Biology subjects

Virta, J. M.

Publications and source records attributed to Virta, J. M..

5 recordsLinked to original sources

bioRxiv2025

Structural analysis of 23S rRNA methylating enzyme Cfr reveals RNA binding determinants for methylation regiospecificity and antibiotic resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPSA phenotype). Cfr catalyses methylation of the C8 position of the A2503 base in 23S rRNA; the recognition site of the above antibiotic classes. Along with RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfrs dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb the PhLOPSA resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in-cellulo 23S rRNA methylated species, and in-cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multi-drug resistance activity. In particular, we found that Cfrs Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure and inhibition of this important resistance determinant.

microbiology↗

bioRxiv2025

Modulation of the 14-3-3σ/C-RAF autoinhibited complexby molecular glues

Molecular glues, compounds that bind cooperatively at protein-protein interfaces are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional "undruggable" targets. Here, we focus on the native protein-protein interaction (PPI) of C-RAF, a key component of the MAPK signaling pathway, with the scaffolding protein 14-3-3. Although extensive drug discovery efforts have focused on the MAPK pathway, its central role in oncology and developmental disorders (RASopathies), still requires alternative approaches, moving beyond direct kinase inhibition. Indeed, stabilization of native PPIs is a relatively unexplored territory in this pathway. The function of C-RAF is regulated on multiple levels including dimerization, phosphorylation and complex formation with the hub protein 14-3-3. 14-3-3 prevents C-RAF activation by molecular recognition and binding at the phospho-serine 259. We used a fragment-merging approach to design a molecular glue scaffold that would bind to the composite surface of the 14-3-3/C-RAF "auto"inhibited complex. The synthesized molecular glues stabilized the 14-3-3/C-RAF complex up to 300-fold in biophysical assays; their glue-based mechanism of action was confirmed with several crystal structures of ternary complexes. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated with biophysical assays. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Validation in cell assays showed on-target engagement, enhanced phosphorylation levels of the C-RAF pS259 site, reduced RAF dimerization and reduced ERK phosphorylation. Overall, this approach enables chemical biology studies on a C-RAF site that is intrinsically disordered prior to 14-3-3 binding and has not been targeted previously. These molecular glues will be useful as chemical probes and starting points for further drug discovery efforts to elucidate the effect of native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.

biochemistry↗

bioRxiv2024

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3{sigma} client proteins CRAF, TAZ, and estrogen receptor (ER), which represent three specific binding modes. We have measured stabilization of 14-3-3{sigma}/client complexes by molecular glues with EC50 values between 100 nM and 1 M in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3{sigma} allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3{sigma} clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/573792v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f1531org.highwire.dtl.DTLVardef@a862cdorg.highwire.dtl.DTLVardef@ee8f6dorg.highwire.dtl.DTLVardef@a780e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A Systematic Approach to the Discovery of Protein-Protein Inter-action Stabilizers

Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3{sigma}. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ER, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3{sigma}/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3{sigma} provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

biochemistry↗

bioRxiv2020

Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

biochemistry↗