Publication highlights

This work reveals that amino acids, rather than fructose 1,6-bisphosphate, are the relevant cellular regulators of pyruvate kinase M2 (PKM2), a critical node in cancer metabolism. It further elucidates the molecular mechanism of PKM2 regulation by amino acids with a new algorithm that predicts allosteric pathways in proteins, a major and difficult problem in structural biology.

This study was the first to demonstrate that RAD51 paralogues bind to and structurally remodel the pre-synaptic RAD-51-ssDNA filament to a stabilised, “open”, and flexible conformation, which facilitates strand exchange with the template duplex. We showed that RAD51 paralogues act by binding the end of the presynaptic filament, which induces a conformational change that stabilises RAD-51 bound to ssDNA and primes the filament for strand exchange. These observations established for the first time the underlying mechanism of HR stimulation by Rad51 paralogues and revealed a new paradigm for the action of HR mediator proteins.

Evidence suggested that the telomere adopts a lasso-like t-loop configuration, which safeguards chromosome ends from being recognised as DNA double strand breaks. However, the regulation and physiological importance of t-loops in end-protection was uncertain. This study uncovered a phospho-switch in TRF2 that coordinates the timely assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response. These results were the first to definitively establish the t-loop as a physiologically important structure required to suppress checkpoint activation at telomere ends.

Despite evolutionarily conservation of molecular mechanisms, the speed of development varies substantially between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme of motor neuron differentiation runs twice as fast in mouse as in human. We provide evidence that a two-fold increase in protein stability and cell cycle duration in human cells compared to mouse can account for the slower pace of human development, indicating that global differences in kinetic parameters play a major role in interspecies differences in developmental tempo. This study establishes a new experimental system in which to address fundamental questions.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC-MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC-MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC-MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

D-cycloserine is an antibiotic used for decades to treat drug resistant tuberculosis. Its inhibition mechanism came into question when in a previous paper we determined alanine racemase activity in “fully inhibited” cells. This study demonstrated a previously unknown path during the assumed irreversible inhibition of alanine racemase that leads to the destruction of the antibiotic, meaning that alanine racemase is not irreversibly inhibited by the drug. The paper highlights the complexity of studying the chemical mechanisms of inhibition of enzymes and points to a novel strategy to design D-cycloserine analogues with improved properties.

Here, we described a cryo-EM structure of a retroviral intasome in a functional complex with a nucleosome. The structure revealed a multivalent interface of the viral integration machinery and chromatin, involving both gyres of nucleosomal DNA and histones. Whilst the histone octamer remains intact, the DNA is lifted from its surface to allow for strand transfer at highly preferred integration sites. These data provided a unique snapshot of an enzyme recognizing and acting upon nucleosomal DNA. The structure was the first to illustrate nucleosome flexibility facilitating a biological process and, as such, had far-reaching implications for chromosome biology.

HIV integrase inhibitors represent some of the most impactful antimicrobial inhibitors. The second-generation drugs display improved barriers to the emergence of resistance, which spearheaded their worldwide rollout. Yet not even the most advanced compounds are immune to viral resistance. Our results explained the mechanism of viral resistance associated with the most common drug resistance mutations. Furthermore, we established the key difference between the first and second-generation strand transfer inhibitors, which will inform further development of this drug class.

Oesophageal adenocarcinoma shows high genetic heterogeneity making the identification of cancer drivers challenging. We developed a machine learning algorithm to identify cancer drivers in 261 oesophageal adenocarcinomas. Although most predicted drivers were rare or patient-specific, they all perturbed well-known cancer pathways. Using the recurrence of the same pathway perturbations rather than individual genes, we stratified patients into six groups different for their clinical features. We validated experimentally the contribution of these genes to disease progression and revealed acquired dependencies exploitable in therapy. This study described a new way to identify cancer drivers that we have recently further developed for application in precision oncology.

The MCM replicative helicase is loaded onto duplex DNA as a double hexamer. Here we use time-resolved cryo-EM to show that ORC binds to its high affinity binding site to load the first MCM hexamer. ORC then releases this site and it, or another ORC molecule then binds the B2 element, which contains a degenerate ORC binding site. This binding is stabilised by a novel interaction between the Orc6 subunit of ORC and the N-terminus of the MCM hexamer. ORC then recruits and loads the second hexamer by the same mechanism as the first hexamer. We employed newly developed in silico reconstitution approaches to describe the full context of the helicase loading reaction, studied on a near-native, chromatinised origin of replication. This study radically changes our approach to investigating chromosome replication with cryo-EM.

This paper decribes how we were able to successfully repurpose the Crick to increase the capacity for Sars-CoV-2 testing in unpredented times.

This important paper showed very high levels of infection amongst healthcare workers in a local hospital. It has influenced government policy – asymptomatic healthcare workers are to be screened as per our recommendation (announced October 12th).

Metastatic melanoma is a lethal disease, in part because of rapid acquisition of resistance to therapy. Using genetically engineered mouse models, we demonstrate that the activating RAC1 P29S mutation, present in up to 5% of melanoma patients, cooperates with BRAF as a driver of melanoma initiation and promotes BRAF inhibitor resistance. The critical RAC1 effector pathway in melanoma is shown to be the transcription factor complex SRF/MRTF, which initiates a switch to a mesenchymal-like state characterized by therapy resistance. Therapeutic targeting of SRF/MRTF may have potential to reverse BRAF inhibitor resistance in melanoma patients bearing the oncogenic RAC1 P29S mutation

KRAS is the most commonly mutated oncogene in human lung cancer, but direct targeting of RAS proteins has proved difficult. A recently developed inhibitor of G12C mutant KRAS protein inhibits lung cancer progression in mouse models but does not provide durable regressions. By studying signalling pathways required for survival of KRAS mutant cells, we demonstrate a strong and selective potentiation of the effects of G12C KRAS inhibitors when mTOR and/or IGF1R are also inhibited. Using mutant specific G12C KRAS inhibitors rather than MEK inhibitors in these combinations is associated with greater specificity and lower toxicity. We propose that adding IGF1R and mTOR inhibitors will increase the impact of G12C KRAS inhibitors in clinical trials.

Cryo-EM has the potential to study any native conformation of a macromolecule. However, the sample preparation time is high, compared to the timescale of most protein interactions and conformational changes. In this paper, we established a robust method of time-resolved cryo-EM sample preparation. We produced high-quality samples for microscopy while speeding up the process of making them by several orders of magnitude. This allowed samples to be collected within 30ms of the initiation of a biochemical reaction, within the timeframe of many critically important and interesting processes. This enables a whole new class of experiments in structural biology research.