Watching molecular machines in action
Recently, single-particle cryo-EM has blossomed to become one of the main methods of structural biology. While the technique could simply visualise low resolution “blobs” a few years ago, it is now capable of yielding high-resolution structures in a routine fashion.
To achieve this, the molecules are shock-frozen in a thin layer of ice and imaged in a transmission electron microscope (TEM), resulting in 2D projections of individual molecules. Computational analysis of several hundred thousand of those images leads to a 3D structure. While such structures can give detailed insights into the function of a given macromolecular complex, there could be more information to find in the data.
Different to the other main structural techniques, the signals from each individual molecule can easily be distinguished in the TEM images. With these signals, it is possible to apply sophisticated sorting techniques to calculate an ensemble of different conformations which the molecular machine adopts at once. This enables us to create movies of macromolecular machines in action. In addition, we can use statistical thermodynamics approaches to extract the thermodynamic and kinetic properties of the system.
However, the underlying experimental and computational approaches are still sparse and incomplete. Their completion is one of the main efforts of the Haselbach lab. We use single molecule techniques to complement sparse kinetic data extracted through cryo-EM.
Despite their intricate design, molecular machines can fail. Using the described biophysical methods, we are trying to understand the molecular response of molecular machines on stress and disease. In our initial study, we focused on the molecular adaption of the human 26S proteasome towards chemical inhibitors. Proteasomal degradation is a multi-step process involving substrate recognition, de-ubiquitination and unfolding carried out by the regulatory particle and proteolysis performed by the core structure. Even though it has been well documented that the proteolytic activity of the core is shut down by using covalent inhibitors, the effect on the holoenzyme was unknown.
We found a major allosteric effect that translates the binding of a single inhibitor to the proteolytic site to a restriction of the conformational space of the regulatory particle that is more than 15 nanometres away. We hypothesise that this can be sensed by different rescue factors that bind to the proteasome, resulting in a release of the applied stress.
Although the use of chemical inhibitors is clinically relevant, it is an artificial set-up. Our future aim is to understand natural causes of malfunctioning macromolecular machines and the processes involved in the repair of malfunctioning machines. We continue to focus on the 26S proteasome.
Here especially, the unfolding and translocation of the substrate into the proteolytic chamber is prone to error. In extreme cases, the entrance to the 20S proteasome is clogged by a substrate and degradation can no longer take place. A few prominent examples of proteasome stalling are the surface antigen of the Eppstein-Barr virus or the amyloid-forming proteins huntingtin or α-synuclein, which might directly clog the proteasome. Also, more general physiological situations such as oxidative stress, heat stress or arsenide intoxication can lead to a failure of the proteasome.
Due to its involvement in many processes, failure of the proteasome can lead to fatal consequences for the cell or even the organism. There are several rescue mechanisms known. In most cases, external protein factors bind to the periphery of the complex and change the degradation rate. However, in all cases is poorly understood how this is achieved mechanistically. Thus, we aim to use our biophysical method set to find the mechanisms that rescue stalled molecular machines.
- Haselbach D, Komarov I, Agafonov DE, Hartmuth K, Graf B, Dybkov O, Urlaub H, Kastner B, Luehrmann R, Stark H (2018). Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex.
Cell 172, 1–11, doi: 10.1016/j.cell.2018.01.010
- Haselbach D, Schrader J, Lambrecht F, Henneberg F, Chari A, Stark H (2017). Long-range allosteric regulation of the human 26S proteasome by 20S proteasome-targeting cancer drugs. Nat Commun; 8:15578. doi: 10.1038/ncomms15578.
- Bertram K, Agafonov DE, Dybkov O, Haselbach D, Leelaram MN, Will CL, Urlaub H, Kastner B, Lührmann R, Stark H (2017). Cryo-EM Structure of a Pre-catalytic Human Spliceosome Primed for Activation. Cell; 170(4):701-713.e11. doi: 10.1016/j.cell.2017.07.011.
- Chari A, Haselbach D, Kirves JM, Ohmer J, Paknia E, Fischer N, Ganichkin O, Möller V, Frye JJ, Petzold G, Jarvis M, Tietzel M, Grimm C, Peters JM, Schulman BA, Tittmann K, Markl J, Fischer U, Stark H. ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.
- Monecke T, Haselbach D, Voß B, Russek A, Neumann P, Thomson E, Hurt E, Zachariae U, Stark H, Grubmüller H, Dickmanns A, Ficner R (2013). Structural basis for cooperativity of CRM1 export complex formation. Proc Natl Acad Sci U S A;110(3):960-5. doi: 10.1073/pnas.1215214110. Epub 2012 Dec 31.