Biology subjects

Perica, T.

Publications and source records attributed to Perica, T..

4 recordsLinked to original sources

bioRxiv2025

Mechanism of MEK1 phosphorylation by the N-terminal acidic motif mediated asymmetric BRAF dimer

The RAF/MEK/ERK signaling cascade regulates cell proliferation and differentiation and is frequently dysregulated in cancer. Approximately 90% of RAF-mutant cancers harbour mutations in B-type of Rapidly Accelerated Fibrosarcoma (BRAF). Its proto-oncogenicity has been attributed to a four-residue N-terminal acidic (NtA) motif. While a long-standing model proposes that the NtA promotes an activating asymmetric RAF dimerization, this idea has lacked structural support. Here, we present the first X-ray crystal structure of an NtA-mediated asymmetric BRAF dimer bound to its substrate Mitogen-activated Protein kinase Kinase (MEK1). Further cellular and biochemical analyses reveal that this asymmetric dimer is not an intermediate along the BRAF activation pathway, but rather a catalytically active state of the kinase. We validate this with the structure of BRAF bound to Ser222-phosphorylated MEK1, capturing the complex in a post-catalytic state. This combination of structural and cellular analysis establishes the relevance of NtA-driven asymmetry in BRAF activation and resolves a long-standing disconnect between RAF cancer genetics and structural biology.

biochemistry↗

bioRxiv2022

Complete mutational mapping of a GTPase switch in vivo reveals novel allosteric regulation

Allosteric regulation is central to protein function in cellular networks1. However, despite technological advances2,3 most studies of allosteric effects on function are conducted in heterologous environments2,4,5, limiting the discovery of allosteric mechanisms that rely on endogenous binding partners or posttranslational modifications to modulate activity. Here we report an approach that enables probing of new sites of allosteric regulation at residue-level resolution in essential eukaryotic proteins in their native biological context by comprehensive mutational scanning. We apply our approach to the central GTPase Gsp1/Ran. GTPases are highly regulated molecular switches that control signaling, with switching occurring via catalyzed GTP hydrolysis and nucleotide exchange. We find that 28% of 4,315 assayed mutations in Gsp1/Ran are highly deleterious, showing a toxic response identified by our assay as gain-of-function (GOF). Remarkably, a third of all positions enriched for GOF mutations (20/60) are outside the GTPase active site. Kinetic analysis shows that these distal sites are allosterically coupled to the active site, including a novel cluster of sites that alter the nucleotide preference of Gsp1 from GDP to GTP. We describe multiple distinct mechanisms by which allosteric mutations alter Gsp1/Ran cellular function by modulating GTPase switching. Our systematic discovery of new regulatory sites provides a functional map relevant to other GTPases such as Ras that could be exploited for targeting and reprogramming critical biological processes.

biophysics↗

bioRxiv2020

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 disruption1,2. 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.

systems biology↗

bioRxiv2020

Biophysical basis of cellular multi-specificity encoded in a model molecular switch

Molecular switches are central to signal transduction in protein interaction networks. One switch protein can independently regulate distinct cellular processes, but the molecular mechanisms enabling this functional multi-specificity remain unclear. Here we integrate system-scale cellular and biophysical measurements to study how a paradigm switch, the small GTPase Ran/Gsp1, achieves its functional multi-specificity. We make 55 targeted point mutations to individual interactions of Ran/Gsp1 and show through quantitative, systematic genetic and physical interaction mapping that Ran/Gsp1 interface perturbations have widespread cellular consequences that cluster by biological processes but, unexpectedly, not by the targeted interactions. Instead, the cellular consequences of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle, and cycle kinetics are allosterically tuned by distal interface mutations. We propose that the functional multi-specificity of Ran/Gsp1 is encoded by a differential sensitivity of biological processes to different kinetic parameters of the Gsp1 switch cycle, and that Gsp1 partners binding to the sites of distal mutations act as allosteric regulators of the switch. Similar mechanisms may underlie biological regulation by other GTPases and biological switches. Finally, our integrative platform to determine the quantitative consequences of cellular perturbations may help explain the effects of disease mutations targeting central switches.

systems biology↗