A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

Abstract

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5′ tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5′ leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

Protein embeddings and deep learning predict binding residues for various ligand classes

“…We observed similar results for the ribonuclease P protein component (UniProt ID: Q9X1H4): Using the PDB structure 6MAX 29,33 with a resolution of 1.42Å, this protein is annotated to have seven residues binding to a small molecule while bindEmbed21DL did not predict any of those with a high probability above 0.95. In fact, the available functional annotations clearly suggest this protein to be binding to nucleic acids and the small molecule bound according to the PDB structure 6MAX seems to mainly serve as inhibitor for RNA-binding 33 . Four residues were also predicted to bind to nucleic acids above a probability of 0.95 (Fig.…”

“…Overall, 10 of 13 (77%) residues annotated as DNA-binding in 5T5K were also predicted by bindEmbed21DL (shown in lighter red; blue residues indicate experimental annotations which were not predicted). B: For the ribonuclease P protein component (UniProt ID: Q9X1H4), four residues were predicted with a probability ≥0.95 (indicated in dark red), none of these matched the annotations in the PDB structure 6MAX 29,33 . However, those four residues were considered as binding according to the two low-resolution structures 3Q1Q (3.8Å) 29,34 (visualized) and 3Q1R (4.21Å) 29,34 .…”

“…We observed similar results for the ribonuclease P protein component (UniProt ID: Q9X1H4): the PDB structure 6MAX 32,37 (resolution 1.42Å) annotated this protein with seven residues binding to a small molecule; none of the 7 were predicted at p≥0.95. Indeed, the available functional annotations clearly suggest nucleic acid-binding; the small molecule bound in 6MAX seems to mainly inhibit RNA-binding 37 . Four residues were predicted to bind nucleic acids reliably (p≥0.95, Fig.…”

Dissecting Monomer-Dimer Equilibrium of an RNase P Protein Provides Insight Into the Synergistic Flexibility of 5’ Leader Pre-tRNA Recognition

“…To understand the mechanism of RNase P holoenzyme activation and ptRNA cleavage, it is essential to study the structure and binding interaction between RNase P protein and 5' ptRNA leader. We chose to work with RNase P from Thermotoga maritima because extensive structure, biochemical analyses, and small molecule screening studies have been performed on the RNase P holoenzyme (Paul et al, 2001;Buck et al, 2005a;Torres-Larios et al, 2005;Reiter et al, 2010;Reiter et al, 2012;Madrigal-Carrillo et al, 2019). We determined that the functionally relevant P protein monomer predominates at low (153 μM) concentrations, enabling NMR titrations to probe the binding interactions of the P protein and 5' leader RNA.…”

“…In addition, much less is known about how P protein flexibility contributes to 5' leader binding and catalytic activation. We chose to explore the solution structure, protein flexibility, and 5' leader binding properties of the RNase P protein from Thermotoga maritima because extensive structure, biochemical analyses, and small molecule screening studies have been performed on this system (Paul et al, 2001;Krivenko et al, 2002;Buck et al, 2005a;Torres-Larios et al, 2005;Reiter et al, 2010;Reiter et al, 2012;Madrigal-Carrillo et al, 2019). The high-resolution structure of the Thermotoga maritima RNase P protein, hence termed P protein, crystallized as a tetramer and its oligomeric state occluded the 5' leader binding through lattice contacts at the P protein's N-terminus (Kazantsev et al, 2003).…”