A pocket full of water molecules – how actin filaments drive the movement of the cell

Actin filaments are protein fibers that make up the inner skeleton of the cell. As the active elements of our cells, actin filaments support cell fusion, movement, and are involved in many other cellular processes. Importantly, they are also a major component of muscle cells. The structural complexity of these filaments has fascinated scientists since their discovery in the 1940s – and has opened a sea of ​​unanswered questions behind their ability to facilitate many of the cell’s processes.

Researchers at the Max Planck Institute for Molecular Physiology in Dortmund have for the first time succeeded in making hundreds of water molecules visible in the actin filament, which represents a quantum leap in actin research.

Using the technique of cryo-electron microscopy (cryo-EM), Stefan Raunser’s group shows in unprecedented detail how actin proteins are lined up in a filament, how ATP – the cell’s energy source – sits in the protein pocket, and where individual water is Molecules position themselves and react with ATP.

“We answer fundamental questions of life that scientists have been trying to answer for several decades,” says Raunser. In eukaryotic cells, actin proteins are abundant and tend to associate (polymerize) into filaments.

These filaments form the network that makes up the cell’s cytoskeleton and controls various cell processes through movement. Immune cells, for example, use actin filaments to move and hunt bacteria and viruses.

The researchers already knew that the dynamics of the filaments are regulated by ATP hydrolysis – the reaction of ATP with water, which cleaves a phosphate group and generates energy. However, what has remained unanswered so far are the precise molecular details behind this process.

Too flexible, too big? – not for cryo-EM

Because actin filaments are too flexible or too large for X-ray crystallization and nuclear magnetic resonance, cryo-EM was the only technique capable of obtaining detailed images. In 2015, Raunser’s team used cryo-EM to image a novel three-dimensional atomic model of the filaments with a resolution of 0.37 nanometers. In 2018, his group described the three different states actin proteins assume in the filament: bound to ATP, bound to ADP in the presence of the cleaved phosphate, bound to ADP after the phosphate is released.

How water molecules move

In their recent study published in NatureRaunser and his colleagues were able to set a new resolution record: they obtained all three actin states with a resolution of around 0.2 nanometers and made previously invisible details visible. The three-dimensional maps not only show all the amino acid side chains of the proteins, but also reveal where hundreds of water molecules are located.

By comparing these new structures to those of isolated actin, they were able to deduce how water molecules move. During polymerization, water molecules in the ATP pocket shift in such a way that only a single water molecule remains in front of ATP, ready to attack a phosphate and initiate hydrolysis.

The accuracy achieved through this approach can help further research in this field: “Our high-resolution model can advance scientists in the design of small molecules for light microscopic research on tissues and ultimately for therapeutic applications,” says Raunser.

A door opener?

The authors also shed light on the ultimate fate of the phosphate. Previously, scientists believed that there is a back door in the ATP pocket that remains open after ATP hydrolysis to facilitate the phosphate exit. However, the new cryo-EM structures show no trace of open backdoors. Therefore, the release mechanism remains a mystery.

“We believe that there is a door, but it probably opens for a moment,” comments Raunser, who now wants to show how the phosphate escapes using mathematical simulations and time-resolved cryo-EM methods. Apparently, these exciting discoveries have opened the door for scientists to dig deeper in hopes of uncovering even more details behind the processes by which actin filaments contribute to cell movement.