The complex interplay between the neck and hinge domains in kinesin-1 dimerization and motor activity

Abstract

Kinesin-1 dimerizes via the coiled-coil neck domain. In contrast to animal kinesins, neck dimerization of the fungal kinesin-1 NcKin requires additional residues from the hinge. Using chimeric constructs containing or lacking fungal-specific elements, the proximal part of the hinge was shown to stabilize the neck coiled-coil conformation in a complex manner. The conserved fungal kinesin hinge residue W384 caused neck coiled-coil formation in a chimeric NcKin construct, including parts of the human kinesin-1 stalk. The stabilizing effect was retained in a NcKinW384F mutant, suggesting important pi-stacking interactions. Without the stalk, W384 was not sufficient to induce coiled-coil formation, indicating that W384 is part of a cluster of several residues required for neck coiled-coil folding. A W384-less chimera of NcKin and human kinesin possessed a non-coiled-coil neck conformation and showed inhibited activity that could be reactivated when artificial interstrand disulfide bonds were used to stabilize the neck coiled-coil conformation. On the basis of yeast two-hybrid data, we propose that the proximal hinge can bind kinesin's cargo-free tail domain and causes inactivation of kinesin by disrupting the neck coiled-coil conformation.

Figure 1.

Summary of wild-type and mutant NcKin constructs. The domain organization of kinesin-1 is indicated at the top of the figure, and below are enlarged schemes and primary sequences of relevant regions. Parts from NcKin are orange, and human kinesin blue; domains are not drawn in scale. The positions of the residues W384, Y362, and P342 that have been mutated in this study are indicated in red. The construct names indicate the origin of the kinesin used (NcKin), followed by the fusion point (italic number) if it is a chimera; then the length of the construct in subscript (hinge/hTail), finally any exchanged amino acid residue(s), if applicable. Constructs used for two-hybrid assays were essentially comparable to these protein backgrounds, and differed only in some details (Material and Methods). The short constructs based on NcKin_hinge were used for kinetic and dimerization assays and tested in cross-linking studies as P342C mutants. The longer NcKin_hTail-based constructs stuck readily to glass surfaces and were used for multiple motor gliding assays, as well as kinetic and, in the P342C mutant background, cross-linking studies. The primary sequences at the bottom show a sequence alignment of wild-type kin-esin-1s (NcKin, N. crassa; AnKin, Aspergillus nidulans; NhKin1, Nectria hematococca; SrKin, Syncephalastrum racemosum; UmKin2, Ustilago maydis; HsKHC, Homo sapiens), and the amino acid sequences of chimera NcKin-379 and -384. The phase of the neck coiled-coil is indicated (abcdefg positions), the similarity between the fungal and the human sequences by asterisks for amino acid identity in all cases, and dots for similarity. ¹⁾ The NcKin_hTail construct can also be regarded as chimera NcKin-433. It behaved indistinguishable from full-length wild-type NcKin_hTail. For reasons of readability and by analogy to the NcKin_hinge construct, it is referred to as NcKin_hTail wild-type-like reference construct in this article.

Figure 2.

Cross-linking of P342C mutants. The mutants are fractionated on a nonreducing SDS-gel under different redox-conditions: –, no reagent, oxidation by air; ox, +0.2 mM DTNB; red, +2–5 mM DTT; res, +0.2 mM DTNB 5′, 5 mM DTT 4 h. Under oxidizing conditions (air, DTNB) a bandshift to ∼200 kDa is visible for P342C and the chimeras NcKin-379_hTailP342C and NcKin-384_hTailP342C to a nearly complete extent, indicating an interchain cross-link (see also text). The cross-link is reversible as it is seen by the appearance of a 60-kDa band (corresponding to one kinesin polypeptide chain) instead of the 200-kDa band under reducing conditions (DTT). No cross-link occurred with the negative control without P342C and with the double mutant P342C,Y362K.

Figure 3.

Interaction between the kinesin tail and the hinge domain. The interaction between the NcKin tail and hinge domains was tested in a yeast two-hybrid assay. The kinesin tail from amino acid 725–928 was used as bait, different N-terminal kinesin constructs as preys. The interactions were detected by the activation of the LacZ reporter gene, which leads to the expression of β-galactosidase. The β-galactosidase expression level was assayed enzymatically with CPRG as a substrate (bar chart, n = 5–15 independent transformants; error bars, SD) and by blue coloration of the colonies on X-α-Gal containing plates (inset). The longest kinesin prey construct, NcKin_Tail, showed the strongest signal on X-α-Gal LW–selection plates, in CPRG assays, and grew on LWHA-plates. The next shorter construct, NcKin_hinge, was still positive on X-α-Gal plates and in the enzymatic assay, but failed to grow on LWHA-plates. All shorter prey constructs, as well as the point mutant NcKin_TailW384F and the chimera NcKin-379_Tail, were highly variable on X-α-Gal plates but were clearly negative in the enzymatic assay. The NcKin-384_Tail chimera was also negative, suggesting additional structural requirements for a robust interaction.

Figure 4.

Possible role of neck/hinge dynamics for the regulation of fungal kinesins. On state: The neck is in a two-stranded coiled-coil state due to stabilization via W384 and adjacent residues (XXX). The hinge region is folded backward to enable interaction of W384 with the neck domain, requiring large spatial freedom that is achieved by the highly flexible neck-hinge junction (Seeberger et al., 2000). Dimerization of the neck provides mechanochemical coupling of the two heads and thus effective and processive movement, powered by fast ATP hydrolysis in the head domain. Off state: In absence of cargo, the tail domain in the full-length protein folds back (compact conformer) and captures W384 and its close vicinity (XXX). This greatly destabilizes the coiled-coil structure, and the neck domain transits in an unfolded state. The lack of head-head coupling stops processive movement of the motor. The ATPase activity is down-regulated because of the inhibitory interaction of Y362 with the motor core.