Modeling dynein dynamics and its interactions with microtubules and microtubule-associated proteins using molecular dynamics simulations

Abstract

Cellular transport is essential for maintaining organization within the cell, like logistics in a city, where microtubules (MTs) function as pathways connecting different areas. Motor proteins, kinesin and dynein, transport cellular cargo along these MT tracks, with kinesin mostly moving toward the plus (+) end and dynein toward the minus (−) end. Both proteins rely on ATP for energy, but they have different structures and mechanisms of action. Mutations that impair their transport can lead to neurodegenerative and developmental disorders. MT-associated proteins (MAPs) regulate this transport by interacting with MTs. In comparison, the abnormal expression of tau can cause an impairment in synaptic vesicle transport, while MAP7 assists motility of kinesin-1 while repressing those of kinesin-3. Kinesin motor proteins move cargo toward the plus (+) end of MTs (anterograde), while dynein moves cargo toward the minus (−) end (retrograde). Over 40 kinesins facilitate transport to the plus end, while a single dynein type (dynein-1) is responsible for retrograde transport. Dynein-2, however, handles intraflagellar transport in cilia. Both dyneins share a conserved structure, with ATPase activity powering their movement. Cytoplasmic dynein consists of two identical heavy chains (DHCs) and several smaller polypeptides. Each DHC has a motor domain (head) with a ring of six AAA modules and a tail domain for dimerization. ATP hydrolysis at AAA1 drives dynein motility, while the MT-binding domain (MTBD) is connected to the catalytic domain via a coiled-coil stalk. The dynein mechanochemical cycle involves four states, where ATP binding triggers MT release, and ATP hydrolysis drives the linker's movement from a bent to straight conformation, generating the force needed for dynein to step along MTs. This cycle allows dynein to transport cargo along MTs efficiently. High-resolution structures (PDB 7Z8G and 7Z8F) were used to examine the ATP-induced release of dynein from MTs. The simulations demonstrated high structural stability throughout a 3000 ns molecular dynamics (MD) simulation. The root mean square deviation (RMSD) values showed minimal deviations. Additionally, throughout the simulations, the dynein stalk and MTBD demonstrated similar angular behavior, supporting the structural integrity of dynein in its ADP-bound state. The conformational changes of dynein's linker during the priming stroke, which is one of the critical events in its mechanochemical cycle, were modeled and analyzed. To investigate the dynamics and energetics of the dynein linker, conventioal MD, steered MD (SMD), and umbrella sampling simulations were conducted on human dynein-2 in its primed state for the power stroke. The simulations demonstrated that the linker can assume a bent or semi-bent conformation, with a 5.7 kT energy barrier separating the two states. It was also shown that in the pre-powerstroke state, the linker cannot revert to its straight conformation due to steric clash with the AAA+ ring. The simulations also revealed that, when isolated from the AAA+ ring, the linker's free energy minimum is positioned near the semi-bent conformation, indicating that the linker stores energy during bending and releases it during the powerstroke. The structure of human cytoplasmic dynein-2 (PDB: 4RH7) was modified by completing missing residues and replacing ADP.Vi at the AAA1 binding site with ATP. The buttress region was cleaved and connected using a GGGG linker, followed by solvation in a water box with neutralized and ionized conditions. MD simulations were performed, totaling 4000 ns, to study the structural changes and dynamics of the cleaved dynein-2 structure. The linker angle was calculated over time, and the results showed deviation from the initial structure for one set of simulation and other three sets of simulations showed minimal deviations. The cleavage of the buttress may have induced conformational changes in the catalytic ring or the stalk, potentially shifting the linker from its bent to semi-bent state. The atomic model of MAP7 on tubulin was constructed using cryo-EM data and modeling tools like VMD and Alphafold2. A total of 4000 ns of MD simulations were performed to study MAP7's interactions with MTs. PCA was used to generate the energy landscape of MAP7, identifying four binding modes. Interaction analysis was conducted based on these binding modes. The four binding modes were revealed by PCA based clustering of MAP7 conformations. Also, intrinsically disordered C-terminal tails of MTs were interacted with the MAP7 MTBD. For Tau, a similar approach was used to model its MTBD, consisting of four repeat sequences (R1-R4). Simulations explored the interactions of Tau's MTBD with MTs, focusing on the structural integrity and dynamics of the Tau-tubulin complex over 4000 ns of MD simulations. The Tau simulations provided insights into how each repeat sequence (R1-R4) interacts with MTs, contributing to MT stability. Similar with the MAP7, intrinsically disordered C-terminal tails of MTs were interacted with the Tau MTBD.