Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors

Abstract

Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)-containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases, and their effectors is unknown. Here, we describe structures of PI4Kβ (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is distinct compared with known structures of Rab complexes and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria.

Fig. 1. Crystal structure of human PI4KIIIβ complex with Rab11a(Q70L)-GTPγS

(A) Full-length PI4KIIIβ (isoform 2) (left) and constructs of PI4KIIIβ and Rab11a(Q70L) used for crystallization (right). The PI4KIIIβ crystallization construct contained three deletions as well as an S294A mutation. (B) Overall architecture of the complex. The PI4K-specific insertion (residues 391-540) in the N-lobe is salmon colored. The large spheres mark a disordered region within which residues 408-507 are deleted. The switch I and switch II regions of Rab11a(Q70L) are represented in orange.

Fig. 2. The PI4KIIIβ/Rab11a interface

(A) Close-up view of the PI4KIIIβ/Rab11a interface. (B) Sequence alignment of interacting regions in Rabs and PI4KIIIβs. Conserved and similar residues are highlighted (red shading and red letters, respectively). Rab11a residues that interact with PI4KIIIβ are indicated with green arrowheads; light blue arrowheads denote PI4KIIIβ residues interacting with Rab11. Rab sequences are grouped into Rabs previously shown to either bind PI4KIIIβ (binders) or not bind PI4KIIIβ (non-binders). (C) Pull-down assays with GST-tagged Rab11A(Q70L)-GTPγS and either wild-type or mutant full-length PI4KIIIβ. The inputs and the bound proteins (lanes 1 and 2, respectively) were analysed on SDS gels stained with InstantBlue. (D) Surface plasmon resonance (SPR) analysis of the full length wild-type PI4KIIIβ binding to the immobilized GST-tagged Rab11a(Q70L) loaded with either GDP (red sensogram) or GTPγS (blue). The affinity of PI4KIIIβ for GST-Rab11a is indicated next to the graphs (data are mean +/− SEM based on five independent experiments). Full details are shown in Fig. S6. Also shown are the sensograms for several PI4KIIIβ mutants binding to Rab11a(Q70L)-GTPγS.

Fig. 3. Ternary complex of PI4KIIIβ with Rab11a-GTP and effector protein FIP3

(A) Pulldown assays with GST-tagged-FIP3 fragment (residues 713-756) and full length PI4KIIIβ either with or without Rab11a(Q70L)-GTPγS. The inputs and the bound proteins were analyzed on SDS gels stained with InstantBlue. (B) Crystal structure of the ternary complex of PI4KIIIβ, Rab11a-GTPγS and the FIP3 RBD domain.

Fig. 4. Inhibitor binding to PI4KIIIβ

(A) Interactions of PI4KIIIβ with PIK93. Dotted lines represent putative hydrogen bonds (prepared by LIGPLOT ()). For each PI4KIIIβ residue, the equivalent residues are shown below for human Vps34, human mTOR, human p110α and P. falciparum PI4KIIIβ (left to right). (B) Ribbon diagram of PI4KIIIβ illustrating sites of P. falciparum resistance mutations (spheres). The helical domain is colored from dark to light blue from N-terminus to the C-terminus. (C) PIK93 bound to PI4KIIIβ. (D) Close-up of the active site, illustrating positions of P. falciparum resistance mutations.