Mapping the Structural and Dynamical Features of Kinesin Motor Domains

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.Author Summary: Kinesin motor proteins transport cargo along microtubule tracks to support essential cellular functions including cell growth, axonal signaling and the separation of chromosomes during cell division. All kinesins contain one or more conserved motor domains that modulate binding and movement along microtubules via cycles of ATP hydrolysis. Important conformational transitions occurring during this cycle have been characterized with extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in conformational change and microtubule interaction modulation remain unclear. Here we describe a comprehensive principal component analysis of available motor domain crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of kinesin motor domains to ATP binding and hydrolysis. Our studies reveal atomic details of conformational transitions, as well as novel nucleotide-dependent dynamical couplings, of distal regions and residues potentially important for the allosteric link between nucleotide and microtubule binding sites.