The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

Autoinhibition of kinesin-1 motor Auto-inhibition state active state Tail binding to head(motor domain) Tail binding to cargo Release ADP Consume ATP No exchange of ADP Diffuse Walking on microtubule Kristen J. Verhey Nature Reviews Molecular Cell Biology 10, 765-777(2009)

Previous research The Autoinhibiton molecular mechanism is unclear One tail domain inhibit acitvating dimer motor David D.Hackey Biochemistry 48,3448-3456(2009) Tail domain (IAK region) Motor domain molecular mechanism MT binding site? ADP binding site ? Kinesin-1 M. F. Stock et al., J. Biol. Chem. 274, 14617 (1999). K. A. Dietrich et al., Proc. Natl. Acad. Sci. U.S.A. 105,8938 (2008). The Autoinhibiton molecular mechanism is unclear → kinesin1 and tail domain complex structure

kinesin-1 motor-tail complex Material & method Method Get structure info from Crystal analysis construct Kinesin -1tail domain The non-fusion tail peptide (NF937-952) 975 amino acid full length Kinesin-1 Motor domain Kinesin-1 1-365 N-terminal (Drophlia Melanogaster) ADP Kinesin-1(KHC)

kinesin-1 motor-tail complex kinesin-1 complex motor domain(dimer) tail domain (one) Comparison Blue motor domain Cyan motor-tail complex coiled-coil position is only major difference

Tail-domain interaction site Tail domain（residues 937 to 952） straight structure like β sheet D185-K944 hydrogen bond H136-P945 C-H π interaction F135,I126-I945 F135,I126-I942 Hydrophobic bond symmetry ?

the tail domain peptide fitting Symmetry of the tail-domain site the tail domain peptide fitting electron-density Center- E944

kinesin-1 motor-tail complex Tail domain（residues 937 to 952） 1,No interaction with other site 2,one motor dimer have one site 3,the interaction site in motor domain have pseudosymmetry kinesin-1 motor domain + Tail domain

double down mechanism If kinesin dimer are crosslinked in PS center Pesudosymmetry center Double locked down Single point attach ATP ADP If kinesin dimer are crosslinked in PS center

Cross-linked kinesin Rerelease of ADP Material and method construct 181th Amino acid 181Ser 181Cys Kinesin-1 1-396 (C-terminal His6 tag ) crosslink SDS PAGE (check crosslink)

decreases by crosslinking Cross-linked kinesin Rerelease of ADP SH→-SH HS- SS→-S-S- +DTT →-S-S-cleavage MANT- ADP release Release of MANT-ADP decreases by crosslinking

conclusion 1,Tail interface crosslink motor domain 2,two point connection prevent kinesin-1 from undocking the neck 3,stop ADP release