Mechanistic enzymology of phosphoryl transfer enzymes including ribozymes
A fundamental principle of biology is that enzymes achieve catalysis by stabilizing reaction transition states. Knowledge of enzyme transition state structure provides deep insight into catalytic mechanism as well as providing design principles for potent inhibitors that are used as drugs to treat cancer, heart disease and combat infection. We are using kinetic isotope effect analyses combined with computational simulations to investigate the mechanisms of key phosphoryl transfer enzymes.
Zhang S, Gu H, Chen H, Strong E, Ollie EW, Kellerman D, Liang D, Miyagi M, Anderson VE, Piccirilli JA, York DM and Harris ME. (2016). Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+. Chem Comm. 52(24):4462-5.
Lee T-S, Radak BK, Harris ME and York DM. (2016). A two metal ion conformational switch pathway for HDV ribozyme activation. ACS Catalysis. 126, 1843-1853.
Koo S, Lu J, Li, N-S, Leung E, Das S, Harris ME, and Piccirilli JA. (2015). Transition state features in the Hepatitis Delta Virus (HDV) ribozyme reaction revealed by atomic perturbations. J Am Chem Soc. 137(28):8973-82.
Radak BK, Lee TS, Harris ME, York DM. (2015). Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM. RNA. (9):1566-77.
Zhang S, Gu H, Chen H, Strong E, Ollie EW, Kellerman D, Liang D, Miyagi M, Anderson VE, Piccirilli JA, York DM and Harris ME. (2016). Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+. Chem Comm. 52(24):4462-5. 2016 Zhang et al Chem Comm
Harris ME, Piccirilli JA and Anderson VE. (2017). Kinetic isotope effect analysis of RNA 2′-O-transphosphorylation. Methods Enzymol. Vol 596, 433-457. 2017 Harris et al Meth Enzymol
Mapping the specificity landscapes of RNA-protein interactions using high throughput sequencing
Gene expression depends on the ability of RNA binding proteins to associate with their target binding sites amidst a vast excess of non-cognate binding sites. Understanding RBP specificity is made even more complicated by the fact that many RBPs functionally associate with a range of different RNA binding sites in the transcriptome. We developed methods to measure the binding affinities and reaction rate constants for thousands of RNAs simultaneously. Mining these high density biochemical data sets obtained for a range of different RBPs is providing important new insights into the functional specificity and binding mechanism of essential cellular RBPs.
Niland CN, Anderson D, Jankowsky E and Harris ME. (2017). The contribution of the C5 protein subunit of Escherichia coli ribonuclease P to specificity for precursor tRNA is modulated by proximal 5′ leader sequences. RNA. in press.
Jankowsky E and Harris ME. (2017). Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing. Methods. in press.
Jain N, Lin H-C, Harris ME and Tolbert B. (2017). Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution. Proc Natl Acad Sci. in press.
Lin H-C, Zhao J, Niland CN, Tran B, Jankowsky E and Harris ME. (2016). Comprehensive analysis of the C5 protein specificity landscape reveals RNA structure and sequence preferences that direct ribonuclease P substrate specificity. Cell Chem Biol. 23(10):1271-1281.
Niland CN, Jankowsky E and Harris ME. (2016). Optimization of High Throughput Sequencing Kinetics (HTS-Kin) for the simultaneous determination of enzymatic rate constants for thousands of RNA substrates. (2016). Anal Biochem. 510:1-10.
Niland CN, Zhao J, Lin H-C, Jankowsky E and Harris ME. (2016). The global specificity landscape for ribonuclease P processing of pre-tRNA 5′ leader sequences. ACSChem Biol. 11(8):2285-92.
Bioinformatics combined with chemical biology and high resolution structure provide insights into RR allostery
Allosteric regulation and inhibition of human ribonucleotide reductase
Ribonucleotide reductase generated deoxynucleotides for DNA synthesis and regulates nucleotide pools. RR is a widespread and conserved enzyme that is a key experimental system for exploration of protein allostery, and an important chemotherapeutic target. Development of small molecule allosteric regulators and inhibitors of human RR is an important goal. In collaboration with the laboratory of Dr. Chris Dealwis we developed a protocol for identifying inhibitors of RR that combines in silico docking, biochemical assays of binding and activity, and cell toxicity studies. We successfully identified multiple compounds that target the different nucleotide binding sites and oligomerization interface on RR. In parallel, we have pursued studies to understand the structure and molecular dynamics that underlie allosteric communication. We identified the key functional groups on nucleotide effectors that drive communication of chemical information essential for allosteric regulation of substrate specificity as well as uncovering long range communication between the two different allosteric binding sites on RR. These results identified important structural and biochemical features of RR allosteric regulation and have identified multiple useful scaffolds for optimization using medicinal chemistry.
Ahmad M, Huff S, Pink J, Alam I, Zhang A, Perry K, Harris ME, Misko T, Porwal SK, Oleinick NL, Viswanathan R, Miyagi M, Dealwis CG. (2015). Identification of non-nucleoside human ribonucleotide reductase inhibitors. J Med Chem. 58(24):9498-509.
Knappenberger AJ, Ahmad FM, Dealwis CG and Harris ME. (2016). Nucleotide effector analogs allosterically regulate human ribonucleotide reductase and identify chemical determinants of substrate specificity. Biochemistry. 55(41):5884–5896.
Ahmad MF, Alam I, Huff SE, Pink J, Flanagan SA, Shewach D, Misko TA, Oleinick NL, Harte WE, Viswanathan R, Harris ME, and Dealwis CG. (2017). Potent competitive inhibition of human ribonucleotide reductase by a non-nucleoside small molecule. Proc Natl Acad Sci 114(31):8241-8246.
Knappenberger AJ, Grandhi S, Sheth R, Ahmad MD, Viswanathan R, and Harris ME (2017). Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase. J Biol Chem. 292(40):16463-16476.
Huff S, Ahmad MF, Yang M, Agrawal P, Pink J, Harris ME, Dealwis C, Viswanathan R (2018). Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase. JMed Chem. 61(3):666-680.