Ubiquitination is a versatile signalling mechanism in eukaryotes, which regulates critical aspects of cellular metabolism, like cell cycle progression, DNA replication and damage repair, chromatin remodelling, transcription, protein aggregation and quality control, vesicular trafficking and circadian clock regulation. Ubiquitination itself is a tightly regulated process and the biochemical pathways regulating its specificity and signalling outcomes present promising drug targets for many human pathologies including cancer, neurodegeneration, cardio-vascular diseases and infection.
Cullin-RING E3 ligases (CRLs) comprise nearly half of all cellular ubiquitin ligases and account for nearly 20% of all cellular ubiquitination. All CRLs are regulated by only a few factors which collectively reshape the cellular CRL pool and enable various rapid adaptive cellular responses to internal and external cues.
Despite a wealth of biochemical and cellular data, many mechanistic questions remain open due to the scarcity of structural information of functionally important states. For instance, we don’t know how a specific CRL is assembled following a signalling event, how it subsequently initiates ubiquitination, and then extends a ubiquitin chain, and we have only very limited insights into how it is eventually inactivated. The underlying biochemical events are either too short-lived and/or mediated by very low affinity interactions to be easily tractable by conventional structural determination techniques.
My group has developed a method that largely overcomes these limitations by allowing the direct observation of biochemical processes at atomic spatial- and millisecond time-resolution by combining microfluidics and cryo-electron microscopy (cryo-EM). We utilize microfluidics devices to mix and incubate biochemical samples on sub-second timescales and then rapidly spray-plunge-freeze the reactants for subsequent three-dimensional structure determination by cryo-EM. Iterating the procedure at increasing incubation times after sample mixing, allows the visualization of a biochemical binding and/or enzymatic reaction as a time-lapse “movie”. In practice this obliterates the need to utilise mutants or crosslinking probes to stabilize catalytic intermediates. Moreover, even very transient or low-affinity interactions are in principle tractable, as long as samples are mixed and frozen faster than the relevant off-rates.
The aim of this PhD project is to apply this novel method to study functionally relevant states of CRLs and thus obtain directly correlated kinetic and structural information. Most of the required reagents are already available in the lab and proof of concept experiments have been performed successfully. We will complement all findings by orthogonal techniques, including well-established biochemical methods as well as advanced fluorescence-based kinetic assays. Ultimately, we will validate the key findings in relevant in vivo systems.
The time-resolved cryo-EM method has broad applications and a second research focus of our group is applying it to elucidate the structural basis of DNA double strand break repair by homologous recombination.
Since only one studentship is available, the above project serves as an example and the exact topic of the PhD project can be determined according to the best match with the candidate’s interests. Thus, outstanding candidates from different scientific backgrounds are strongly encouraged to apply.
1. Lydeard, J. R., Schulman, B. A. and Harper, J. W. (2013)
Building and remodelling Cullin-RING E3 ubiquitin ligases.
EMBO Reports 14: 1050-1061. PubMed abstract
2. Mosadeghi, R., Reichermeier, K. M., Winkler, M., Schreiber, A., Reitsma, J. M., Zhang, Y., Stengel, F., Cao, J., Kim, M., Sweredoski, M. J., Hess, S., Leitner, A., Aebersold, R., Peter, M., Deshaies, R. J. and Enchev, R. I. (2016)
Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle.
eLife 5: e12102. PubMed abstract
3. Scott, D. C. and Schulman, B. A. (2018)
SCF E3 ligase substrates switch from CAN-D to Can-ubiquitylate.
Molecular Cell 69: 721-723. PubMed abstract
4. Scott, D. C., Rhee, D. Y., Duda, D. M., Kelsall, I. R., Olszewski, J. L., Paulo, J. A., de Jong, A., Ovaa, H., Alpi, A. F., Harper, J. W. and Schulman, B. A. (2016)
Two distinct types of E3 ligases work in unison to regulate substrate ubiquitylation.
Cell 166: 1198-1214 PubMed abstract
5. Chen, B. and Frank, J. (2016)
Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule.
Microscopy 65: 69-79. PubMed abstract