Emerging maps of allosteric regulation in cellular networks.

Allosteric regulation is classically defined as action at a distance, where a perturbation outside of a protein active site affects function. While this definition has motivated many studies of allosteric mechanisms at the level of protein structure, translating these insights to the allosteric regulation of entire cellular processes - and their crosstalk - has received less attention, despite the broad importance of allostery for cellular regulation foreseen by Jacob and Monod. Here, we revisit an evolutionary model for the widespread emergence of allosteric regulation in colocalized proteins, describe supporting evidence, and discuss emerging advances in mapping allostery in cellular networks that link precise and often allosteric perturbations at the molecular level to functional changes at the pathway and systems levels.

A complete allosteric map of a GTPase switch in its native cellular network.

Allosteric regulation is central to protein function in cellular networks. A fundamental open question is whether cellular regulation of allosteric proteins occurs only at a few defined positions or at many sites distributed throughout the structure. Here, we probe the regulation of GTPases-protein switches that control signaling through regulated conformational cycling-at residue-level resolution by deep mutagenesis in the native biological network. For the GTPase Gsp1/Ran, we find that 28% of the 4,315 assayed mutations show pronounced gain-of-function responses. Twenty of the sixty positions enriched for gain-of-function mutations are outside the canonical GTPase active site switch regions. Kinetic analysis shows that these distal sites are allosterically coupled to the active site. We conclude that the GTPase switch mechanism is broadly sensitive to cellular allosteric regulation. Our systematic discovery of new regulatory sites provides a functional map to interrogate and target GTPases controlling many essential biological processes.

Systems-level effects of allosteric perturbations to a model molecular switch.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.