Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3

Abstract

SummaryKinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms.

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

“…Binding of GST-JIP3 LZ2 to His 6 -KLC1 extTPR is an enthalpy-driven process with a dissociation constant K D of ~3.5 μM in which two TPR domains bind to a JIP3 LZ2 coiled-coil dimer ( Figure 5D ). This stoichiometry is consistent with the recently published crystallographic analysis of the KLC2 TPR -JIP3 LZ2 complex that shows two TPR domains symmetrically bound to the JIP3 LZ2 coiled-coil ( Cockburn et al, 2018 ) and also native mass spectrometry (MS) analysis ( Figure 5—figure supplement 1 ). No significant changes in K D are observed when His 6 -KLC1 extTPR is replaced by either His 6 -KLC1 extTPR -JIP1 C-term ( Figure 5E ) or His 6 -KLC1 TPR -JIP1 C-term ( Figure 5F ) thus indicating that the binding of JIP3 LZ2 to KLC1 TPR is independent of either JIP1 C-term and of the conformational change it elicits or the LFP-acidic region.…”

“…Alternatively, the LFP-acidic linker remains bound and there is an as yet uncharacterized activation pathway triggered by JIP1/JIP3 binding that is more reliant on the additional direct adaptor interactions with KHC. Our ITC and mass spectrometry data suggest the possibility that could be driven by steric effects resulting from the JIP3 LZ2 -mediated dimerization of KLC1 TPR within the kinesin-1 tetramer, a notion further substantiated by the recent publication of a KLC2 TPR -JIP3 LZ2 structure that shows two TPR domains (in their ‘open’ conformation) symmetrically bound to the JIP3 LZ2 coiled-coil ( Cockburn et al, 2018 ). However, our data strongly argue that a functional JIP1-JIP3 transport complex would have its TPR domains in their closed conformation induced by Y-acidic binding.…”

Motor–cargo adaptors at the organelle–cytoskeleton interface

“…The kinesin-1 light chain TPR domain can interact directly with a leucine zipper domain (a subclass of coiled-coil) within the JIP3 adaptor protein. This cooperates with peptide motif recognition by JIP1 [30][31][32] (Figure 2a). Both proteins also directly engage the KHCs [33,34].…”

“…Both proteins also directly engage the KHCs [33,34]. JIP3 may promote dimerization of the kinesin light chain TPR domains within the tetramer to promote activation [30]. Alternatively, this could provide a mechanism to cross-link multiple motors into a larger assembly, analogous to the recruitment of multiple dyneins discussed below (Figure 2b).…”

Kinesin-1 motility traced by an activity-based precipitating dye

“…34 In cells, kinesin-1 is inactive and only gets activated upon cargo binding. 35,36 Kinesore interacts with kinesin-1 at the kinesin light chain-cargo interface (Ki = 49 µM for aiKLC2 TPR : SKIP WD complex), mimicking the effect of cargo binding and resulting in kinesin-1 activation. The enhanced motion causes profound rearrangement of the MT network.…”