Half-Site Inhibition of Dimeric Kinesin Head Domains by Monomeric Tail Domains

Abstract

The two heavy chains of kinesin-1 are dimerized through extensive coiled coil regions and fold into an inactive conformation through interaction of the C-terminal tail domains with the N-terminal motor (head) domains. Although this potentially allows a dimer of tail domains to interact symmetrically with a dimer of head domains, we report here that only one of the two available monomeric tail peptides is sufficient for tight binding and inhibition of a dimer of head domains. With a dimeric tail construct, the other tail peptide does not make tight contact with the head dimer and can bind a second head dimer to form a complex containing one tail dimer and two head dimers. The IAK domain and neighboring positively charged region of the tail is sufficient for tight half-site interaction with a dimer of heads. The interaction of tails with monomeric heads is weak, but a head dimer produced by the dimerization of the neck coil is not required because an artificial dimer of head domains also binds monomeric tail peptides with half-site stoichiometry in the complete absence of the native neck coil. The binding of tail peptides to head dimers is fast and readily reversible as determined by FRET between mant-ADP bound to the head dimer and a tail labeled with GFP. The association and dissociation rates are 81 µM−1s−1 and 32 s−1 respectively. This half-site interaction suggests that the second tail peptide in a folded kinesin-1 might be available to bind other molecules while kinesin-1 remained folded.

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

“…The calculated K d from the functional assay was 0.076 AE 0.003 μM, approximately twofold higher than the value obtained by fluorescence anisotropy. The results of this functional assay are reasonably consistent with prior work demonstrating similar functional inhibition of kinesin-1 heads by shorter tail constructs (10,31). The difference in the affinity measured by anisotropy vs. MT-stimulated ATPase may reflect some nonspecific binding in the anisotropy assay due to the hydrophobicity of the fluorescent probe, which is fairly common.…”

“…1B). Correspondingly, a stretch of basic residues in the kinesin-1 tail (aa 927-937) is required for tight binding to the head (31). For our experiments, we used a truncated tail construct (aa 790-975) N-terminally fused to maltose-binding protein for enhanced solubility (Tail975; Fig.…”

“…Hackney et al showed that head dimerization is required for high affinity head-tail binding, and tail-mediated inhibition of motor activity occurs at a stoichiometry of 1 tail∶2 heads (31). This indicates that the tail may make stabilizing interactions by nestling in between the two heads.…”

“…When kinesin-1 is autoinhibited, the KHC tails interact directly with the KHC heads (14). The tail HM site mediates tight binding to the head, so that an adjacent inhibitory motif can block ADP release (10,14,(31)(32)(33). While existing results agree that the KLCs are also involved in regulation, they appear to be contradictory as to whether the KLCs activate (16) or inhibit (17,18) kinesin-1 motor activity.…”

“…A conserved region in the KHC tail, which we refer to as the head/MT-binding (HM) site, mediates two distinct functions: (i) binding to the enzymatic KHC heads for inhibition of motor activity (31) and (ii) binding to MTs (11,28). When kinesin-1 is autoinhibited, the KHC tails interact directly with the KHC heads (14).…”