Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods

We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of α and C-terminal end of β) of erythroid spectrin. The values of the inverse line width parameter (ΔH0-1) from a family of SpoI-1-368Δ peptides scanning residues 21-30 exhibited a periodicity of ∼3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C′, a helix with a heptad pattern sequence. The residues with low ΔH0-1 values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the α spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the ΔH0-1 values from a family of spin-labeled SpβI-1898-2083Δ peptides also exhibited a periodicity of ∼3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high ΔH0-1 values and a hydrophobic side with low ΔH0-1 values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in αβ spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies. © 2005 American Chemical Society

Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD+ and triclosan

Structure of the ternary complex of F. tularensis enoyl-acyl carrier protein reductase reveals the structure of the substrate binding loop whose electron density was missing in an earlier structure, and demonstrates a shift in the position of the NAD+ cofactor

Structure of dihydroorotase from Bacillus anthracis at 2.6 Å resolution

A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure

Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD plus and triclosan

Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD+ has been solved to a resolution of 2.1 A. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD+ reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD+ cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors

Cole Relaxation Frequency as a Prognostic Parameter for Breast Cancer

We previously reported successful classification of breast cancer versus benign tissue using the Cole relaxation frequency measured on tissue excised during breast surgery as part of a study at two urban hospitals in the U.S. Midwest. Using that health system’s cancer registry, we have discovered retrospectively that outcomes for patients who participated in the initial study can be classified correctly in 3 well-differentiated categories: nonrecurrent (NR); recurrent with no metastasis (RNM); and recurrent with metastasis (RM). As Cole relaxation frequency increases, the classification moves from NR to RNM and finally to RM. Multivariate analysis showed a significant association of “time-cancer-free” for all patients in these recurrent categories, with P-values ranging between 0.0001 to 0.0047. Thus, this follow-up report shows the potential feasibility of using Cole relaxation frequency as a prognostic parameter in a larger prospective study

Cole Relaxation Frequency as a Prognostic Parameter for Breast Cancer

We previously reported successful classification of breast cancer versus benign tissue using the Cole relaxation frequency measured on tissue excised during breast surgery as part of a study at two urban hospitals in the U.S. Midwest. Using that health system’s cancer registry, we have discovered retrospectively that outcomes for patients who participated in the initial study can be classified correctly in 3 well-differentiated categories: nonrecurrent (NR); recurrent with no metastasis (RNM); and recurrent with metastasis (RM). As Cole relaxation frequency increases, the classification moves from NR to RNM and finally to RM. Multivariate analysis showed a significant association of “time-cancer-free” for all patients in these recurrent categories, with P-values ranging between 0.0001 to 0.0047. Thus, this follow-up report shows the potential feasibility of using Cole relaxation frequency as a prognostic parameter in a larger prospective study

Benzimidazole-Based FabI Inhibitors: A Promising Novel Scaffold for Anti-staphylococcal Drug Development

The enoyl-ACP reductase (FabI) enzyme is a well validated target for anti-staphylococcal drug discovery and development. With the goal of finding alternate therapeutics for drug-resistant strains of Staphylococcus aureus, such as methicillin-resistant S. aureus (MRSA), our previously published series of benzimidazole-based inhibitors of the FabI enzyme from Francisella tularensis (FtFabI) have been evaluated against FabI from S. aureus (SaFabI). We report here the preliminary structure-activity relationship of this series and the prioritization of compounds toward lead optimization. Mutational studies have identified key residues that contribute toward stabilizing the inhibitors in the active site of FabI. Mutations that do not significantly impact enzyme function but destabilize inhibitor binding are more likely to occur in nature as organisms evolve to evade the action of antibiotics leading to resistance. Identifying these residues provides guidance for minimizing susceptibility to resistance. Additionally, we have identified compounds that elicit antibacterial activity through off-target effects and observe that close analogs can display differing modes of action (on-target vs off-target) and need to be individually evaluated early on to prioritize compounds for lead optimization. Overall, our data suggest that the benzimidazole scaffold is a promising scaffold for anti-staphylococcal drug development

Expression, Purification and Characterization of Enoyl‐ACP Reductase II, FabK, from Porphyromonas gingivalis

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS‐II, are distinct from their mammalian counterparts, FAS‐I, in terms of both structure and enzymatic mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. One such enzyme, enoyl‐acyl carrier protein (ACP) reductase II, FabK, is a key, rate‐limiting enzyme in the FAS‐II pathway. The bacterial organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the U.S. population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS‐II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping‐Pong Bi‐Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for high‐throughput, 384‐well format. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x‐rays to high resolution