Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif

Fernando Vilela,Magali Aumont-Nicaise,Paola Llinas,Jessica Andreani,Julie Ménétrey,Christophe Velours,Mélanie Chenon,Aurélien Thureau,Valérie Campanacci,Olena Pylypenko

doi:10.1038/s41598-019-52537-3

Abstract

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases.

Highlights

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin[1] motors
Our study reveals that the N-terminal part of JIP3/4 is the binding site for the kinesin1-KHC7 and Dynein-DLIC25 which are microtubule-based motors with opposite directionality, and for myosin 5a, an actin-based motor
This highlights that the RH1-Leucine Zipper I (LZI) tandem, like the Leucine Zipper II (LZII) coiled coil located in the median part of the protein, are important and versatile protein-protein interaction modules for multiple cytoskeletal motors

Summary

Introduction

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin[1] motors Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The Leucine Zipper II (LZII) coiled coil located in the median part of JIP3/4 is well characterized It encompasses the binding sites for (i) the light chain of kinesin 1 (KLC)[6,9], (ii) the p150glued subunit of the dynactin complex[8,9] and (iii) the small GTPase Arf[69,23]. To characterize the N-terminal part of JIP3/4 which encompasses a RH1 (RILP homology 1) domain followed by the leucine zipper I (LZI) coiled coil, we perform bioinformatical, biophysical and structural characterizations of this region. We provide evidence that the RH1 domain is a cytoskeletal motor-binding motif and that both JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases

Methods

Results

Conclusion