The mechanism of kinesin inhibition by kinesin-binding protein

Abstract

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

“…Secondarystructure prediction suggested that KBP is built from 19 helices, but clear 'sausage-like' density for only eight helices was observed. Ultimately, the KBP structure was indeed found to have 19 helices as predicted, arranged in a right-handed -solenoid (Atherton et al, 2020). When this final structure was fitted into the KBP density from unsupported ice, eight helices were a good fit, another five fitted poorly and there was missing density for another six (Fig.…”

“…KBP-KIF15_MD6S or KBP-KIF1A_MD complexes were purified via IMAC using the 6ÂHis tag on the kinesin constructs after incubation with a tenfold excess of KBP, as described previously (Atherton et al, 2020).…”

“…Particles were initially processed with cryoSPARC2 (Punjani research papers et al, 2017), cisTEM (Grant et al, 2018) and RELION version 3.0 (Zivanov et al, 2018) before data combination, duplicate removal and final processing in RELION version 3.1 (Zivanov et al, 2018). 3D reconstructions were sharpened with negative B factors applied to the gold-standard FSC 0.143 cutoff as described previously (Atherton et al, 2020).…”

“…Full-length human KBP (residues 1-621 in a PSTCm1 expression vector with kanamycin resistance and an N-terminal thrombin-cleavable 6ÂHis tag) was expressed in Escherichia coli Rosetta2 cells (Novagen) and purified using immobilized metal-affinity chromatography (IMAC) and sizeexclusion chromatography (SEC) as described previously (Atherton et al, 2020;Kevenaar et al, 2016).…”

“…KBP is important in a number of cellular processes, including neuronal development, spermatogenesis and mitosis, and its mutation causes Goldberg-Shprintzen syndrome (GOSHS; Alves et al, 2010;Brooks et al, 2005;Dafsari et al, 2015;Drerup et al, 2016;Lehti et al, 2013;Lyons et al, 2008;Malaby et al, 2019;Salehpour et al, 2017;Valence et al, 2013). KBP functions as a selective inhibitor of microtubule attachment of a subset of kinesin motor proteins (Kevenaar et al, 2016;Wozniak et al, 2005), and our recent structural work describes the inhibitory mechanism of KBP (Atherton et al, 2020). Here, we describe a number of effects on our samples derived from protein-surface interactions during cryo-EM sample preparation that we hope will be informative to other researchers in the field: (i) partial denaturation of KBP, likely due to interactions with the air-water ISSN 2059-7983 interface, (ii) the protection of KBP from partial denaturation by adherence to a graphene oxide (GO) substrate away from the air-water interface, (iii) changes to KBP angular distributions introduced by adherence to GO, (iv) aggregation in KBP-kinesin motor domain (MD) complexes resulting from interactions with a carbon grid support, which was not observed on a gold grid support, and (v) different behaviors of two KBP-kinesin motor domain (MD) complexes on a GO substrate.…”