Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

Abstract

Background:Myosins convert the energy of ATP hydrolysis into force production. Results: Substitution of the metal-interacting serine in switch-1 with cysteine renders the motor sensitive to manganese. Conclusion: This technology provides a reversible structural linkage between the nucleotide pocket and actin-binding region in the myosin motor domain. Significance: Understanding the ATPase mechanism requires a description of allosteric communication in molecular motors.

Switch-1 Instability at the Active Site Decouples ATP Hydrolysis from Force Generation in Myosin II

“…In contrast, the S237C mutant retained relatively tight binding in the presence of MgATP ( f b = 0.68 ± 0.05), and detachment was rescued in the presence of MnATP ( f b = 0.22 ± 0.09). These results are consistent with previously published studies with M761 WT and S237C constructs without the α-actinin repeats, and confirm that the S237C substitution in M761-2R appears to modulate the actomyosin interaction base on the divalent metal present in solution (Cochran et al, 2013). This suggests that serine (-OH) in the WT switch-1 binds Mg 2+ and Mn 2+ sufficiently tightly to affect the release from F-actin, while the cysteine (-SH) in S237C can only bind Mn 2+ well enough for efficient F-actin release.…”

“…Hence exchanging divalent metals provided a direct and experimentally reversible link between switch-1 and the actin binding cleft. Interestingly, the basal ATPase rate of wild type (WT) myosin in the presence of divalent metals other than Mg 2+ is very high, which is similar to the high basal rates observed with the S237C mutant (Cochran et al, 2013). In addition, substituting Ca 2+ leads to high basal ATPase rates that were inhibited in the presence of F-actin for both WT and S237C.…”

“…In a previous study, we were able to control the enzymatic activity and F-actin binding of a non-motile truncation of Dictyostelium discoideum myosin II (M761) by substituting the divalent metal coordinating switch-1 serine (S237) with cysteine, which diminishes its interaction with the Mg +2 metal, and thus inhibits its ATPase activity (Cochran, Thompson, & Kull, 2013). Substituting Mg +2 with Mn +2 , which strongly interacts with cysteine, restores metal interaction, ATPase activity, and actin binding.…”

“…Given that switch loop movement and product release are tightly coupled to the power stroke and force generation, we hypothesized that destabilizing switch-1 movement would negatively affect the tight coupling of ATP hydrolysis to force production that results in motility. To test this hypothesis, we fused two α-actinin repeats (2R) that function as an artificial lever arm (Anson, Geeves, Kurzawa, & Manstein, 1996) to M761 and M761(S237C), the truncated myosin II constructs used in our previous study (Cochran et al, 2013). Cosedimentation and steady-state ATPase assays confirmed that the α-actinin repeats did not change the behavior of the motor constructs.…”

Altered chemomechanical coupling causes impaired motility of the kinesin-4 motors KIF27 and KIF7

“…To distinguish these possibilities, we measured the microtubule-stimulated ADP release for KIF7 using a mant-ADP release assay. When bound in the nucleotide pocket of a kinesin motor, mant-ADP displays a higher fluorescence than when free in solution ( Cochran et al, 2013 ), and the microtubule-stimulated mant-ADP release rate can thus be measured as a decrease in fluorescence ( Nitta et al, 2008 ; Cochran et al, 2013 ; Chen et al, 2017 ). We found that KIF7 was capable of binding to mant-ADP as the initial fluorescence intensity was higher in the presence of KIF7 ( Fig.…”