eBIC produces first 3D structure of the complete human dynein

Cryo-EM 3D Reconstruction of Full-Length Human Dynein-1

2017 MRC Laboratory of Molecular Biology. Published by Elsevier Inc.

 A team of researchers have used Diamond’s electron Bio-Imaging Centre (eBIC) to help produce the first 3D structure of complete human cytoplasmic dynein-1, an essential molecular motor.

Writing in the journal Cell, they present a cryo-electron microscopy (cryo-EM) structure of the cytoplasmic dynein-1 complex in an inhibited state and explain how dynein is activated by another protein complex called dynactin.

“Dynein is very flexible complex and so it’s structure is difficult to resolve to high resolution.” explained Dr Andrew Carter, a lead author of the paper who is based at the Medical Research Council’s Laboratory of Molecular Biology. “Collecting data at eBIC really helped us to solve the structure and understand the overall architecture of the whole dynein complex.”

Dynein is essential for ensuring the insides of cells are organised properly. It works by binding to cargos including proteins, RNA and membrane compartments, then walking along the cells microtubule skeleton. Microtubules provide a cellular ‘motorway’ that dynein shares with another family of molecular motors called kinesins. These motors are coordinated and regulated to ensure the cells components are in the right place at the right time.

Using cryo-EM, the researchers were able to reveal the full three-dimensional structure of dynein in an inhibited state where it cannot easily bind the microtubule. In order to become an active transport machine, dynein binds to another large protein complex called dynactin and a cargo adaptor protein. The authors also studied the structure of this triple complex by cryo-EM to understand how dynein is activated by dynactin binding.

The researchers found that dynein is normally kept in an inhibited state called the ‘phi-particle’. By solving the 3D structure of the phi-particle, the authors show how the parts of dynein which produces force - the motor domains – are bound together and explain how this causes inhibition. In collaboration with Alex Bird’s lab in Dortmund, the researchers show that disrupting the phi-particle in cells changes dynein’s location and causes problems during cell division. It is also possible that disrupting the phi-particle contributes to rare brain disorders which originate from abnormal development of the brain’s cortical layer.

“It is great to see these results, particularly given the challenges in solving the structure of large, flexible complexes like dynein,” added Daniel Clare, a Senior EM Scientist based at eBIC. “These kinds of ground breaking results are exactly why we established eBIC and we hope this is just the tip of the iceberg.”
The team went on to investigate how dynein is activated. The activity of the dynein complex can be monitored by attaching a fluorescent dye to one part of the protein and adding it to a small glass chamber coated with purified microtubules. In a fluorescence microscope, the movement of single dynein complexes walking along microtubule tracks can be observed and measured.

Using this ‘single molecule assay’ in combination with mutagenesis and electron microscopy allowed the researchers to find out that dynactin is the crucial factor which stimulates dynein activity. They found that dynactin activates dynein by flipping the dynein motors into the correct alignment to begin walking along the microtubule.

A number of human mutations that cause malformation of cortical development (MCD) map to the interface between dynein motor domains and so may work by disrupting the phi-particle. Although MCD mutations are very rare, five different mutations map to this same interface. Dynein is also known to be involved with many diseases, for example viral infections, and a better understanding of how dynein works might lead to new ideas for the treatment of these diseases.

“We now have a much better understanding of how dynein is auto-inhibited and activated but many structural and mechanistic questions remain, such as ‘How it can carry so many different cargos?’ ” concluded Dr Carter. “Cryo-EM is a great technique to address these questions because it can deal with large complexes which are inherently flexible. We’ll continue to use the latest hardware and software developments in cryo-EM, in combination with in vitro or cellular assays, to improve our understanding of these fascinating molecular machines.”

Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.

Zhang, K et al. Cell , Volume 169 , Issue 7 , 1303 - 1314.e18

DOI: http://dx.doi.org/10.1016/j.cell.2017.05.025