Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex.

The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein-DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used (31)P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.

Rational reprogramming of fungal polyketide first-ring cyclization.

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-ß-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

Biochemical and structural studies of 6-carboxy-5,6,7,8-tetrahydropterin synthase reveal the molecular basis of catalytic promiscuity within the tunnel-fold superfamily.

6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds.

(Z)-Stereoselective Synthesis of Mono- and Bis-heterocyclic Benzimidazol-2-ones via Cascade Processes Coupled with the Ugi Multicomponent Reaction.

Several novel cascade reactions are herein reported that enable access to a variety of unique mono- and bis-heterocyclic scaffolds. The sequence of cascade events are mediated through acid treatment of an Ugi adduct that affords 1,5-benzodiazepines which subsequently undergo an elegant rearrangement to deliver (E)-benzimidazolones, which through acid-promoted tautomerization convert to their corresponding (Z)-isomers. Moreover, a variety of heterocycles tethered to (Z)-benzimidazole-2-ones are also accessible through similar domino-like processes, demonstrating a general strategy to access significantly new scaffold diversity, each containing four points of potential diversification. Final structures of five scaffolds have been definitively proven by X-ray crystallography.

The Synthesis of Stable, Complex Organocesium Tetramic Acids through the Ugi Reaction and Cesium-Carbonate-Promoted Cascades.

Two structurally unique organocesium carbanionic tetramic acids have been synthesized through expeditious and novel cascade reactions of strategically functionalized Ugi skeletons delivering products with two points of potential diversification. This is the first report of the use of multicomponent reactions and subsequent cascades to access complex, unprecedented organocesium architectures. Moreover, this article also highlights the first use of mild cesium carbonate as a cesium source for the construction of cesium organometallic scaffolds. Relativistic DFT calculations provide an insight into the electronic structure of the reported compounds.

Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking.

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The ß1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

(Z)-N-tert-Butyl-2-(4-meth-oxy-anilino)-N'-(4-meth-oxy-phen-yl)-2-phenyl-acetimidamide.

In the crystal of the title compound, C26H31N3O2, pairs of N-H⋯O hydrogen bonds link molecules, forming inversion dimers, which enclose an R 2 (2)(20) ring motif. One N atom does not form hydrogen bonds and lies in a hydro-phobic pocket with closest inter-molecular contacts of 4.196 (2) and 4.262 (2) Å.

tert-Butyl 4-(3,4-dichloro-anilino)piperidine-1-carboxyl-ate.

In the title compound, C(16)H(22)Cl(2)N(2)O(2), the substituted piperidine ring adopts a chair conformation with both substituents in equatorial positions. In the crystal, N-H⋯O and C-H⋯O hydrogen bonds connect mol-ecules into ribbons along the a-axis direction.

3-(4-Bromo-phen-yl)-1-butyl-5-[1-(2-chloro-6-methyl-phen-yl)-1H-tetra-zol-5-yl]imidazolidine-2,4-dione.

In the title mol-ecule, C21H20BrClN6O2, the chloro-substituted benzene ring forms a dihedral angle of 77.84 (7)° with the tetra-zole ring and the bromo-substituted ring forms a dihedral angle of 43.95 (6)° with the imidazole ring. The dihedral angle between the tetra-zole and imidazole rings is 67.42 (8)°. The terminal methyl group of the butyl substituent is disordered over two sets of sites, with refined occupancies 0.67 (3) and 0.33 (3). In the crystal, there is a short Br⋯N contact of 3.183 (2) Å.

Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Šwith the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.

2,4-Diphenyl-6-trifluoro-methyl-2,3-dihydro-1H,5H-pyrrolo-[3,4-c]pyrrole-1,3-dione.

The asymmetric unit of the title compound, C(19)H(11)F(3)N(2)O(2), contains two crystallographically unique mol-ecules which differ in the rotation of a phenyl ring and a -CF(3) substituent. The dihedral angles involving the pyrrole ring and the attached phenyl ring are 62.82 (8) and 71.54 (7)° in the two molecules. The difference in the rotation of the CF(3) groups with respect to the pyrrolo rings to which they are attached is 23.5(1)°. For one mol-ecule, there is a close contact between an H atom and the centroid of the phenyl ring of an adjacent mol-ecule (2.572 Å). A similar contact is lacking in the second mol-ecule. In the crystal, N-H⋯O inter-actions connect adjacent mol-ecules into a chain normal to (01[Formula: see text]). Crystallographically unique mol-ecules alternate along the hydrogen-bonded chains.

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds.

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a "stepping-stone" method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and lambda. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and lambda. The domains show 40% sequence identity but differ by switching of alpha-helix to beta-sheet in a C-terminal region spanning approximately 25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.

Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor.

The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 A resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.

Structure of the Y94F mutant of Escherichia coli thymidylate synthase.

Tyr94 of Escherichia coli thymidylate synthase is thought to be involved, either directly or by activation of a water molecule, in the abstraction of a proton from C5 of the 2'-deoxyuridine 5'-monophosphate (dUMP) substrate. Mutation of Tyr94 leads to a 400-fold loss in catalytic activity. The structure of the Y94F mutant has been determined in the native state and as a ternary complex with thymidine 5'-monophosphate (dTMP) and 10-propargyl 5,8-dideazafolate (PDDF). There are no structural changes ascribable to the mutation other than loss of a water molecule hydrogen bonded to the tyrosine OH, which is consistent with a catalytic role for the phenolic OH.

Crystal structures of N-tert-butyl-3-(4-fluoro-phenyl)-5-oxo-4-[2-(tri-fluoro-meth-oxy)phen-yl]-2,5-di-hydro-furan-2-carboxamide and 4-(2H-1,3-benzodioxol-5-yl)-N-cyclo-hexyl-5-oxo-3-[4-(tri-fluoro-meth-yl)phen-yl]-2,5-di-hydro-furan-2-carboxamide.

The title compounds, C22H19F4NO4, (I), and C25H22F3NO5, (II), each contain a central nearly planar di-hydro-furan-one ring. The r.m.s. deviation from planarity of these rings is 0.015 Šin (I) and 0.027 Šin (II). The mol-ecules are T-shaped, with the major conformational difference being the O-C-C-O torsion angle [-178.9 (1) in (I) and 37.7 (2)° in (II)]. In the crystal of (I), mol-ecules are linked by N-H⋯O hydrogen bonds, forming chains along [001] while in (II) mol-ecules are linked by N-H⋯O hydrogen bonds, forming chains along [010]. In (II), the tri-fluoro-methyl substituent is disordered over two sets of sites, with refined occupancies of 0.751 (3) and 0.249 (3).

IMPROVED SYNTHESIS OF 10-(2-ALKYLAMINO-2-OXOETHYL)-1,4,7,10-TETRAAZACYCLODODECANE-1,4,7-TRIACETIC ACID DERIVATIVES BEARING ACID-SENSITIVE LINKERS.

Alkylation of the hydrobromide salts of 1,4,7-tris(methoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane and 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane with appropriate α-bromoacetamides, followed by hydrolysis, provides convenient access to 10-(2-alkylamino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivatives that contain acid-sensitive functional groups. The utility of the method is demonstrated by improved syntheses of two known DOTA monoamides bearing acid-sensitive ω-tritylthio alkyl chains in much higher yields based on cyclen as the starting material.

Conformations of the apo-, substrate-bound and phosphate-bound ATP-binding domain of the Cu(II) ATPase CopB illustrate coupling of domain movement to the catalytic cycle.

Heavy metal P1B-type ATPases play a critical role in cell survival by maintaining appropriate intracellular metal concentrations. Archaeoglobus fulgidus CopB is a member of this family that transports Cu(II) from the cytoplasm to the exterior of the cell using ATP as energy source. CopB has a 264 amino acid ATPBD (ATP-binding domain) that is essential for ATP binding and hydrolysis as well as ultimately transducing the energy to the transmembrane metal-binding site for metal occlusion and export. The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion. Through crystal structures of the apo- and phosphate-bound ATPBDs, with limited proteolysis and fluorescence studies of the apo- and substrate-bound states, we show that the isolated ATPBD of CopB cycles from an open conformation in the apo-state to a closed conformation in the substrate-bound state, then returns to an open conformation suitable for product release. The present work is the first structural report of an ATPBD with its physiologically relevant product (phosphate) bound. The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation. These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.

A new use for a familiar fold: the X-ray crystal structure of GTP-bound GTP cyclohydrolase III from Methanocaldococcus jannaschii reveals a two metal ion catalytic mechanism.

GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg2+ for catalytic activity and is activated by monovalent cations such as K+ and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 A resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp-->Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.

Two structures of a lambda Cro variant highlight dimer flexibility but disfavor major dimer distortions upon specific binding of cognate DNA.

Previously reported crystal structures of free and DNA-bound dimers of lambda Cro differ strongly (about 4 A backbone rmsd), suggesting both flexibility of the dimer interface and induced-fit protein structure changes caused by sequence-specific DNA binding. Here, we present two crystal structures, in space groups P3(2)21 and C2 at 1.35 and 1.40 A resolution, respectively, of a variant of lambda Cro with three mutations in its recognition helix (Q27P/A29S/K32Q, or PSQ for short). One dimer structure (P3(2)21; PSQ form 1) resembles the DNA-bound wild-type Cro dimer (1.0 A backbone rmsd), while the other (C2; PSQ form 2) resembles neither unbound (3.6 A) nor bound (2.4 A) wild-type Cro. Both PSQ form 2 and unbound wild-type dimer crystals have a similar interdimer beta-sheet interaction between the beta1 strands at the edges of the dimer. In the former, an infinite, open beta-structure along one crystal axis results, while in the latter, a closed tetrameric barrel is formed. Neither the DNA-bound wild-type structure nor PSQ form 1 contains these interdimer interactions. We propose that beta-sheet superstructures resulting from crystal contact interactions distort Cro dimers from their preferred solution conformation, which actually resembles the DNA-bound structure. These results highlight the remarkable flexibility of lambda Cro but also suggest that sequence-specific DNA binding may not induce large changes in the protein structure.