Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A

Jinqi Ren,Lin Huo,Wenjuan Wang,Yong Zhang,Wei Li,Jizhong Lou,Tao Xu,Wei Feng

doi:10.1074/jbc.m115.689091

Abstract

Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3.

Highlights

Intracellular transport is a fundamental biological process that governs the delivery and distribution of cellular components and that is often powered by cytoskeleton-dependent molecular motors [1,2,3]
The data from molecular dynamics simulations further suggested that the introduction of Pro-390 back into the neck coil (NC)-CC1 tandem breaks the continuous coiled-coil dimer but endows it with the intrinsic flexibility to couple NC with the CC1-FHA tandem to form a stable dimer
CC1 and the FHA domain within the CC1-FHA tandem are integrated by a covalent linker to form an elongated parallel dimer (Fig. 2A), which is consistent with the previous structural studies of the CC1-FHA tandem of KIF1A [24]

Summary

Experimental Procedures

Protein Expression and Purification—DNA sequences encoding mouse KIF13A fragments including the NC-CC1 tandem (residues 355– 445), the CC1-FHA tandem (residues 386 – 559), the NC-CC1-FHA tandem (residues 355–559), and various mutants were each cloned into a modified version of the pET32a vector. Crystallization and Data Collection—Native crystals of the CC1-FHA tandem (10 mg/ml in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) and native and selenomethionine crystals of the NC-CC1(⌬Pro-390) mutant (15 mg/ml in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) were obtained using the sitting-drop vapor diffusion method at 16 °C. The overall quality of the final structural models of the CC1-FHA tandem and the NC-CC1(⌬Pro-390) mutant was assessed by PROCHECK [30]. Cell Culture, Imaging, and Data Analysis—The KIF13A fragments including MD (residues 1–354), MD-NC (residues 1–385), MD-NC-CC1 (residues 1– 445), MD-NC-CC1-FHA (residues 1–559), and various mutants were each cloned into a pEGFP-N3 vector. Molecular Dynamics Simulations—Based on the structures of the CC1-FHA and NC-CC1(⌬Pro-390) dimers, the initial

Diffraction data

PDB ID

Results

Discussion

Wei Feng