Insights into p53-Dependent Apoptotic Signaling and Cell Fate vis-a-vis Functional Cooperation among BCL-xL, Cytoplasmic p53, and PUMA

Following DNA damage, nuclear p53 induces the expression of PUMA (p53 upregulated modulator of apoptosis), a BH3‑only protein that binds and inhibits the anti‑apoptotic BCL‑2 repertoire, including BCL‑xL. Structural investigations of PUMA and the BCL‑xL×PUMA BH3 domain complex by X‑ray crystallography and nuclear magnetic resonance (NMR) spectroscopy reveal a novel, PUMA‑induced, domain‑swapped dimerization of BCL‑xL that requires a π‑stacking interaction between PUMA W71 and BCL‑xL H113. PUMA is an intrinsically disordered protein, but upon interaction with BCL‑xL, PUMA W71 and the PUMA BH3 domain residues fold into an alpha helix and subtly remodel BCL‑xL to trigger its dimerization. Wild type PUMA or a PUMA mutant incapable of promoting BCL‑xL dimerization (PUMA W71A) equivalently inhibit the anti‑apoptotic BCL‑2 repertoire to sensitize for death receptor‑activated apoptosis, but only wild type PUMA promotes p53‑dependent DNA damage‑induced apoptosis. Biochemical and cellular data demonstrate that PUMA‑mediated structural remodeling and dimerization of BCL‑xL modulates its affinity for cytosolic p53, providing a detailed mechanism of BCL‑xL, cytosolic p53, and PUMA functional cooperation. Our data suggest that within the BCL‑2 family, ligand binding‑induced, domain‑swapped dimerization is a critical control point to increase signal transduction complexity within the apoptotic pathways

Bak Conformational Changes Induced by Ligand Binding: Insight into BH3 Domain Binding and Bak Homo-Oligomerization

Recently we reported that the BH3-only proteins Bim and Noxa bind tightly but transiently to the BH3-binding groove of Bak to initiate Bak homo-oligomerization. However, it is unclear how such tight binding can induce Bak homo-oligomerization. Here we report the ligand-induced Bak conformational changes observed in 3D models of Noxa·Bak and Bim·Bak refined by molecular dynamics simulations. In particular, upon binding to the BH3-binding groove, Bim and Noxa induce a large conformational change of the loop between helices 1 and 2 and in turn partially expose a remote groove between helices 1 and 6 in Bak. These observations, coupled with the reported experimental data, suggest formation of a pore-forming Bak octamer, in which the BH3-binding groove is at the interface on one side of each monomer and the groove between helices 1 and 6 is at the interface on the opposite side, initiated by ligand binding to the BH3-binding groove

Conformationally-flexible and moderately electron-donating units-installed D–A–D triad enabling multicolor-changing mechanochromic luminescence, TADF and room-temperature phosphorescence

A novel twisted donor–acceptor–donor (D–A–D) π-conjugated compound that contains flexible and moderately-electron-donating units has been designed and synthesized. It exhibited not only multi-color-changing mechanochromic luminescence and thermally activated delayed fluorescence, but also, unexpectedly, room-temperature phosphorescence in a host layer

Accurate prediction of dynamic protein-ligand binding using P-score ranking

Protein–ligand binding prediction typically relies on docking methodologies and associated scoring functions to propose the binding mode of a ligand in a biological target. Significant challenges are associated with this approach, including the flexibility of the protein–ligand system, solvent-mediated interactions, and associated entropy changes. In addition, scoring functions are only weakly accurate due to the short time required for calculating enthalpic and entropic binding interactions. The workflow described here attempts to address these limitations by combining supervised molecular dynamics with dynamical averaging quantum mechanics fragment molecular orbital. This combination significantly increased the ability to predict the experimental binding structure of protein–ligand complexes independent from the starting position of the ligands or the binding site conformation. We found that the predictive power could be enhanced by combining the residence time and interaction energies as descriptors in a novel scoring function named the P-score. This is illustrated using six different protein–ligand targets as case studies.</p

Accurate prediction of dynamic protein-ligand binding using P-score ranking

Protein–ligand binding prediction typically relies on docking methodologies and associated scoring functions to propose the binding mode of a ligand in a biological target. Significant challenges are associated with this approach, including the flexibility of the protein–ligand system, solvent-mediated interactions, and associated entropy changes. In addition, scoring functions are only weakly accurate due to the short time required for calculating enthalpic and entropic binding interactions. The workflow described here attempts to address these limitations by combining supervised molecular dynamics with dynamical averaging quantum mechanics fragment molecular orbital. This combination significantly increased the ability to predict the experimental binding structure of protein–ligand complexes independent from the starting position of the ligands or the binding site conformation. We found that the predictive power could be enhanced by combining the residence time and interaction energies as descriptors in a novel scoring function named the P-score. This is illustrated using six different protein–ligand targets as case studies.</p

Fragment Dissolved molecular dynamics: A systematic and efficient method to locate binding sites.

Fragment-based drug discovery (FBDD) has been popular in the last decade, but some drawbacks, such as protein denaturation or ligand aggregation, have not yet clearly overcome in the framework of biomolecular simulations. In this work a systematic and semi-automatic method is presented as a novel proposal, named fragment dissolved Molecular Dynamics (fdMD), to improve research in future FBDD projects. Our method employs simulation boxes of solvated small fragments, adding a repulsive Lennard-Jones potential term to avoid aggregation, which can be easily used to solvate the object of interest. This method has the advantage of solvating the target with a low number of ligands, thus preventing this way denaturation of the target, while simultaneously generating a database of ligand-solvated boxes that can be used with other targets. A number of scripts are made available to analyze the results and obtain the descriptors proposed as a means of trustfully discard spurious binding sites. To test our method, four sets of different complexity have been solvated with ligand boxes and four molecular dynamics runs of 200 ns length have been run for each system, which have been extended up to 1 μs when needed. The reported results point that the selected number of replicas are enough to identify the correct binding sites irrespective of the initial structure, even in the case of proteins having several close binding sites for the same ligand. Among the proposed descriptors, average MMGBSA and average KDEEP energies emerge as the most robust ones

Engineering Enzymes and Pathways for Alternative CO2 Fixation and Glyoxylate Assimilation

Natural CO2 fixation is mainly associated with the Calvin-Benson-Bassham (CBB) cycle found in many photoautotrophic organisms, e.g. cyanobacteria. The CBB cycle as well as its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) evolved in a atmosphere, that contained mainly CO2 and merely any O2. With emerging oxygenic photosynthesis and the oxygenation of the atmosphere, RuBisCO became increasingly inefficient. Its inefficiency to discriminate between both substrates, CO2 and O2, led to the evolution of carbon concentrating mechanisms (CCMs) and photorespiration. The latter is a metabolic route to remove the toxic side product of the oxygenase reaction, 2-phosphoglycolate (2PG) and recycle it into useable metabolites. During canonical photorespiration, at least one molecule of CO2 would be released per two molecules of 2PG, reducing on biomass production at a notable margin. Among a variety of different approaches to mitigate this problem, examples for two of them will be discussed in this thesis. Synthetic photorespiration will be adressed via two chapters on the nature-inspired 3-hydroxypropionate (3OHP) bypass. Synthetic CO2 fixiation will be features in one chapter about substrate selectivity in the new-tonature crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle. Photosynthetic organisms not always completely recycle photorespiratory 2PG, but also dephosphorylate and excrete glyoxylate to the surrounding medium. Other bacteria, like the thermophile Chloroflexus aurantiacus can feed on these acids and evolved a pathway, the 3OHP bi-cycle to metabolize them without the loss of CO2. This inspired a synthetic photorespiration pathway, the 3OHP bypass. The first attempt to introduce this pathway into the cyanobacterium Synechococcus elongatus were performed by Shih et al. Chapter 3 features the continued efforts to improve the 3OHP bypass in S. elongatus. A improved selection scheme, based on a carboxysome knockout strain and the pathway based detoxification of propionate were utilized to evolve a part of the 3OHP bypass in a turbidostat setup. The high CO2 requiring strain improved its tolerance from 0.5% to 0.2% within 125 days. Among the 3OHP bi-cycle enzymes are some catalysts with unique properties, like the intramolecular CoA transferase, mesaconyl-C1-C4-CoA CoA transferase (Mct). Chapter 4 is dedicated to a structural analysis on why this enzyme can be exclusively intramolecular. It has a narrow active site, that allows the CoA moiety of mesaconylCoA to blocks external acids from entering. A protein structure with trapped intermediates and kinetic analysis with external acids support this claim. Additionally we investigated a promiscuous succinic semialdehyde dehydrogenase (SucD) that is featured in synthetic CO2 fixation pathways, as described in chapter 2. SucD from Clostridium kluyveri is promiscuous to other CoA esters and especially active with mesaconyl-C1-CoA, another intermediate of the CETCH cycle. This side reaction will slowly drain mesaconyl-CoA from the pool of intermediates and lead to the accumulation of mesaconic semialdehyde. The specificity was addressed by solving the crystal structure of CkSucD and closing the active site by the substitution of an active site lysin to arginine. The mutation decreased site activity from 16% to 2%, but the overall efficiency decreased. In another SucD from Clostridium difficile, the same mutation had a comparable effect, changing the sidereaction from 12% to 2%, while conserving the overall efficiency. The designed enzyme is a wortwhile replacement for future iterations of the CETCH cycle

Structural and Biochemical Studies of Glyoxylate Shunt Enzymes as Drug Targets of Mycobacterium tuberculosis

As the world population battles drug-resistant tuberculosis (TB), there is an urgent need for novel anti-tubercular drugs. This dissertation documents the studies of glyoxylate shunt enzymes, isocitrate lyase (ICL) and malate synthase (GlcB), in Mycobacterium tuberculosis (Mtb) as drug targets for the therapeutic development of TB. Two different drug discovery approaches were used. A mechanism based approach was utilized for isocitrate lyase, while a fragment based approach was applied for malate synthase, and both approaches employed X-ray crystallography as a primary technique. Through the mechanism based approach, an ICL inhibitor complexed crystal structure was solved to 2.6 Å resolution after the treatment with itaconate. From the structure, the active site cysteine (Cys191) underwent covalent modification to form an S-methylsuccinyl adduct. The inhibitory mechanism was based on the direct nucleophilic attack on the itaconate vinyl group by Cys191 after activation via a nearby general base. Additional crystal structure of ICL following the inactivation by 2-vinyl isocitrate (2-VIC) at 1.8 Å resolution confirmed the formation of an S-homopyruvoyl adduct of Cys191. The structure was consistent with the proposed inhibitory mechanism where 2-VIC first bound in the active site in the same manner as the substrate isocitrate. A base catalyzed aldo cleavage of the C2-C3 bond of 2-VIC then produced 2-vinyl glyoxylate and the aci-succinate. Cys191 was deprotonated to generate succinate, as in the lyase mechanism, followed by the Michael addition of Cys191 thiolate to 2-vinyl glyoxylate to form the final S-homopyruoyl adduct. A fragment based approach was used to advance drug discovery and further probe the active site of Mtb GlcB. Two libraries of 1580 fragments were screened against GlcB using differential scanning fluorimetry (DSF) to identify binding hits, and 18 complexed crystal structures were solved at 1.9-2.5 Å resolutions. The fragment bound GlcB crystal structures captured the conformations of the active site, which have not been reported for Mtb GlcB. The movements of two loops around the active site gave rise to a second portal to the surface and the narrowing of the active site tunnel. This series of conformational changes was hypothesized as a pathway for substrate-product exchange. The structures of the enzyme at various stages of product formation and dissociation, as well as an apo enzyme structure, were further elucidated to confirm the hypothesis. As a result, a detailed, mechanism driven substrate-product exchange in catalysis was formulated. One novel interaction from the fragments and the enzyme was further incorporated into the existing phenyl-diketo acid (PDKA) inhibitor, providing new drug designs. The resulting lead molecule was 100 times more potent compared to the parent PDKA, and was shown to make the same interaction and induce the same movement in the active site as the original fragment. The comprehensive knowledge from the structural studies of the two glyoxylate shunt enzymes provided new information that could lead to a greater understanding of Mtb’s physiology and guide the discovery of more effective treatments of TB

Arsenite-Induced Alterations of DNA Photodamage Repair and Apoptosis After Solar-Simulation UVR in Mouse Keratinocytes in Vitro

Our laboratory has shown that arsenite markedly increased the cancer rate caused by solar-simulation ultraviolet radiation (UVR) in the hairless mouse skin model. In the present study, we investigated how arsenite affected DNA photodamage repair and apoptosis after solar-simulation UVR in the mouse keratinocyte cell line 291.03C. The keratinocytes were treated with different concentrations of sodium arsenite (0.0, 2.5, 5.0 μM) for 24 hr and then were immediately irradiated with a single dose of 0.30 kJ/m(2) UVR. At 24 hr after UVR, DNA photoproducts [cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6-4PPs)] and apoptosis were measured using the enzyme-linked immunosorbent assay and the two-color TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay, respectively. The results showed that arsenite reduced the repair rate of 6-4PPs by about a factor of 2 at 5.0 μM and had no effect at 2.5 μM. UVR-induced apoptosis at 24 hr was decreased by 22.64% at 2.5 μM arsenite and by 61.90% at 5.0 μM arsenite. Arsenite decreased the UVR-induced caspase-3/7 activity in parallel with the inhibition of apoptosis. Colony survival assays of the 291.03C cells demonstrate a median lethal concentration (LC(50)) of arsenite of 0.9 μM and a median lethal dose (LD(50)) of UVR of 0.05 kJ/m(2). If the present results are applicable in vivo, inhibition of UVR-induced apoptosis may contribute to arsenite’s enhancement of UVR-induced skin carcinogenesis

Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors

Resistance against currently used antitubercular therapeutics increasingly undermines efforts to contain the worldwide tuberculosis (TB) epidemic. Recently, benzothiazinone (BTZ) inhibitors have shown nanomolar potency against both drug-susceptible and multidrug-resistant strains of the tubercle bacillus. However, their proposed mode of action is lacking structural evidence. We report here the crystal structure of the BTZ target, FAD-containing oxidoreductase Mycobacterium tuberculosis DprE1, which is essential for viability. Different crystal forms of ligand-free DprE1 reveal considerable levels of structural flexibility of two surface loops that seem to govern accessibility of the active site. Structures of complexes with the BTZ-derived nitroso derivative CT325 reveal the mode of inhibitor binding, which includes a covalent link to conserved Cys387, and reveal a trifluoromethyl group as a second key determinant of interaction with the enzyme. Surprisingly, we find that a noncovalent complex was formed between DprE1 and CT319, which is structurally identical to CT325 except for an inert nitro group replacing the reactive nitroso group. This demonstrates that binding of BTZ-class inhibitors to DprE1 is not strictly dependent on formation of the covalent link to Cys387. On the basis of the structural and activity data, we propose that the complex of DrpE1 bound to CT325 is a representative of the BTZ-target complex. These results mark a significant step forward in the characterization of a key TB drug target