In kinesins mechanochemical cycle,ATPs binding to the nucleotide-free leading head is exquisitely gated so that futile hydrolysis is effectively avoided.Experiments show that,when both kinesin heads bind to a microtubule,ATP cannot bind to kinesins leading head when the neck linker (NL) of this head has a backward orientation.How NLs backward orientation is maintained needs understanding on a structural basis.By using steered molecular dynamics and mutation simulations,we investlgate the backward-pointing conformation of the leading beads NL under different inter-head tensions.We find that the NL cannot keep in a strict backward orientation solely by the inter-head tension.LYS325 (amino acid sequence in 2KIN) has an assistant locking function which locks the NL and β0 to the β-domain.This locking function ltas an enhanced positive cooperation with the inter-head tension.When the inter-head tension is weakened,this locking function can be broken,resulting in a loose backward orientation of the NL.The difference between the strict and loose backward orientation of the NL might be a crucial factor in the gating mechanism.These results are consistent with relevant experiments and proposals.