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Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule.

ABSTRACT: Nucleotide-free kinesin motor domains from several kinesin families convert reversibly to a refractory conformation that cannot rapidly rebind ADP. In the absence of glycerol, the refractory conformation of Drosophila kinesin motor domains is favored by 50-fold with conversion of the active to the refractory species at ?0.052 s(-1) and reactivating in the presence of ADP at ?0.001 s(-1). This reactivation by ADP is due to conformational selection rather than induced fit because ADP is not bound to the refractory species at concentrations of ADP that are sufficient to saturate the rate of reactivation. Glycerol stabilizes the active conformation by reducing the rate of inactivation, while having little effect on the reactivation rate. Circular dichroism indicates a large conformational change occurs on formation of the refractory species. The refractory conformation binds ANS (8-anilino-1-napthalenesulfonic acid) with a large increase in fluorescence, indicating that it has molten globule character. High ANS binding is also observed with the refractory forms of Eg5 (a kinesin-5) and Ncd (a kinesin-14), indicating that a refractory conformation with molten globule characteristics may be a common feature of nucleotide-free kinesin motor domains.

SUBMITTER: Hackney DD

PROVIDER: S-EPMC5159748 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule.

Hackney David D DD McGoff Marshall S MS

Archives of biochemistry and biophysics 20160826

Nucleotide-free kinesin motor domains from several kinesin families convert reversibly to a refractory conformation that cannot rapidly rebind ADP. In the absence of glycerol, the refractory conformation of Drosophila kinesin motor domains is favored by 50-fold with conversion of the active to the refractory species at ∼0.052 s(-1) and reactivating in the presence of ADP at ∼0.001 s(-1). This reactivation by ADP is due to conformational selection rather than induced fit because ADP is not bound ...[more]

PMID: 27576140

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Project description:Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4-α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4-α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.

Dataset Information

Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule.

Publications

Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule.

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