LIGAND-MEDIATED CONTROL OF THE GLUCOKINASE REGULATORY PROTEIN
Brian G. Miller and Juliana Martinez
Department of Chemistry and Biochemistry, Florida State University, Tallahassee FL 32306
The glucokinase regulatory protein (GKRP) plays an essential role in glucose homeostasis by acting as a competitive inhibitor of human glucokinase (GCK) and triggering its localization to the hepatocyte nucleus upon glucose deprivation. Metabolites such as fructose 6-phosphate and sorbitol 6-phosphate promote assembly of the GCK-GKRP complex, whereas fructose 1-phosphate and functionalized piperazines with potent in vivo antidiabetic activity disrupt the complex. Here, we establish the molecular basis by which these natural and synthetic ligands modulate the GCK-GKRP interaction. We demonstrate that GKRP displays conformational heterogeneity at the N-terminus and deleting this region eliminates the ability of sorbitol 6-phosphate to promote the GCK-GKRP interaction. Stabilizing ligands favor an extended N-terminus, which sterically positions two arginine residues for optimal coulombic interaction with a pair of carboxylate side chains from GCK. Conversely, disruptors promote a more compact N-terminus in which an interfacial arginine residue is stabilized in an unproductive orientation through a cation-π interaction with tyrosine 75. Elucidating the mechanistic origins of ligand-mediated control over the GCK-GKRP interaction is expected to impact the development and future refinement of therapeutic agents for diabetes and cardiovascular disease, both of which result from improper GKRP regulation of GCK.
Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway
Naga Sandhya Guntaka, Steven D. Bruner
Department of Chemistry, University of Florida, Gainesville, Florida 32611
Small molecule microbial secondary metabolites by regulating host-microbe interactions play an important role in all aspects of disease etiology and treatment. Colibactin is a secondary metabolite linked to the progression and pathogenesis of colorectal cancer (CRC) and inflammatory bowel disease (IBD) by inducing DNA damage in host cells. The chemical details of the colibactin and the biosynthetic pathway are emerging but clearly are unusual and noncanonical. Our research addresses a key aspect of colibactin biosynthesis, the occurrence of multiple metabolites and the biosynthetic rationale for this. Recent studies suggest an atypical role of ClbQ, a type II editing thioesterase in releasing pathway intermediates from the assembly line (1) and genetic deletion of ClbQ has been shown to abolish colibactin cytotoxic activity (2). Presented is an interdisciplinary approach to address the role of ClbQ, using enzyme structure, organic synthesis of substrates/intermediates and mechanistic analysis. The 2.0 Å crystal structure and biochemical characterization of ClbQ reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate between carrier proteins in the pathway. As reported in earlier studies (1), late-stage cyclized intermediates are not the preferred substrates for ClbQ. However, late-stage linear precolibactin intermediates are hydrolyzed. Our data, combined with previous reports, support a novel role of ClbQ in facilitating the promiscuous offloading of premature precolibactin metabolites and suggest novel insights into colibactin biosynthesis.
- Li, Z.-R., Li, J., Gu, J.-P., Lai, J. Y. H., Duggan, B. M., Zhang, W.-P., Li, Z.-L., Li, Y.-X., Tong, R.-B., Xu, Y., Lin, D.-H., Moore, B. S., and Qian, P.-Y. (2016) Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin. Nat. Chem. Biol. 12, 773–775.
- Cougnoux, A., Dalmasso, G., Martinez, R., Buc, E., Delmas, J., Gibold, L., Sauvanet, P., Darcha, C., Déchelotte, P., Bonnet, M., Pezet, D., Wodrich, H., Darfeuille-Michaud, A., and Bonnet, R. (2014) Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype. Gut. 63(12), 1932-42.
Mechanism of proton transfer in Class A β-lactamase function and inhibition by avibactam
Yu Chen, Orville Pemberton
Department of Molecular Medicine, University of South Florida
The hydrolysis of β-lactam antibiotics by Class A β-lactamases proceeds through an acylation step where the catalytic Ser70 forms an acyl-enzyme bond with the β-lactam substrate, and a deacylation step where the covalent linkage is cleaved by the catalytic water activated by Glu166. For the acylation reaction, the origin of the proton transferred to the β-lactam ring nitrogen has been debated with both Lys73 and Lys234 proposed as the source. Meanwhile, whereas the acyl-enzyme intermediate is usually transient for β-lactam substrates, the recent FDA-approved β-lactamase inhibitor avibactam is able to form an acyl-enzyme complex stable against deacylation by the catalytic water. Here we present a 0.83 Å resolution X-ray complex crystal structure of CTX-M-14 Class A β-lactamase with avibactam, shedding light on both the proton transfer process of the acylation reaction and the stability of the avibactam covalent complex. Particularly, we are able to determine the hydrogen atom positions of key catalytic residues, and demonstrate that both Lys73 and Glu166 are neutral. These observations suggest that avibactam is able to trap the acylation reaction at the very last stage after the proton is transferred, via Ser130, from Lys73 to the substrate, but before Lys73 can extract a proton from Glu166 and activate the latter to serve as the general base for the deacylation reaction. Our results have important implications for the catalytic mechanism of Class A β-lactamases and inhibitor discovery against these enzymes.
MECHANISMS OF ACTIVATED HUMAN GLUCOKINASE DISEASE VARIANTS
Shawn M. Sternisha and Brian G. Miller
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306
Human glucokinase (GK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. GK is being actively pursued as a therapeutic target for diabetes. Gain-of-function mutations in the glk gene result in hyperactive enzyme variants that cause persistent hyperinsulinemic hypoglycemia. Past biochemical and biophysical studies support the postulate that activated disease variants can be segregated into two mechanistically distinct classes. In α-type activation, GK displays an increased affinity for glucose and the 1H-13C HMQC spectrum of 13C-Ile labeled enzyme resembles the glucose-bound state. Conversely, in β-type activation glucose affinity is largely unchanged and the 1H-13C spectrum reveals no change in the enzyme structure. Here, we use a combination of viscosity variation studies, chemical quench-flow, and hydrogen-deuterium exchange mass spectrometry to uncover the mechanistic basis for both activation types. Our work elucidates the molecular basis of GK disease variants and provides insights into the nature of GK’s unique kinetic cooperativity.
Drug Candidate Discovery: Targeting Bacterial Topoisomerase I Enzymes for Novel Antibiotic Leads
Shayna Sandhaus, Yuk-Ching Tse-Dinh
Florida International University
The global community is facing a crisis—antibiotics are often ineffective due to the emergence of multi-drug resistant bacterial pathogens. The need for new antibiotics acting against novel bacterial cell targets is dire. Bacterial topoisomerase I (TopoI) is an attractive target for new antibiotics, since it should be vulnerable to bactericidal topoisomerase poison inhibitors in every bacterium, and its function is known to be required for the survival of certain bacterial pathogens including Mycobacterium tuberculosis. Selective and potent inhibitors of bacterial TopoI can be useful as new antibiotic leads. Bacterial TopoI relaxes supercoiled DNA by using its active-site tyrosine residue to attack the phosphodiester backbone of the DNA, forming a covalent intermediate and cleaving one strand of the DNA. It then passes the other strand through the break and rejoins the DNA to increase the DNA linking number by one. Catalytic inhibitors of topoisomerase I may prevent the enzyme from binding or cleaving the DNA, while poison inhibitors can stabilize the DNA-enzyme covalent intermediate, thus causing the accumulation of DNA breaks, leading to bacterial cell death. This project seeks novel inhibitors of bacterial topoisomerase I in various bacterial strains of global health significance. Two main assays are used to find antibacterial compounds that target TopoI—an enzyme inhibition assay (a gel-based assay that monitors the formation of relaxed DNA in the presence of inhibiting compounds), and a growth inhibition assay (an assay that monitors the growth of bacteria in the presence of topoisomerase inhibitors). Several promising compounds have been found from various screens that inhibit bacterial TopoI well, and are able to prevent bacterial cell growth. Many of the discovered compounds are effective against M. tuberculosis topoisomerase I, and can prevent the growth of M. smegmatis, a non-pathogenic homolog of M. tuberculosis. The use of diverse approaches such as in silico docking studies and mixture-based compound screening has been successful at finding novel inhibitors of bacterial topoisomerase I, and may bring us one step closer to new and effective antibiotics.
Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing
Michael E. Harris1, Hsuan-Chun Lin1, Jing Zhao1, Courtney N. Niland2, and Eckhard Jankowsky2
1Department of Chemistry, University of Florida
2Department of Biochemistry, Case Western Reserve University
RNA-binding proteins (RBPs) are typically involved in non-equilibrium cellular processes, and specificity can arise from differences in ground state, transition state or product states of the binding reactions for alternative RNAs. We used newly developed high throughput methods to measure and analyze the RNA association kinetics and equilibrium binding affinity for all possible sequence combinations in precursor tRNA binding site of C5, the essential protein subunit of Escherichia coli ribonuclease P. The results show that the RNA sequence specificity of C5 arises due to favorable RNA-protein interactions that stabilize the transition state for association and bound ES complex. Specificity is further impacted by unfavorable RNA structure involving the C5 binding site in the ground state. The results illustrate a comprehensive quantitative approach for analysis of RNA binding specificity, and show how both RNA structure and sequence preferences of an essential protein subunit direct the specificity of a ribonucleoprotein enzyme.