Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or diseasemodifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanismbased and neuroprotective strategy for PD.P D is primarily caused by selective degeneration of midbrain dopamine (mDA) neurons and is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65 (1-3). Currently available pharmacological treatments [e.g., L-3,4-dihydroxyphenylalanine (L-DOPA)] are largely symptomatic and lose their efficacy over time, with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments (2, 3).During the last two decades, many intrinsic signals and extrinsic transcription factors have been identified to play critical roles for mDA neuron development (4-6). In particular, development of mDA neurons is dependent on two major signaling molecules, Sonic hedgehog (Shh) and wingless-type MMTV integration site family, member 1 (Wnt1), and their downstream factors. These two critical pathways (i.e., Shh-FoxA2 and Wnt1-Lmx1a) merge to control the expression of the orphan nuclear receptor related 1 protein (Nurr1) (7), suggesting that Nurr1 is a key regulator of mDA neurons. Indeed, Nurr1 [also known as nuclear receptor subfamily 4, group A, member 2 (NR4A2)] is essential not only for development (8-10) but also for maintenance of mDA neurons in adult brains (11). In addition, a recent study demonstrated that Nurr1 plays critical roles ...

Cystathionine β-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of HS biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the di-zinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.Metalloenzymes are some of the most powerful catalysts in the world. Understanding how the protein/metal partnership can give rise to dramatic rate enhancement will broaden the scope and understanding of enzyme evolution, protein engineering, and synthetic catalyst design. Two well known theories provide an evolutionary framework in the description of enzymatic rate enhancment: Arieh Warshel's preorganization theory (1-3) and the strain theory (4, 5) as first proposed by R.J.P Williams and B.L. Vallee.Based on decades of chemical and computational research, Warshel's results indicate that enzymes provide preorganized networks, optimized by evolutionary pressure, paid for by folding energy, and aimed at enhancing the electrostatic force between charges. These preorganized features enhance the electrostatic effect, and lower the activation energy barrier, by orienting dipoles toward the stabilization of functional charges and the charged transition states (1-3).Enzymatic rate enhancement by the use of "strained" groups is an idea first postulated by R.J.P. Williams and B.L. Vallee. Based on the unusual absorption, EPR, magnetic, redox, and ligand binding properties of metalloenzymes, Vallee and Williams proposed that large molecules, like proteins, could hold functional groups (metals, cofactors, side-chains) in strained conformations as a strategy toward rate enhancement. They defined the entatic state as an energized state supported by the stable protein fold (4, 5). Figure 1 illustrates a simple exampl...

Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.Shigella flexneri is a gram-negative facultative intracellular pathogen that causes bacillary dysentery and diarrhea by infection of colonic epithelial cells. During human disease, the organism traverses multiple environmentally varied niches within the host. Following ingestion, transit through the intestine to the colon exposes S. flexneri to a broad range of pH and salt conditions. Protection of gram-negative bacteria from the environment is mediated in part by the presence of the outer membrane, the composition of which may be altered in response to environmental signals encountered during infection. The outer membrane is an asymmetric lipid bilayer in which the inner leaflet is composed of phospholipids and the outer leaflet of lipopolysaccharide (LPS), a complex molecule that comprises three distinct moieties: lipid A (endotoxin), core oligosaccharide, and O-antigen polysaccharide. Lipid A, which is the major lipid constituent of the outer leaflet of the outer membrane, anchors the saccharide moieties to the outer membrane. In Escherichia coli and S. flexneri, lipid A is a glucosamine disaccharide modified by six acyl chains: primary acyl chains at the 2, 3, 2Ј, and 3Ј positions and secondary acyl chains attached to the 2Ј and 3Ј primary chains. Attachment of the secondary acyl chains to the 2Ј and ...