Comparison of the active sites of (a) HDV ribozyme and (b) HH ribozyme. HDVr and HHr active states are thought to use Mg2+ binding modes to enable different catalytic strategies (general base “γ” catalysis in HDVr and general acid “δ” catalysis in HHr). The range of transition states observed thus far for non-enzymatic reactions, RNase A and HDVr are beginning to revel how catalytic modes direct RNA 2′-O-transesterification reactions along distinct mechanistic pathways. From Weismann et al. JACS 2023

  • Comparative analysis of transition state stabilization by ribonucleases and ribozymes.

We are using precise atomic level modifications in collaboration with the York Lab and Piccirilli Lab to determine how enzymes can redirect reaction pathways towards alternative transition states. We are continuing to adapt concepts and methods from biophysical chemistry that open up new and important phosphoryl transfer enzymes to investigation by isotope effects and allow us to address challenging questions in mechanistic enzymology.  Our recent studies reveal exciting differences in the transition state stabilized by ribozymes and protein ribonucleases, inspiring us to explore the mechanistic landscape of biological catalysis of RNA strand cleavage.

Molecular recognition of RNA is inherently complex due to the fact that both sequence and structure together dictate binding affinity. In addition, the RNA sequence ‘context’ surrounding functional binding sites can enhance or inhibit binding. HTS-Kin provides the foundational biochemical measures to construct quantitative and comprehensive models of specificity.  Testing and refining the predictive nature of such models is essential for applying them to understand biology and principles that direct specificity in vivo. Jankowsky and Harris Methods 2017.

  • Quantitative and comprehensive analysis of ribonuclease specificity. 

The specificity of RNA processing endonucleases is particularly challenging to understand because they typically have broad specificity, which enables them to target multiple alternative RNA substrates. To confront this challenge, we developed in collaboration with the Jankowsky Lab a general approach
(High Throughput Sequencing Kinetics -HTS-Kin) using bacterial ribonuclease P (RNase P) as a model system.  HTS-KIN measures rate distributions for RNase cleavage of randomized pools of alternative substrates. Our primary method for mining these data are hybrid models that combine position weight matrices (PWM) that score contributions of individual nucleotides, and interaction coefficients (IC) that express interactions between nucleotides. Current aims are to extend HTS-KIN and PWM+IC modeling to biomedically important RNases and adapt our approach to analyze in vivo specificity.
  • Active site of E. coli RNase P with ptRNA bound. The 5′ leader of ptRNA is positioned by stacking interactions allowing variable sequences to engage catalytic interactions. Distal leader nucleotides form H-bonds with conserved P RNA nucleotides and electrostatic interactions with rnpA protein. Zhu et al Nat Comm. 2023.

  • Structure and inhibition of RNase P enzymes from pathogenic bacteria

In collaboration with the Taylor Lab we are using insights from specificity modeling together with cryoEM to understand at an atomic level the structures RNase P enzymes from pathogens. Our motivation is to leverage the detailed structural, kinetic, and biochemical understanding of these enzymes as a basis for discovery of small molecule inhibitors.  Recent success in determining the structure of the E. coli RNase P-ptRNA complex revealed a dynamic holoenzyme and substrate induced conformational changes involved in binding as well as potential target sites.  Now, we are determining the structures of RNase P enzymes from ESKAPE pathogens and in collaboration with the Pyle Lab applying HTS to discover small molecule inhibitors as potential antimicrobial agents.

Active site of coronavirus Nsp15 (PDB:7N33).  Quantitative analysis of Nsp15 kinetics supports a metal ion independent acid/base mechanism. Although well established that Nsp15 cleaves RNA 3′ to U, we lack a complete understanding of its sequence and structure specificity necessary to understand its biological function. Huang et al JBC 2023.

  • Specificity and catalysis by coronavirus Nsp15 endonuclease.

Understanding the functional properties of SARS-CoV-2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics have not been fully defined.  We are using the tools of mechanistic enzymology to define the catalytic mechanism of Nsp15, and employing deep mutational scanning to comprehensively define its substrate specificity.  In the long terms we aim to identify in vivo substrates of Nsp15 and understand its roles in viral pathogenesis.

Human RR is activated by ATP and inactivated by dATP. Inactivation is linked to hexamer formation, while the mechanism of ATP activation is less well understood. Trapping inactive conformations presents several viable strategies for designing small molecule inhibitors as potential chemotherapeutics. Huff et al. Biomolecules 2022

  • Allosteric regulation and inhibition of ribonucleotide reductase. 

Ribonucleotide reductase (RR) is an essential and ubiquitous enzyme that converts ribonucleotide diphosphates to 2’-deoxynucleotide triphosphates, and serves as a key control point for maintaining balanced pools of dNTPs during DNA synthesis. Inhibition of RR is chemotherapeutic and several drugs currently used to treat multiple types of cancer are inhibitors of human RR.  In collaboration with the Roitberg Lab we are combining molecular dynamics, mutagenesis, and enzyme kinetics to identify allosteric networks involved in human RR regulation.  Together with the Viswanathan Lab we are using the insights derived from fundamental studies of human RR allostery to identify new classes of small molecule inhibitors as novel anticancer agents.