved in ARF1, namely Thr 79 (B), Thr 53–Lys 58 (C) and Asn 60 (D). The structure of ARF1–GTP (green) is superimposed on the structure of ARF6–GTP in the ARF6–JIP4-LZII complex (blue). Chain C of JIP4-LZII is shown in yellow.

Figure 7

ARF6–JIP4 interactions at the membrane. (A) Model of the ARF6–(JIP4)₂–ARF6 heterotetramer at the membrane obtained when both ARF6 molecules are anchored to the membrane. ARF6 molecules are shown in blue with switch II in red and the interswitch in green. The myristoylated amphipatic N-terminal helix of ARF6 that is critical for interaction with membrane is indicated as a blue cylinder lying against the membrane. The orientation of ARF6 molecules is modelled such that both N-terminal parts are close to the membrane. (B) Model of the ARF6–(JIP4)₂–ARF6 heterotetramer at the membrane considering that JIP4 is positioned tangentially with respect to the membrane. In this model, one ARF6 molecule is oriented with its N-terminal part close to the membrane, whereas that of the second ARF6 molecule is turned toward the cytosol, suggesting that only one ARF6 molecule interacts with JIP4 at the membrane. (C) Model of an ARF6–(JIP4)₂ heterotrimer at the membrane.