The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function

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MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function

Analysis of the kinetics of binding of Protein Kinase A Inhibitor alpha (PKIa) to cAMP-dependent protein kinase a catalytic subunit (PKA-C)

In the excel file are summarized the TR-FRET raw data used for the analysis of the binding kinetic for PKIa to ATP-saturated PKA-C. All the experiments were acquired at Biophysical Technology Center (BMBB Department, University of Minnesota, Minneapolis, MN) by GL and JM within The analysis of the TR-FRET data was performed by GL and JM.TR-FRET raw data used for the analysis of the binding kinetic for full-length protein kinase inhibitor (PKIa) to ATP-saturated cAMP-dependent protein kinase A (PKA-C). The experiments are part of a publication on eLIFE: "Multi-state Recognition Pathway of the Intrinsically Disordered Protein Kinase Inhibitor by Protein Kinase A", where we investigated the structural and kinetics changed that PKIa undergoes upon interaction with PKA-CNIH GM 100310 to G.