Neurodegenerative diseases are mostly incurable conditions that result in progressive degeneration or death of nerve cells. Prominent examples such as Spinal Muscular Atrophy (SMA) are caused by mutations in the cytoplasmic dynein multi-protein complex. Dynein 1 participates in a wide array of cellular activities, ranging from the cargo transport of proteins, RNA, and vesicles to nuclear migration and cell division. Processive movement of dynein along microtubules requires the binding to the essential co-activator dynactin and specific adaptor proteins, which recruit cargo and facilitate movement by forming a stable ternary complex with dynein and dynactin. Our understanding of these interactions is limited and many questions remain: How do cargo adaptors bring dynein and dynactin together? How does this lead to activation of the motor and the initiation of transport? Which elements determine the adaptor specificity? How do the disease-causing mutations modify these interactions? Importantly, it is becoming increasingly evident that binding to light intermediate chain of dynein 1 (`LIC1') is a common feature of functionally distinct adaptors. We will focus our investigations on the interaction between the C-terminal tail of the LIC1 with the adaptors Spindly, BICD2 and Hook3, which each have important but distinct functions in transport. These studies are complicated by the fact that the disordered nature of C- terminal LIC1 makes it difficult to obtain a comprehensive dynamic view at atomic resolution from X-ray or EM techniques. However, we will take the innovative approach of using nuclear magnetic resonance (NMR) as a nano-scale `magnifying glass' that pinpoints interaction sites within the proteins at atomic resolution. Compared to other methods, NMR is uniquely suited to study disordered proteins and to characterize highly dynamic interactions. We propose four aims: (1) Structural and dynamical characterization of free C-terminal dynein LIC1. (2) Characterization of dynein LIC1 interaction with Spindly, BicD2 and Hook3, and their binding competition. (3) Prediction and testing of effects of mutations on dynein LIC1 - adaptor interaction. (4) Structural & dynamic basis of interaction triangle between dynein LIC1, Spindly and ROD/Zw10/Zwilch complex. Our experiments are therefore expected to provide detailed molecular insight into how human dynein LIC1 engages with structurally diverse dynein adaptors. Through collaboration, we will extend our findings into animal models. Overall, this research is significant because the presence of pathogenic point mutations in dynein and adaptor proteins suggests that these insights will also help to understand the diseases caused by malfunction of the dynein-driven cargo transport and will offer therapeutic intervention targets.
Malfunction of the cytoplasmic multi-protein dynein motor is implicated in various neurodegenerative diseases. Processive movement of dynein along microtubules requires binding to the essential co- activator dynactin and specific motor adaptor proteins. We will elucidate the generalities and specificities of the molecular mechanisms of the highly dynamic interaction between the light intermediate chain of dynein 1 and various adaptors at atomic resolution by NMR and other biophysical methods.
