Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin alpha1

Abstract

Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin α1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P(2)). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

Fig. 1.

Structures of CENTA1 in apo- and IP4-bound forms. (A) Cartoon diagram of CENTA1 in apo-form (gray) aligned with IP4-bound form: ArfGAP (1–119, green), PH1 (131–238, magenta), PH2 (253–359, salmon red), interdomain linkers (120–130, 239–252, yellow), C-terminal helix (360–370, red). IP4 molecules and CENTA1 zinc-finger cysteines are shown in sticks. Zinc is shown as an orange sphere. (B) Bottom-up view of the ± 10.0 kT/e electrostatic potential surface of CENTA1 in the IP4-bound structure. (C) and (D) Ligplot representations of the interaction between IP4 and PH1, PH2 domains respectively. Residues from the KX_n(K/R)XR motif are marked with stars.

Fig. 2.

Biochemical and biophysical characterization of phosphoinositides-CENTA1 interaction. (A) PIP array assay. Lanes A–G, each spot contains 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 pmol of corresponding phosphoinositides on the membrane. (B) T_agg titration curve of wild-type and mutant CENTA1 at different IP4 concentrations. (C) Relative aggregation temperature of wild-type and mutant CENTA1 to that of R149CR271C double mutant. Values for IP4 are fitted ΔT_max results from titration. Values for PIP3 and PI(3,4)P₂ are ΔT_agg in the presence of 100 μg/mL phosphoinositides (109 and 123 μM, respectively).

Fig. 3.

Structure of CENTA1 bound to KIF13B-FHA domain in the asymmetric unit.

Fig. 4.

Model of the KIF13B-CENTA1 transport machinery. (A) Structural model of KIF13B/CENTA1/IP4 complex. Electrostatic potential surface of centaurin molecules was calculated from IP4-bound CENTA1 coordinate. KIF13B-FHA domains are shown as ribbons. IP4s bound to the PH domains were modeled from the CENTA1/IP4 complex structure. The C-terminal helix of CENTA1 that appeared upon FHA or IP4 binding is shown as a blue ribbon. (B) Assembly of KIF13B/CENTA1 transport machinery.