Figure 3. Nucleotide binding to a kinesin head is inhibited when the NL is oriented backward.

(A) k₁ values of the apo condition were subtracted from that of 1 mM ADP to calculate the nucleotide-binding induced MT release rate from the weakly-bound state. k_1-ADP − k_1-apo of NL-pulled kinesins was 35 s⁻¹ at −1.5 pN, and decreased to ~0 s⁻¹ at higher negative forces. (B) k₂ values of the apo condition were subtracted from that of 1 mM ADP to calculate the nucleotide-binding induced MT release rate from the strongly-bound state. k_2-ADP − k_2-apo of NL-pulled kinesins remained nearly constant at 3.0 s⁻¹ under negative forces, whereas k_2-ADP − k_2-apo of head-pulled kinesins increased from 3.4 s⁻¹ at −1.8 pN to 14.2 s⁻¹ at −6.7 pN.