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. 2018 Jan 5;293(1):215-225.
doi: 10.1074/jbc.M117.816017. Epub 2017 Nov 20.

Structural analyses of key features in the KANK1·KIF21A complex yield mechanistic insights into the cross-talk between microtubules and the cell cortex

Zhuangfeng Weng  1 , Yuan Shang  2 , Deqiang Yao  3 , Jinwei Zhu  4 , Rongguang Zhang  5
Affiliations

Structural analyses of key features in the KANK1·KIF21A complex yield mechanistic insights into the cross-talk between microtubules and the cell cortex

Zhuangfeng Weng et al. J Biol Chem. .

Abstract

The cross-talk between dynamic microtubules and the cell cortex plays important roles in cell division, polarity, and migration. A critical adaptor that links the plus ends of microtubules with the cell cortex is the KANK N-terminal motif and ankyrin repeat domains 1 (KANK1)/kinesin family member 21A (KIF21A) complex. Genetic defects in these two proteins are associated with various cancers and developmental diseases, such as congenital fibrosis of the extraocular muscles type 1. However, the molecular mechanism governing the KANK1/KIF21A interaction and the role of the conserved ankyrin (ANK) repeats in this interaction are still unclear. In this study, we present the crystal structure of the KANK1·KIF21A complex at 2.1 Å resolution. The structure, together with biochemical studies, revealed that a five-helix-bundle-capping domain immediately preceding the ANK repeats of KANK1 forms a structural and functional supramodule with its ANK repeats in binding to an evolutionarily conserved peptide located in the middle of KIF21A. We also show that several missense mutations present in cancer patients are located at the interface of the KANK1·KIF21A complex and destabilize its formation. In conclusion, our study elucidates the molecular basis underlying the KANK1/KIF21A interaction and also provides possible mechanistic explanations for the diseases caused by mutations in KANK1 and KIF21A.

Keywords: KANK1/KIF21A complex; ankyrin repeat; cell adhesion; kinesin; microtubule; microtubule-cell adhesion cross-talk; scaffold protein; structural biology.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Characterization of the interaction between KANK1 and KIF21A. A, schematic diagram showing the domain organizations of KANK1 and KIF21A. The interaction between KANK1 and KIF21A is indicated by a two-way arrow. Talin, liprins, and IRSp53 bind to the KN domain and the coiled-coil domain of KANK1, respectively. The middle coiled-coil domain forms an intramolecular interaction with the motor domain of KIF21A. CFEOM1-causing mutants (e.g. R954W, indicated by a red asterisk) would release the autoinhibited conformation of KIF21A. B, mapping the KANK1/KIF21A interaction via ITC-based measurements. n.d., not detectable. C, ITC assay showing that the KBD of KIF21A bound with the ANK domain of KANK1 with a Kd value of ∼0.58 μm. D, analytical gel filtration analysis showing that KANK1 ANK and KIF21A KBD form a 1:1 stoichiometric complex. SDS-PAGE analysis of the protein components from the eluted peaks (12–16 ml) is also shown. mau, milliabsorbance units.
Figure 2.
Figure 2.
Overall structure of the KANK1·KIF21A complex. A and B, ribbon diagram (A) and cylinder (B) representations of the KANK1·KIF21A complex structure. In the drawing, the capping domain and ANK repeats of KANK1 are shown in light blue and orange, respectively. The KBD peptide of KIF21A is shown in green. C, superposition of the structures of the KANK2-ANK (PDB entry 4HBD, light blue) and the KANK1·KIF21A complex (this work). D, omit map of the KIF21A KBD peptide bound to KANK1 ANK. The map is countered at the level of 1.0 σ in PyMOL.
Figure 3.
Figure 3.
Detailed interface of the KANK1·KIF21A complex. A and B, the KANK1/KIF21A interface is divided into two sites corresponding to site I (the polar interaction site) (A) and site II (the hydrophobic interaction site) (B). The interaction details between KANK1 and KIF21A in two sites are shown in stereo view. C, the combined surface and ribbon representations of the KANK1·KIF21A complex. The hydrophobic residues, positively charged residues, and negatively charged residues of KANK1 are colored yellow, blue, and red, respectively. D, structure-based sequence alignment of the KBD peptide of KIF21A from different species. In this alignment, the absolutely conserved and conserved residues are colored red and green, respectively. Residues involved in binding to KANK1 are annotated below as blue dots. E and F, summary of ITC-based measurements of binding affinities between wild type or mutants of KANK1 ANK and KIF21A KBD. n.d., not detectable.
Figure 4.
Figure 4.
The binding specificities of KANK family proteins to KIF21A KBD. A–C, ITC-based measurements of binding affinities between the KIF21A KBD and the ANK domains from the KANK family proteins, KANK2 (A), KANK3 (B), and KANK4 (C). n.d., not detectable.
Figure 5.
Figure 5.
The capping domain is essential for the intact KANK1/KIF21A binding. A, the combined surface and ribbon representations of the coupling interface between the capping domain and the ANK repeats of KANK1. B, stereo views of the molecular details of the capping domain/ANK repeats interface. C, ITC assay showing that the ANK repeats alone bound to the KIF21A KBD peptide with a Kd value of ∼64.9 μm.
Figure 6.
Figure 6.
The effects of disease-associated mutations on the formation of the KANK1·KIF21A complex. A, the combined ribbon and stick-dot representations showing the detailed roles of disease-causing mutations in KANK1 ANK and KIF21A KBD. In this drawing, the side chains of key residues associated with diseases are shown in the stick mode, and the related hydrogen bonds and salt bridges are indicated by black dashed lines. B, effects of various disease-associated mutations on KANK1·KIF21A complex formation based on ITC assays. n.d., not detectable.
Figure 7.
Figure 7.
Phosphomimicking mutants of KIF21A impair their bindings to KANK1 ANK. A, consensus phosphorylation sites (PKA and Akt) located in the KBD fragment of KIF21A. B–D, ITC-based measurements of binding affinities between the ANK domain of KANK1 and different phosphomimicking mutants of KIF21A KBD.
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