Biology subjects

Tolbert, B. S.

Publications and source records attributed to Tolbert, B. S..

5 recordsLinked to original sources

bioRxiv2023

Thermodynamic Coupling of the tandem RRM domains of hnRNP A1 underlie its Pleiotropic RNA Binding Functions

The functional properties of RNA-binding proteins (RBPs) require allosteric regulation through inter-domain communication. Despite the foundational importance of allostery to biological regulation, almost no studies have been conducted to describe the biophysical nature by which inter-domain communication manifests in RBPs. Here, we show through high-pressure studies with hnRNP A1 that inter-domain communication is vital for the unique stability of its N- terminal domain containing a tandem of RNA Recognition Motifs (RRMs). Despite high sequence similarity and nearly identical tertiary structures, the two RRMs exhibit drastically different stability under pressure. RRM2 unfolds completely under high-pressure as an individual domain, but when appended to RRM1, it remains stable. Variants in which inter-domain communication is disrupted between the tandem RRMs show a large decrease in stability under pressure. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered C-terminus to engage in protein-protein interactions and more importantly, they also influenced the RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

biochemistry↗

bioRxiv2023

Enterovirus Evolution Reveals the Mechanism of an RNA-Targeted Antiviral and Determinants of Viral Replication

Selective pressures on positive-strand RNA viruses provide opportunities to establish target site specificity and mechanisms of action of antivirals. Here, Enterovirus-A71 revertant viruses with resistant mutations in the SLII IRES domain (SLIIresist) were selected at low doses of the antiviral DMA-135. The EV-A71 revertant viruses were resistant to DMA-135 at concentrations that robustly inhibit replication of wild-type virus. EV-A71 IRES structures harboring the suppressor mutations induced efficient expression of reporter Luciferase mRNA in the presence of non-cytotoxic doses of DMA-135 whereas DMA-135 dose-dependently inhibited Luciferase expression from the wild-type IRES element. NMR studies indicate that the resistant mutations change the structure of SLII at the bulge loop binding site of DMA-135 and at part of an extended surface recognized by host RNA-binding protein AUF1. Comparisons of biophysical analysis of complexes formed between AUF1, DMA-135, or either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. Further studies demonstrate that the hnRNP A1 protein retains binding affinity for SLIIresist, illustrating that DMA-135 inhibition and viral resistance do not perturb the SLII-hnRNP A1 arm of the regulatory axis. Taken together, this work demonstrates how viral evolution under selective pressures of small molecules can elucidate RNA binding site specificity, mechanisms of action, and provide additional insights into the viral pathways inhibited by the antiviral DMA-135.

molecular biology↗

bioRxiv2022

R-BIND 2.0: An Updated Database of Bioactive RNA-Targeting Small Molecules and Associated RNA Secondary Structures

Discoveries of RNA roles in cellular physiology and pathology are raising the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an "RNA-privileged" chemical space. Biennial updates of the database and the establishment of a website platform (rbind.chem.duke.edu) have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA Structure Search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different to that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.

biochemistry↗

bioRxiv2020

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.

microbiology↗

bioRxiv2020

Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

We herein report an RNA-targeting antiviral small molecule that reduces replication of the human enterovirus 71 (EV71) via stabilization of an inhibitory small molecule-RNA-protein ternary complex. The EV71 virus poses serious threats to human health, particularly in regions of Southeast Asia, and no FDA approved drugs or vaccines are available. We first screened an RNA-biased small molecule library using a peptide-displacement assay to identify ligands for the stem loop II structure of the EV71 internal ribosomal entry site, which was previously shown to impact viral translation and replication. One ligand, DMA-135, decreased viral translation and replication in cell-based studies in a dose-dependent manner with no significant toxicity. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein that then represses translation. This mechanism was further supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.

biochemistry↗