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Dynein Motor Domain Shows Ring-Shaped Motor, Buttress Print

Movement is fundamental to life. It takes place even at the cellular level where cargo is continually being transported by motor proteins. These tiny machines convert the energy gained from hydrolysing ATP into a series of small conformational changes that allow them to literally “walk” along microscopic tracks. Motor proteins (in the kinesin and myosin families) have been extensively studied by x-ray crystallography, but until recently there was little molecular structural information for dyneins, another type of motor protein. A group from the University of California, San Francisco, working at ALS Beamline 8.3.1 has reported the 6-Å-resolution structure of the motor domain of dynein in yeast. It reveals details of the ring-shaped motor as well as a new, unanticipated feature called the buttress that may play an important role in dynein’s mechanical cycle.

Yeast Yields Enable Protein Study

The transportation of materials around a cell is essential for its function and survival. Tiny motor proteins like dynein make this movement possible, transporting a vast number of different cargos around the cell. Dynein is also relevant to antiviral medicine, as some viruses target and hijack dynein, using it to get into a cell’s center.

Using x-ray crystallography, researchers can determine the structure of proteins and extrapolate how they work. Dynein was difficult to study prior to this report. With an average protein size of ~360 amino acids, dynein is larger and more complex than other motor proteins, and getting enough protein for crystallography was somewhat of a challenge.

To overcome this problem, UCSF researchers turned to baker’s yeast, where the dynein gene is not essential, and through a process called homologous recombination they could easily manipulate the gene. But the yield of protein from yeast was low, prompting researchers to try fed-batch fermentation, an approach used to make commercial yeast. In this method, a sugar solution is fed to the yeast at a slow, but exponentially increasing rate. This prevents build up of toxins and allows the yeast to grow to 10 times their normal density. A 75-liter fed-batch growth produced 14 kg of yeast and required a new freezer be purchased to store it all. With this massive increase in starting material, enough protein was produced to find optimal conditions required for crystallization, accommodating the modeling study.

 

Overview of the dynein motor domain, showing the ring of six AAA domains, with the linker domain spanning across the ring surface from AAA1 to the base of the stalk. The stalk was truncated in the crystal structure to remove the top two thirds.

 

Model for part of the fourth AAA domain of dynein built into a 6-Å-resolution electron density map (blue chicken wire representation).

Like other motor proteins, dynein produces movement by coupling ATP binding and hydrolysis with changes in shape. ATP binds to a dynein’s motor domain, causing it to release from the microtubule track to which it’s bound and resetting a mobile part of the structure called the linker domain into a different position. Then dynein rebinds the microtubule, triggering the hydrolysis of ATP and a conformational change in the position of the linker domain, generating force. Finally ADP is released from the motor domain, allowing another ATP molecule to bind.

Dynein differs from other motor proteins in two ways. Instead of having a single ATP-binding domain, dynein comprises a ring of six AAA+ domains (ATPases Associated with diverse cellular Activities). At least two of these domains are sites of hydrolysis, which raises the question of how many ATP molecules dynein actually uses. Second, the microtubule-track binding site is in a separate domain from the ATP binding site, found at the end of a long alpha helical projection called the stalk.

The structure of dynein’s microtubule-binding domain was previously determined at ALS Beamline 8.3.1 (see ALS Science Highlight How Dynein Binds to Microtubules). Now, the x-ray crystal structure for its motor domain has been solved at the same beamline using phases determined from a polytungstate cluster (W12) heavy-atom derivative. Because of the conserved structure of AAA domains and the high alpha helix content of the dynein, researchers were able to build a model and assign a position to all the secondary structure elements. This model shows the mobile linker domain arching over the ring of AAA domains. It also shows that the linker is on the same face of the ring as a set of highly conserved inserts in the AAA domains, suggesting that as the linker moves it may contact different AAA domains. The ring itself was remarkably asymmetric, with some AAA domains packed close together and others gaping open. Notably, dynein’s main ATP binding site, between AAA1 and AAA2, was in an open conformation, consistent with the fact that the structure was solved in the absence of ATP and ADP.

An intriguing part of the newly modeled structure is the buttress: a coiled-coil hairpin that extends out of AAA5 to contact the microtubule-binding stalk. The presence of the buttress was unanticipated even though, in hindsight, both electron microscopy and coiled-coil prediction software previously hinted at its existence. It is perfectly placed to link ATP-driven rearrangements of the AAA ring to conformational changes that propagate along the stalk to change the affinity of the microtubule-binding domain for its track. The buttress therefore seems to be a key element in the coupling of ATP hydrolysis to dynein’s movement along microtubules.

Model for the whole dynein motor domain including the stalk (yellow) and its interaction with the buttress (orange). The view is from the face of the AAA ring opposite from the linker. The region in green is based on an idealised stretch of coiled coil and the previously determined structure (see ALS Science Highlight How Dynein Binds to Microtubules) of the dynein microtubule-binding domain.

 

Right: Andrew Carter and Carol Cho collecting data at ALS Beamline 8.3.1 together with technician Lan Jin. Andrew and Carol worked together in Ron Vale’s lab at UCSF on the dynein structure for over five years, resulting in the work reported here. Left: Carol Cho harvesting some of the 14 kg of yeast grown by fed-batch fermentation.

 


 

Research conducted by A.P. Carter (UC San Francisco and Medical Research Council Library of Molecular Biology) and C. Cho, L. Jin, and R.D. Vale (UC San Francisco).

Research funding: U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES). Operation of the ALS is supported by DOE BES.

Publication about this research: A.P. Carter, C. Cho, L. Jin, and R.D. Vale, "Crystal structure of the dynein motor domain," Science 331, 6021 (2011).

ALS Science Highlight #238

 

ALSNews Vol. 326