Nav: Home

Direct visualization of dynamic structures of protein disaggregation molecular machines

June 01, 2018

When proteins are exposed stresses like a heat shock, they lose their native structure and form toxic insoluble aggregations. Bacterial molecular chaperone ClpB and its yeast homologue Hsp104 have an ability to disentangle and reactivate the aggregated proteins. ClpB forms hexameric ring structure and its protomer consists of two ATPase cores, AAA1 and AAA2, and two additional domains, N-domain and M-domain. Using the chemical energy of ATP hydrolysis, ClpB ring threads the aggregated proteins through its central pore to disentangle them. The disaggregation activity of ClpB is regulated by the rod-shaped M-domains surrounding the periphery of ClpB ring. However, the molecular-level mechanisms, such as how ATP binding and hydrolysis change the ClpB structure and how the changes induce disaggregation, are not known. Although the three-dimensional structure of ClpB/Hsp104 has been determined by X-ray crystallography and cryo-electron microscopic single-particle analysis, information about the dynamics of individual molecules were required to understand the mechanism.

By using a high-speed atomic force microscopy (HS-AFM), Uchihashi and co-workers succeeded in observing structural changes of ClpB molecules with a 100-ms temporal resolution for the first time. In the presence of ATP, ClpB forms hexameric closed- and open-ring (Fig. 1). The number of subunits in the ring and the polymorphism of the ring structures were further confirmed by native mass spectrometry and negative-staining electron microscopy analysis, respectively. During the HS-AFM observation, these two conformations converted each other, and the higher the ATP concentration, the greater the population of the closed ring. In addition, closed rings were further classified into "round" whose height was almost uniform, "spiral" in which the height changed continuously like a spiral staircase, and "twisted-half-spiral" in which two half-spiral structures faced each other (Fig. 1). The twisted-half-spiral conformation suggested that the hexameric ring consisted of two trimers. This was also supported by sedimentation velocity analytical ultracentrifugation. These three conformations also converted each other, and it turned out that the higher the ATP concentration, the higher the frequency of the conversions. These observations revealed that the ATP binding induced the closed ring formation and its hydrolysis caused significant structural changes between the round, the spiral, and the twisted-half-spiral conformations.

From the observations of ClpB mutants that were inhibited ATP binding or hydrolysis on the AAA1 and/or the AAA2, individual roles of these two domains on the structural dynamics were clarified. ATP binding to the AAA1 induces oligomerization of ClpB, and the hexameric state was stabilized by ATP binding to the AAA2. The structural changes between the round, the spiral, and the twisted-half-spiral forms were caused by ATP hydrolysis at AAA2 (Fig. 2). Moreover, the mutual structural changes of the closed rings were drastically decreased in an M-domain mutant that lost disaggregation activity but retained ATPase activity suggesting that the structural changes played an important role in the disaggregation reaction.

Protein aggregation is closely related to various diseases including Alzheimer's disease. The formation of protein aggregation is also problematic in the use of proteins in medical and industrial fields. The results of this research have a potential to contribute to treatment of such diseases and/or maintenance of useful proteins. Furthermore, ClpB belongs to AAA+ protein family that contains various important proteins contributing to such as DNA replication, membrane fusion, protein degradation, and circadian clock maintenance. The members of this family shared AAA+ domains as ATPase cores, and the results of this research can be expected to lead to elucidation of the common mechanism of these AAA+ family proteins.
-end-


National Institutes of Natural Sciences

Related Proteins Articles:

Coupled proteins
Researchers from Heidelberg University and Sendai University in Japan used new biotechnological methods to study how human cells react to and further process external signals.
Understanding the power of honey through its proteins
Honey is a culinary staple that can be found in kitchens around the world.
How proteins become embedded in a cell membrane
Many proteins with important biological functions are embedded in a biomembrane in the cells of humans and other living organisms.
Finding the proteins that unpack DNA
A new method allows researchers to systematically identify specialized proteins called 'nuclesome displacing factors' that unpack DNA inside the nucleus of a cell, making the usually dense DNA more accessible for gene expression and other functions.
A brewer's tale of proteins and beer
The transformation of barley grains into beer is an old story, typically starring water, yeast and hops.
More Proteins News and Proteins Current Events

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Rethinking Anger
Anger is universal and complex: it can be quiet, festering, justified, vengeful, and destructive. This hour, TED speakers explore the many sides of anger, why we need it, and who's allowed to feel it. Guests include psychologists Ryan Martin and Russell Kolts, writer Soraya Chemaly, former talk radio host Lisa Fritsch, and business professor Dan Moshavi.
Now Playing: Science for the People

#537 Science Journalism, Hold the Hype
Everyone's seen a piece of science getting over-exaggerated in the media. Most people would be quick to blame journalists and big media for getting in wrong. In many cases, you'd be right. But there's other sources of hype in science journalism. and one of them can be found in the humble, and little-known press release. We're talking with Chris Chambers about doing science about science journalism, and where the hype creeps in. Related links: The association between exaggeration in health related science news and academic press releases: retrospective observational study Claims of causality in health news: a randomised trial This...