The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells

Abstract

Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between β0 at the N-terminus (the coverstrand) and β9 in the first half of the necklinker. The CNB was shown to contribute to force generation by single kinesin-1 motors under purified conditions and has been predicted to function in force generation across the kinesin superfamily. However, differences in coverstrand and necklinker sequences make the role of the CNB across different kinesin families unclear. We adapted artificial cargo systems to delineate key features of the CNB during multi-motor transport against low and high loads in cells. Our results indicate that in teams, kinesin-1 motors can robustly transport cargo against high- and low-load in cells. Molecular modeling was used to predict mutations in the coverstrand and necklinker that disrupt and shorten beta-sheet formation. Experimental testing of these mutations shows that impaired beta-sheet formation compromised the capacity for teams of kinesin-1 motors to transport high-load cargo but not low-load cargo to the periphery of cells. Surprisingly, truncation of the region N-terminal to the coverstrand, predicted to have a minimal role in CNB formation, severely crippled the capacity for teams of kinesin-1 motors to transport both low- and high-load cargoes in cells. We plan to extend these studies to additional kinesin families to identify distinct motility dependencies on formation of the CNB.