Browsing by Author "Dunkle, Jack A."
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Item The beta-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning(Elsevier, 2023) Gogar, Rajleen K.; Carroll, Franki; Conte, Juliana, V; Nasef, Mohamed; Dunkle, Jack A.; Frantom, Patrick A.; University of Alabama TuscaloosaUnder oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-depen-dent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is pro-tected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "11-latch" structural element that includes the alpha 6 helix, a glycine-rich loop, a 11-hairpin, and a cis-proline residue. To identify a functional role for the 11-latch, we used site -directed mutagenesis to obtain the N99D and N99A SufS var-iants. N99 is a conserved residue that connects the alpha 6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a dis-torted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high -affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demon-strate that the 11-latch does not affect the chemistry of persul-fide formation but does protect it from undesired reductants. The data also indicate the 11-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.Item The determination of Cr(III)'s mode of binding dna(University of Alabama Libraries, 2019) Brown, Silas Earl; Vincent, John B.; University of Alabama TuscaloosaChromium(VI) complexes are potent mutagens and carcinogens when inhaled, while the potential of these complexes to generate similar effects when taken orally is an area of active debate. The focus on this work is to investigate how chromium binds to DNA on a molecular level. The exact mechanism(s) of action of this activity is unknown, but potential mechanisms can be grouped into two categories. The first is mechanisms associated with redox chemistry during reduction of Cr(VI). Numerous studies have been focused on studying this potential mechanism. The second mechanism is based on the generated Cr(III) binding to DNA to form binary and ternary complexes. Virtually no data on the molecular level structure of these Cr(III)- DNA complexes exists. Such studies are complicated by the spectroscopic and magnetic properties of Cr(III). Second, previous studies have used plasmid DNA, DNA polymers, calf thymus DNA, or DNA isolated from cultured cells, which because of their size and complexity, present numerous potential Cr-binding sites with a range of binding constants. What is required to determine the preferential sites for Cr-binding and to characterize the structure of these sites is the use of DNA oligomers significantly smaller in size whose base sequences can be carefully designed and which can be synthesized in appreciable quantities. Results of spectroscopic and magnetic studies (1H and 31P nuclear magnetic resonance spectroscopy including multidimensional techniques, pulsed electron paramagnetic resonance spectroscopy, and infrared spectroscopy) to characterize the binding of Cr(III) to such DNA oligomers indicate that Cr(III) as [Cr(H2O)5]3+ can bind specifically to the guanine N7 position of B-form double stranded DNA without direct interaction with the phosphate backbone and resulting in minimal distortions in iii the structure of the DNA. A potential Cr(III)-based inter-strand crosslink of DNA has been characterized. Preliminary steps to synthesize and characterize ternary Cr(III)-small molecule- DNA compounds have been investigated.Item Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues(American Society of Biochemistry and Molecular Biology, 2019) Blahut, Matthew; Wise, Courtney E.; Bruno, Michael R.; Dong, Guangchao; Makris, Thomas M.; Frantom, Patrick A.; Dunkle, Jack A.; Outten, F. Wayne; University of South Carolina Columbia; University of Alabama TuscaloosaIron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli. Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5 '-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for C alpha deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.Item Electron paramagnetic resonance studies of drug binding in cytochrome P450 enzymes(University of Alabama Libraries, 2019) Lockart, Molly Marie; Bowman, Michael K.; University of Alabama TuscaloosaCytochrome P450 enzymes (CYPs) are heme-containing monooxygenase enzymes that exist in nearly every living organism. They are responsible for oxidizing a wide variety of substrates in biosynthetic and detoxification pathways and are a common target for drug design. CYP-drug binding modes are traditionally characterized using optical difference spectra, but these binding assignments oftentimes miss atypical CYP-drug binding and do not account for mixtures of binding modes. Electron paramagnetic resonance (EPR) spectroscopy can shed light on CYP-drug binding. A mixture of continuous wave (CW) EPR and pulsed EPR methods provides some valuable insight into how common drugs and drug fragments interact with the active site heme. First, the two human isoforms that contribute significantly to drug metabolism, CYP3A4 and CYP2C9, are studied in complex with a variety of drugs. The results demonstrate that CW EPR parameters can reveal drug binding modes. Remarkably, this research finds an abundance of water-bridged complexes, and many of them coexist in frozen solution with complexes where the drug directly coordinates to the heme. These mixtures of binding modes have significant ramifications for drug design. Additionally, CYP3A4 is studied in the context of drug-drug interactions, looking at how common drugs, like acetaminophen, caffeine, midazolam, and carbamazepine can simultaneously occupy the active site when combined. These results find that multiple drugs occupy the active site and that they are distinct from any CYP-single drug complex. A method is established for observing human CYP drug-drug interactions with EPR, and it provides evidence of simultaneous drug binding with common drugs. In addition to human CYPs, this research examines drug binding in CYP51B1, a Mycobacterium tuberculosis CYP isoform. The results find that inhibitor-like compounds form mixtures of bound complexes, including some that retain the axial water. Overall, this research provides several new details about CYP-drug interactions. These results and observations highlight the importance of understanding and characterizing CYP-drug binding with more detailed analyses that provide information on the full range of CYP binding modes.Item Investigation of the RNA-Protein Structure-Function Relationships in the CRISPR-Cas10 Complex and Erythromycin-Resistance RNA Methyltransferases(University of Alabama Libraries, 2021) Nasef, Mohamed; Dunkle, Jack A.; University of Alabama TuscaloosaCRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that type III CRISPR-Cas systems synthesize second messenger molecules, previously unknown to exist in prokaryotes, known as cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal type III systems, cOA generation and its regulation have not been explored for the Gram-positive bacterium, Staphylococcus epidermidis, as well as the specific target RNA sequence requirements. In chapter 2, we demonstrate that this system performs Mg2+ dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions and synthesis is antagonized by Csm3-mediated target RNA cleavage. In chapter 3, we mechanistically investigated the effect of single and multiple mismatches on the activation of cOA synthesis of type III-A Cas10-Csm complex from S. epidermidis. We show that mismatches at specific positions in target RNA significantly reduced the amount of cOAs produced in a sequence context-dependent manner. We also show that the defects are not due to perturbations in binding efficiencies or in RNA cleavage.In chapter 4, we focus on an erythromycin resistance methyltransferases (Erm) found in many Gram-positive pathogens. Erm proteins are S-adenosyl methionine-dependent Rossmann-fold methyltransferases which convert A2058 of 23S rRNA to m62A2058. This modificationsterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multi-drug resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, while ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24 % sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity binding assays. We demonstrate that residues in α4, α5 and the α5-α6 linker are essential for methyltransferase function including: an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine, and basic residues in α5 and the α5-α6 linker which likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.Item Investigations of conserved structure-function relationships and enzymatic evolution(University of Alabama Libraries, 2021) Conte, Juliana Victoria; Frantom, Patrick A.; University of Alabama TuscaloosaThe E.coli SUF pathway is the iron-sulfur cluster biogenesis pathway responsible for assembling iron-sulfur clusters during times of oxidative stress and iron starvation. SufS is the cysteine desulfurase enzyme responsible for the acquisition of sulfur and subsequent transfer to transpersulfurase SufE. Recent studies have provided several crystal structures of SufS including two variants displaying stalled intermediates of the desulfurase reaction. Combining the structural data with biochemical investigations has uncovered several key elements in SufS activity. SufS is a homodimer proposed to use a half-sites mechanism involving conformational changes and communication through dimer interface interactions. Despite the significant contributions made to understanding SufS function, questions still remain about the specific roles of several structural elements involved in activity and regulation. Investigations discussed herein are aimed at probing structural components of SufS, including the beta hairpin structure. The beta hairpin makes up one wall of the active site of the opposite monomer. The proposed regulatory mechanism of SufS uses the beta hairpin dynamics to control access to the active site. In chapter 2 beta hairpin variants are shown to significantly disrupt SufS structure. Variants were unable to bind the PLP cofactor essential to catalysis and instability of the variants resulted in protein aggregation as demonstrated with size exclusion chromatography analysis. Chapter 3 focuses on single substitutions of residues located in and around the active site including Asn99, Arg56, and Arg359. Kinetic assays were conducted to determine defects in desulfurase activity by measuring the rate of alanine production, the product released by the desulfurase reaction. Asn99 variants exhibited a ten-fold decrease in turnover number, confirming Asn99 does play a role in generating an optimal environment for activity. This is likely due to the hydrogen bonds Asn99 forms with the glycine residues in the loop at the base of the beta hairpin, potentially contributing to the regulation of dynamics. Arg56 is located in a dynamic loop above the active site and was previously proposed to contribute to catalysis by hydrogen bonding with the sulfhydryl of the substrate cysteine. Kinetic analysis of both R56A and R56K variants is consistent with Arg56 having a significant role in SufS activity. It is possible that this residue is responsible for the deprotonation of SufE C51 and facilitating persulfide transfer. Kinetic analysis of the Arg359 variants suggest a role in cysteine binding and positioning the substrate for bond cleavage. The work presented here contributes to the current understanding of the SufS mechanism and may be more broadly applicable to other cysteine desulfurases.Item Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases(Oxford University Press, 2017) Walker, Forrest C.; Chou-Zheng, Lucy; Dunkle, Jack A.; Hatoum-Aslan, Asma; University of Alabama TuscaloosaCRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR-Cas system that encodes the Cas10-Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10-Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10-Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10-Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR-Cas immunity and central nucleic acid metabolism.Item Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex(Cold Spring Harbor Lab Press, 2019) Nasef, Mohamed; Muffly, Mary C.; Beckman, Andrew B.; Rowe, Sebastian J.; Walker, Forrest C.; Hatoum-Aslan, Asma; Dunkle, Jack A.; University of Alabama TuscaloosaCRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.Item Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition(Elsevier, 2020) Rowe, Sebastian J.; Mecaskey, Ryan J.; Nasef, Mohamed; Talton, Rachel C.; Sharkey, Rory E.; Halliday, Joshua C.; Dunkle, Jack A.; University of Alabama TuscaloosaErythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m(6) (2)A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in alpha 4, alpha 5, and the alpha 5-alpha 6 linker are essential for methyltransferase function, including an aromatic residue on alpha 4 that likely forms stacking interactions with the substrate adenosine and basic residues in alpha 5 and the alpha 5-alpha 6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.Item Structural evidence for a latch mechanism regulating access to the active site of SufS-family cysteine desulfurases(International Union of Crystallography, 2020) Dunkle, Jack A.; Bruno, Michael R.; Frantom, Patrick A.; University of Alabama TuscaloosaCysteine serves as the sulfur source for the biosynthesis of Fe-S clusters and thio-cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio-cofactors by cleaving the C-alpha-S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe-S biosynthesis. Here, a 1.5 angstrom resolution X-ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a beta-hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active-site beta-hairpin is likely to require adjacent structural elements to function as a beta-latch regulating access to the SufS active site.Item Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS(American Chemical Society, 2019) Dunkle, Jack A.; Bruno, Michael R.; Outten, F. Wayne; Frantom, Patrick A.; University of Alabama Tuscaloosa; University of South Carolina ColumbiaSufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli, this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically, this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five X-ray crystal structures of SufS including a new structure of SufS containing an inward-facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved beta-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data, identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.Item Synthesis of non-nucleoside methyltransferase inhibitors & investigation of pinacol phosphonate ester as a phosphonomethoxy source in antiviral drugs(University of Alabama Libraries, 2021) Chakraborty, Amarraj; Snowden, Timothy S.; University of Alabama TuscaloosaMixed lineage leukemia (MLL) is an incurable form of pediatric cancer. Disruptor of telomeric silencing 1-like (DOT1L), a lysine methyltransferase, is a critical enzyme associated with the initiation and progression of MLL-rearranged acute leukemias. Here we report a synthesis of 14 potential DOT1L inhibitors in a collaborative, early drug discovery investigation. Preparation of these inhibitors involved multiple reaction steps and challenging isolation procedures. However, overall yields for these inhibitors were improved from 3-11% up to 36-42% through optimization of reaction conditions and purification processes. Each target compound and >30 new polyaza heterocycles were characterized with 1H-, 13C-, and 2D-NMR spectroscopy and HRMS spectrometry. Four of the prepared compounds demonstrated confirmed inhibitory activity against DOT1L both in nucleosome and whole cell assays conducted at the University of Michigan Medical School. The most potent DOT1L inhibitor exhibited inhibitory activity with IC50 = 1.0 ± 0.1 μM, which was a 40-fold improvement in potency versus the initial hit. Screening against nine other methyltransferases established target selectivity of the most potent inhibitor and revealed another DOT1L inhibitor with modest inhibitory activity against the protein arginine methyltransferase PRMT3, which is associated with unrelated diseases.Phosphonomethyl ether is a functionality widely present in nucleoside phosphonate-containing antiviral therapeutics including the anti-HIV/ anti-HBV drugs Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafinamide (TAF), and Adefovir. Industrial-scale syntheses of these prodrugs require diethyl p-toluene sulfonyloxymethyl phosphonate (DESMP) to install a phosphonomethyl ether fragment via O-alkylation of a nucleobase segment under basic conditions. This step significantly diminishes overall yield due to complications arising from high water solubility of the corresponding phosphonate diester and partial hydrolysis to the monoester during workup. In collaboration with Professor Anthony J. Arduengo, III and the Medicines for All Institute at VCU, we designed and prepared an innovative phosphonate ester, which may prove advantageous over DESMP in the O-alkylation step. Pinacol sulfonyloxymethyl phosphonate esters were synthesized in three steps using inexpensive reagents and solvents. The optimized conditions facilitate reproducible isolation on moderate scale and limit chemical waste. Products from each step were successfully prepared as solids and were characterized with 1H-, 13C-, and 31P-NMR spectroscopy and HRMS spectrometry. Crystal structures of each product were obtained by X-ray crystallography.