Revealing the mechanism behind the constant motion of organisms from the structure of filamentous actin

What is an organism? “Something in which everything is constantly working and exchanging without interruption; I think this is the true nature of an organism,” says Toshiro Oda, Team Leader of the X-ray Structural Analysis Research Team in the Structural Physiology Research Group at the RIKEN SPring-8 Center.

Muscles contract to move the body, cells migrate and divide themselves in the body, molecules move and exchange constantly in cells—organisms are in constant motion at all levels, and that motion is controlled by a key protein, actin. In 2009, Oda and his colleagues succeeded in clarifying the fine structure of filamentous actin, a sequence of actin subunits, which in turn has revealed the unique features of its functional mechanism.

Why do muscles move? In 1942, the Hungarian scientist Bruno Straub discovered a protein called actin in a muscle. Later, it was found that muscles contract as the motor protein myosin ‘walks’ along actin filaments (F-actin) — polymer sequences of globular actin (G-actin) subunits. The Chemistry of Muscle Contraction, a book by Straub’s mentor Albert Szent-Gyorgyi, had an impact on many scientists all over the world. Stimulated by this book, Fumio Oosawa, Professor Emeritus of Nagoya and Osaka universities, began studying actin and opened a new area known as biophysics, a discipline in which biological phenomena are investigated from the viewpoint of physics.

After studying biophysics at Nagoya University, Oda began studying the actin filament at Matsushita Electric Industrial Co. in 1997. In 1999, he moved to RIKEN and continued his research. “In those days, another motor function of F-actin, other than in muscle, was about to attract wide attention.” In the 1970s, in vitro studies led to the discovery of a phenomenon in which actin molecules polymerize sequentially to one end of F-actin while at the other end they depolymerize. This process results in movement of the F-actin in one direction, allowing the filament to serve as a motor. This phenomenon was later found to be the basis for various biological phenomena in cells in vivo.

“F-actin had long been known to be abundantly present on the back of the cell membrane, where it functions as a cytoskeleton to maintain stable cell morphology. For this reason, F-actin had been viewed as something static. Recent studies have shown, however, that F-actin is also involved in dynamic functions, including cell migration, cell division, intracellular movement of chemical compounds, and memory formation in brain neurons. When a memory is formed, neurons in the brain bind together to form a synapse. In this process, many F-actins assemble at a particular site in the neuron, which in turn pushes the cell membrane from inside to produce projections called spines, which form synapses. Additionally, memory cannot be maintained unless G-actin polymerizes and depolymerizes repeatedly, that is, the G-actin molecules must exchange constantly. ‘Everything is continually working and exchanging without interruption’; I think this is the true nature of organisms. This intrinsic function of organisms to be constantly in movement is supported by F-actin.”

In fact, actin is one of the most abundant proteins in eukaryotic organisms. Its structure is highly conserved across the barriers of biological species. “Actin is an essential protein that is indispensable for organisms.” Although motor proteins like myosin have not been found in prokaryotic organisms, such organisms host many proteins that are similar to actin. “It is thought that the mechanisms of biological motion based on polymerization and depolymerization, like that in F-actin, emerged early in the history of organisms, representing a prototype for the mechanism by which organisms are in continual movement.”

Clarifying the fine structure of F-actin

A wide variety of proteins have been found to bind to F-actin to control the polymerization and depolymerization of G-actin. Some disorders in this control can cause diseases such as cancer metastases. “Currently, amazing research is being done that aims to identify the various functions of cells and the causes of diseases by examining, one by one, the modes of functioning of proteins that bind to F-actin to control G-actin polymerization and depolymerization. I adopted a reverse approach. Although many proteins are involved in the control of F-actin, only one kind of F-actin is subject to control. F-actin must therefore have a mechanism for accepting that control. If this mechanism can be clarified, the common mechanism behind the control of F-actin may become clear. With this idea as my starting point, I began to analyze the fine structure of F-actin in 1997.”

In 1990, Kenneth Holmes and colleagues at the Max Planck Institute for Medical Research in Germany clarified the structure of G-actin, the globular actin subunit, with an atomic resolution of 2.8 angstroms. “F-actin was known to be comprised of two strands of G-actin molecules in sequence convolving around each other. The group in Germany delineated the structure of F-actin by arranging units of the elucidated structure of G-actin in a spiral. The structure was generally sufficient to explain some experimental data, so many people believed the Holmes model to be satisfactory to explain the structure of F-actin. However, the structure did not suffice to explain the essential function of F-actin that enabled it to act as a motor.”

The mechanism of actin control starts with the binding of actin to adenosine triphosphate (ATP), an energy-supplying compound. Multiple G-actins then polymerize together to form long filaments (F-actin). In the filaments, ATP reacts with water and undergoes hydrolysis, causing one phosphoric acid molecule to split off and leaving adenosine diphosphate (ADP). With the energy supplied by the hydrolysis of ATP, the G-actin bound to the ADP depolymerizes from the F-actin. In this way, F-actin acts as a motor. However, the structure proposed by the Holmes model is unable to explain how ATP is hydrolyzed in this process.

“Before solving this problem, it was necessary to clarify the fine structure of F-actin.” Fine structures can be determined at the atomic level using X-ray crystallography if well-formed crystals can be obtained. “As the length of F-actin is variable, however, it is not possible to obtain a suitable, well-formed crystal unless the lengths of the filaments can be made uniform. Although investigations that make use of genetic engineering and other techniques have been conducted to obtain a uniform length of F-actin, none have been successful. Instead, I decided to use X-ray fiber diffraction to analyze the structure of F-actin.”

X-ray fiber diffraction is a technique used to analyze the structures of thin, fibrous substances that are difficult to crystallize. In 1952, UK scientist Rosalind Franklin took X-ray diffraction images of DNA by X-ray fiber diffraction. In 1953, with those images as an important guide, James Watson from the USA and Francis Crick from the UK clarified the double helical structure of DNA, for which they were awarded the Nobel Prize for Physiology or Medicine in 1962. “Among Franklin’s followers was Prof. Dr. Holmes, who clarified the structure of G-actin.”

In X-ray fiber diffraction, molecules of the fibrous substance are first arranged in a specified orientation, and then a liquid crystal phase is produced. The liquid crystal is irradiated with an X-ray beam, and the diffraction patterns are measured to derive the structure. To achieve the right orientation, Oda and his colleagues increased the F-actin concentration and exerted a large magnetic field of 18 tesla. They measured the diffraction patterns produced by exposure to an intense X-ray beam using SPring-8, the large synchrotron radiation facility at RIKEN’s Harima Institute. “However, we could not completely align all of the actin filaments in a particular orientation, so we were unable to derive the fine structure of the F-actin directly from the diffraction patterns obtained from our samples.”

Eventually, however, through repeated trial and error, Oda successfully developed a method for resolving the structure by incrementally modifying a computer model developed with reference to the F-actin structure proposed by Holmes and other data. Diffraction patterns were calculated for the model structure, and the calculated patterns were then compared with those obtained by measurements using SPring-8 by Oda and his colleagues. This process was repeated until the calculated diffraction pattern matched the measured diffraction image. The fine structure of F-actin was thus derived. “In recent years, protein chemistry and computation have made dramatic advances, enabling us to make relatively easy calculations of how proteins are able to deform. This is why we could employ the technique.”

Finally, in January 2009, Oda succeeded in clarifying the structure of F-actin with a resolution of just 3.3 Å (5.6 Å in the radial direction).

The mechanism of ATP hydrolysis revealed

Resolving the fine structure of F-actin revealed important insights. G-actin consists of two domains, which are twisted when the G-actin is isolated. When constructed into F-actin, however, the G-actin untwists and assumes a flat comformation. “ATP is bound to G-actin in a groove between the two domains. G-actin has been structurally analyzed with a high resolution of 1.35 Å, so the position of the water molecule around the ATP is already known. From the fine structure we clarified, it was found that when G-actin untwists into the flat comformation, deformation occurs around the amino acid (glutamine 137) that anchors the water molecule that hydrolyzes the ATP. The resolution of our analytical system, 3.3 Å, was not sufficient to clarify the position of the water molecule, but we assume that the water molecule approaches the ATP as a result of the deformation, and that the ATP-bound domain rotates to allow the ATP to approach the water molecule, causing the ATP and water molecule to react leading to hydrolysis.” Clarification of the fine structure of F-actin made it possible to visualize the mechanism of ATP hydrolysis for the first time.

Elucidating the function of F-actin

It was also found that, with the transition to a flat comformation, actin molecules bind together at their inequalities to assume a structure that is likely to produce a filament. “The structure we clarified was nearly in the center of the actin filament. When another protein binds to one end of the actin, the flat comformation of the actin may further change so as to make the bonds of the actin molecules readily breakable and promote their depolymerization. I think the actin filament cannot function unless the actin in the filament assumes some structure other than a flat comformation. I want to try to determine what structures the actin filament and actin can assume.”

Oda is working on structural analysis of the actin filament in a state in which it is bound by other proteins. This research aims at elucidating the mechanism behind the functioning of the actin filament and the common mechanism by which various proteins control the actin filament.

Aiming at an atomic level resolution―the potential of XFEL

Another problem that needs to be solved is how to further increase the resolution beyond 3.3 Å. “Usually, the arrangement of atoms can only be determined accurately with a resolution higher than 3.0 Å. With our current level of resolution, we cannot clarify how the deformation occurs around the amino acid that anchors the water molecule in the groove.” One promising tool that could be used to achieve structural analysis at higher resolution is the X-ray Free Electron Laser (XFEL) facility being built at the RIKEN Harima Institute. The XFEL, due for completion in fiscal 2010, is an X-ray laser capable of producing a beam more than a billion times brighter that that generated by the SPring-8 synchrotron. “The major reason for our inability to increase the resolution is that F-actin cannot be aligned completely in a particular orientation. The XFEL will potentially allow us to derive the structure at the atomic level from measurements obtained by applying an intense X-ray laser beam to a single F-actin.”

There are also high expectations for clarifying the structure of the actin filament at the atomic level in the context of muscle research. “The structure of the motor protein myosin was clarified at the atomic level in 1993, and research into muscle contraction progressed dramatically thereafter. However, the entire mechanism for muscle contraction cannot be elucidated at the atomic level unless the structure of the actin filament, which acts as a rail, can be clarified at the atomic level.”

Exploring the true nature of organisms

“It took some ten years to clarify the fine structure of F-actin. If I had known that it would take so long, I would probably not have embarked on this research,” says Oda with a smile. “I was prepared to fight a running battle, and I also realized that my research theme would not allow me to write lots of papers one after another. I have to thank Prof. Dr. Yuichiro Maéda of Nagoya University, former chief scientist in the Structural Biochemistry Laboratory at RIKEN, for affording me the opportunity to continue my research. Studying under Professor Emeritus Fumio Oosawa, Prof. Dr. Maéda used to say us, ‘Let’s do research that leads to clarifying the true nature of our subject.’ He himself succeeded in determining the structure of troponin, a protein that serves as a muscle contraction switch, after more than ten years of painstaking work. It is my responsibility to push forward the structural analysis of F-actin. Starting with its structure, I want to clarify the true nature of organisms through the mechanism that allows them to maintain their constant movement.”

About the Researcher

Toshiro Oda

Toshiro Oda, born in 1964, received his BS and MS degrees from the Department of Physics, Nagoya University, in 1988 and 1990, respectively. He earned his PhD in the same department in 1995. He then joined the International Institute for Advanced Research of Matsushita Electric Industrial Co. in Japan as a research associated, and in 1999 moved to the RIKEN Harima Institute and the SPring-8 Center. Between 2000 and 2002, he worked at the Max-Planck Institute for Medical Research in Heidelberg, Germany as a guest researcher. In 2004, Oda became a senior scientist at the Harima Institute, and until 2008 was group leader of the ERATO Actin Filament Dynamics Project. He is currently team leader of the X-ray Structural Analysis Research Team at the SPring-8 Center.

Toshiro Oda
Team Leader
X-ray Structural Analysis Research Team
Structural Physiology Research Group
RIKEN SPring-8 Center