Crystallographic Studies of Steroid-Protein Interactions.

Steroid molecules have a wide range of function in eukaryotes, including the control and maintenance of membranes, hormonal control of transcription, and intracellular signaling. X-ray crystallography has served as a successful tool for gaining understanding of the structural and mechanistic aspects of these functions by providing snapshots of steroids in complex with various types of proteins. These proteins include nuclear receptors activated by steroid hormones, several families of enzymes involved in steroid synthesis and metabolism, and proteins involved in signaling and trafficking pathways.

Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N-(1-imino-2-chloroethyl)-l-ornithine ( K = 1.3 μM, k = 0.

Structure of Salmonella FlhE, conserved member of a flagellar type III secretion operon.

The bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found.

Structural analysis of a fungal methionine synthase with substrates and inhibitors.

The cobalamin-independent methionine synthase from Candida albicans, known as Met6p, is a 90-kDa enzyme that consists of two (βα)8 barrels. The active site is located between the two domains and has binding sites for a zinc ion and substrates L-homocysteine and 5-methyl-tetrahydrofolate-glutamate3. Met6p catalyzes transfer of the methyl group of 5-methyl-tetrahydrofolate-glutamate3 to the L-homocysteine thiolate to generate methionine.

Sulfur incorporation generally improves Ricin inhibition in pterin-appended glycine-phenylalanine dipeptide mimics.

Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties observed in the 'linker space' when N-{N-(pterin-7-yl)carbonylglycyl}-l-phenylalanine 1 was bound to the active site of RTA. The gylcine-phenylalanine dipeptide analogs included both amides and thioamides. Decarboxy gly-phe analog 2 showed a 6.

Synthesis and structure of 16,22-diketocholesterol bound to oxysterol-binding protein Osh4.

We have synthesized 16,22-diketocholesterol, a novel ligand for oxysterol-binding protein Osh4, and determined X-ray structure of the diketocholesterol in complex with Osh4. The X-ray structure shows that α7 helix of Osh4 assumes open conformation while the rest of Osh4, closed conformation, implying this diketocholesterol-bound Osh4 structure may represent a structural intermediate between the two conformations.

Peptide-conjugated pterins as inhibitors of ricin toxin A.

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of ricin toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography.

Revisiting the role of glycosylation in the structure of human IgG Fc.

Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors.

Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis.

Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form.

On the mechanism of dimethylarginine dimethylaminohydrolase inactivation by 4-halopyridines.

Small molecules capable of selective covalent protein modification are of significant interest for the development of biological probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH) (Johnson, C. M.

Characterization of C-alkyl amidines as bioavailable covalent reversible inhibitors of human DDAH-1.

C-Alkyl amidine analogues of asymmetric N(ω),N(ω)-dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for the development of therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part click-chemistry-mediated activity probe, a homologated series of C-alkyl amidines were ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N⁵-(1-Iminopentyl)-L-ornithine was determined to be the most potent compound in vitro (K(d)=7 μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant, as gauged by X-ray crystallography and isothermal titration calorimetry.

Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: toward a targeted polypharmacology to control nitric oxide.

Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as antitumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds.

Crystal structure of the engineered neutralizing antibody M18 complexed to domain 4 of the anthrax protective antigen.

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development.

Structure of NS1A effector domain from the influenza A/Udorn/72 virus.

The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented.

The X-ray structure of ricin A chain with a novel inhibitor.

Ricin is a potent heterodimeric cytotoxin; the B chain binds eucaryotic cell surfaces aiding uptake and the A chain, RTA, reaches the cytoplasm where it enzymatically depurinates a key ribosomal adenine, inhibiting protein synthesis. Ricin is known to be an agent in bioterrorist repertoires and there is great interest in finding, or creating, efficacious inhibitors of the toxin as potential antidotes. We have previously identified two families of bicyclic RTA inhibitors, pterins and purines.

Promiscuous partitioning of a covalent intermediate common in the pentein superfamily.

Many enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here we trap and determine the structure of a stable covalent adduct that mimics this intermediate using a mutant dimethylarginine dimethylaminohydrolase and an alternative substrate. The interactions observed between the enzyme and trapped adduct suggest an altered angle of attack between the nucleophiles of the first and second half-reactions of normal catalysis.

X-ray analysis of phosphoglycerate kinase 2, a sperm-specific isoform from Mus musculus.

Phosphoglycerate kinase 2 (PGK2) is an isozyme of the glycolytic pathway that provides ATP required for sperm motility. It is encoded by an autosomal retrogene that is expressed only during spermatogenesis, concomitant with the inactivation of the X-linked Pgk1 gene. PGK2 from the mouse, Mus musculus, has been overexpressed from a plasmid in bacteria and purified.

The structure of eukaryotic translation initiation factor-4E from wheat reveals a novel disulfide bond.

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m(7) guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight beta-strands, three alpha-helices, and three extended loops.

The structure of the minimal relaxase domain of MobA at 2.1 A resolution.

The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization.

Kinetic and structural analysis of active site mutants of monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

5,10-Methylenetetrahydrofolate dehydrogenase (MTD) catalyzes the reversible oxidation of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate. This reaction is critical for the supply of one-carbon units at the required oxidation states for the synthesis of purines and dTMP. For most MTDs, dehydrogenase activity is co-located with a methenyl-THF cyclohydrolase activity as part of bifunctional or trifunctional enzyme.

Structure of DsbC from Haemophilus influenzae.

Bacterial DsbC proteins are involved in rearranging or reducing mismatched disulfide bonds folding within the periplasm. The X-ray structure of the enzyme from Haemophilus influenzae has been solved and compared with the known structure of the Escherichia coli protein. The proteins act as V-shaped dimers with a large cleft to accommodate substrate proteins.

The structure of ribosome inactivating proteins.

Ribosome Inactivating Proteins, RIPs, depurinate an invariant adenine from the 28S rRNA of eukaryotic ribosomes; they have evolved to near enzymatic perfection for this task. The N-glycosidase fold is conserved in plant and bacterial enzymes. RIPs can form complexes with cell surface recognition proteins that dramatically increase the cytotoxicity of the molecule.

The X-ray structure of Escherichia coli RraA (MenG), A protein inhibitor of RNA processing.

The Escherichia coli protein regulator of RNase E activity A (RraA) has recently been shown to act as a trans-acting modulator of RNA turnover in bacteria; it binds to the essential endonuclease RNase E and inhibits RNA processing in vivo and in vitro. Here, we report the 2.0A X-ray structure of RraA.

Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base.

Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLC(Bc)) to a non-ionizable Asn generate a mutant enzyme (D55N) with 10(4)-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.