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. Proteins found in some bacteria that bind and metabolize steroids have been investigated as well. A survey of the steroid-protein complexes that have been studied using crystallography and the insight learned from them is presented.

A Lysine-Targeted Affinity Label for Serine-ß-Lactamase Also Covalently Modifies New Delhi Metallo-ß-lactamase-1 (NDM-1).

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.

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, N5-(1-imino-2-chloroethyl)-l-ornithine ( KI = 1.3 µM, kinact = 0.34 min-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline ( Ki = 470 µM) and 2-chloroacetamidine ( KI = 310 µM, kinact = 4.0 min-1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N5-(1-imino-2-chloroisopropyl)-l-ornithine ( kinact /KI = 208 M-1 s-1) and N5-(1-imino-2-chlorisopropyl)-l-lysine ( kinact /KI = 440 M-1 s-1), and one that lengthens the linker beyond that found in the substrate, N5-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, KI = 0.19 µM, kinact = 0.22 min-1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 104 M-1 s-1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 102 M-1 s-1), and has a partition ratio of 1 with a >100 000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 µM) with cytotoxicity appearing only at higher concentrations (ED50 = 118 µM). A 1.91 Å resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.

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. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. We isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5Å resolution. The structure reveals a ß-sandwich fold, with no close structural homologs. Possible roles of FlhE, including that of a chaperone, are discussed.

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 (ßα)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. Met6p is essential for fungal growth, and we currently pursue it as an antifungal drug design target. Here we report the binding of L-homocysteine, methionine, and several folate analogs. We show that binding of L-homocysteine or methionine results in conformational rearrangements at the amino acid binding pocket, moving the catalytic zinc into position to activate the thiol group. We also map the folate binding pocket and identify specific binding residues, like Asn126, whose mutation eliminates catalytic activity. We also report the development of a robust fluorescence-based activity assay suitable for high-throughput screening. We use this assay and an X-ray structure to characterize methotrexate as a weak inhibitor of fungal Met6p.

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.

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 6.4-fold decrease in potency (IC50 = 128 µM), yet the analogous thioamide 7 recovered the lost activity and performed similarly to the parent inhibitor (IC50 = 29 µM). Thiourea 12 exhibited an IC50 nearly six times lower than the oxo analog 13. All inhibitors showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated log P values do not correlate to the increase in bioactivity suggesting other factors dominate.

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. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC50 values in the low micromolar range.

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. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Šresolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the C(H)2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between C(H)2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (R(g)) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger R(g) than glycosylated Fc, suggesting a more open C(H)2 orientation under these conditions. Moreover, the R(g) of aglycosylated Fc was reduced by mutations at the C(H)2-C(H)3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

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. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0-2.8Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (ßα)(8)-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.

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.; Linsky, T. W.; Yoon, D. W.; Person, M. D.; Fast, W. J. Am. Chem. Soc. 2011, 133, 1553-1562). Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compound 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Solution studies support an inactivation mechanism in which the active site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resolution X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Molecular modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the solution-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.

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. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction could be estimated. These results suggest that further stabilization of the covalent adduct is a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.

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. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N(5)-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 microM, respectively) and DDAH-1 (990, 52, 7.5, 110 microM, respectively). A 1.9 A X-ray crystal structure of the N5-(1-iminopropyl)-L-ornithine:DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.

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. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 A resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.

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. Both classes have poor solubility which impairs inhibitor development. Here we report the use of 2-amino-4,6-dihydroxy-pyrimidines as RTA inhibitors. Unlike previously observed single ring inhibitor platforms, these displace Tyr 80 and bind deep in the RTA specificity pocket. These compounds are at least 10 times more soluble than pterin-based inhibitors and appear to be useful new class of ricin inhibitors.

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. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.

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. The stable covalent adduct is also capable of further reaction. Addition of imidazole rescues the original hydrolytic activity. Notably, addition of other amines instead yields substituted arginine products, which arise from partitioning of the intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities among the reaction pathways in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this intermediate may also help to illuminate the evolutionary history of these enzymes.

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. It was crystallized in three forms: as the apoenzyme, as a complex with 3-phosphoglycerate (3PG), and as a complex with 3PG and ATP. The crystal structures were solved to 2.7, 2.0, and 2.7 A resolutions, respectively. The overall fold is nearly identical with previously solved mammalian PGK1 molecules. The apoenzyme is in the "open" form; that is the N-terminal domain that can bind 3PG and the C-terminal domain that binds ATP are too far apart for the substrates to interact. Binding 3PG causes a 13 degree rotation that partially closes the structure and causes helix 13, which is disordered in the unliganded structure, to stabilize. Binding ATP leaves the protein in the open configuration but also causes helix 13 to be ordered. Sequence alignment suggests that the active site of PGK2 is essentially identical to that of the cytoplasmic PGK1, but significant differences accumulate on a side of the C-terminal domain away from the active site. These changes may mediate the binding of this isoform to other proteins within the sperm flagellum, while still allowing the hinging action between the domains that is essential to catalytic activity.

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. Surprisingly, the wild-type protein did not crystallize with m(7)GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m(7)GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m(7)GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s(-1).

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. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.