Kinesin-1 holoenzyme assembly coordinates cargo-adaptor recognition with heavy-chain autoinhibition

Kinesin-1 is a major microtubule-based molecular motor that transports diverse cellular cargoes, yet how assembly of its subunits is coupled to cargo recognition and motor regulation remains poorly understood. Mammalian kinesin-1 functions as a heterotetrameric holoenzyme composed of kinesin heavy chains (KIF5s) and kinesin light chains (KLCs), but the structural principles linking holoenzyme assembly to motor regulation remain unclear. Here, using KIF5C as a model system, we define the molecular mechanisms underlying kinesin-1 holoenzyme assembly and cargo-adaptor regulation. We identify a conserved coiled-coil interface between KIF5C and KLC1 that mediates their 2:2 assembly, with quantitative mutagenesis revealing key hydrophobic determinants of complex formation. We further demonstrate that the mitochondrial adaptor TRAK2 directly engages the KIF5C CC4 cargo-binding platform through a defined 2:2 interaction required for mitochondrial recruitment of KIF5C. Although KLC1 and TRAK2 bind distinct regions of KIF5C, KLC1 modulates TRAK2 association through steric and conformational effects, revealing how holoenzyme composition influences cargo-adaptor accessibility. Mechanistically, we identify a previously unrecognized intramolecular interaction between the KIF5C CC1 and CC4 domains that forms a stalk-mediated autoinhibitory latch. TRAK2 and KLC1 release this inhibitory interaction through distinct mechanisms. Together, our study establishes a molecular framework in which kinesin-1 holoenzyme assembly regulates cargo-adaptor recognition and autoinhibitory remodeling, providing mechanistic insight into how molecular motors coordinate cargo engagement with activation.