The P3018S disease variant reveals how dynein's trailing motor sets ensemble velocity.
Mutations in the cytoplasmic dynein heavy chain (DYNC1H1) underlie a range of neurodevelopmental disorders yet how individual variants perturb dynein function remains poorly understood. We characterize the disease-associated P3018S mutation, located in the AAA4 module and within the Lis1-interacting region of the motor. Dynein-dynactin-BicD2 (DDB) complexes containing P3018S dynein move at half the wild-type velocity and generate reduced stall forces but retain the ability to assume the inhibitory phi conformation that Lis1 suppresses, respond to Lis1, and assemble into higher-order force-generating states. Several Schizosaccharomyces species, which lack Lis1, naturally encode a serine at this position, suggesting evolutionary relevance for dynein activation. Using mixed wild-type-mutant assemblies, we find that the dynein occupying the trailing position on dynactin dictates ensemble velocity, and that the leading dyneins LIC enhances trailing-motor velocity by [~]130%, revealing a mechanism for leading-to-trailing motor stimulation within multi-dynein assemblies.