The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected.
The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°.
AtzF; allphanate hydrolase; amidase domain; Pseudomonas sp. strain ADP
As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N9 substitution, or tautomerization arising from N7 substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N9-H···Val46 hydrogen bond, combined with the limited space available around the N9 position. The water-filled pocket under the N7 position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N7 (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N7 position towards the Mg2+-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg2+ centres or Mg2+ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.
Escherichia coli possesses two acyl ornithine aminotransferases, one catabolic (AstC) and the other anabolic (ArgD), that participate in L-arginine metabolism. Although only 58% identical, the enzymes have been shown to be functionally interchangeable. Here we have purified AstC and have obtained X-ray crystal structures of apo and holo-AstC and of the enzyme complexed with its physiological substrate, succinylornithine. We compare the structures obtained in this study with those of ArgD from Salmonella typhimurium obtained elsewhere, finding several notable differences. Docking studies were used to explore the docking modes of several substrates (ornithine, succinylornithine and acetylornithine) and the co-substrate glutamate/α-ketogluterate. The docking studies support our observations that AstC has a strong preference for acylated ornithine species over ornithine itself, and suggest that the increase in specificity associated with acylation is caused by steric and desolvation effects rather than specific interactions between the substrate and enzyme.
An apo form of human arginase I which is suitable for soaking experiments has been crystallized.
Arginase (EC 184.108.40.206) is an aminohydrolase that acts on l-arginine to produce urea and ornithine. Two isotypes of the enzyme are found in humans. Type I is predominantly produced in the liver and is a homotrimer of 35 kDa subunits. Human arginase (hArginase) I is seen to be up-regulated in many diseases and is a potential therapeutic target for many diverse indications. Previous reports of crystallization and structure determination of hArginase have always included inhibitors of the enzyme: here, the first case of a true apo crystal form of the enzyme which is suitable for small-molecule soaking is reported. The crystals belonged to space group P212121 and have approximate unit-cell parameters a = 53, b = 67.5, c = 250 Å. The crystals showed slightly anisotropic diffraction to beyond 2.0 Å resolution.
arginase; seeding; alternative reservoirs
The structure of human vascular adhesion protein 1 in space group C2 with seven molecules in the asymmetric unit has been refined at a resolution of 2.9 Å.
Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyte–endothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a = 394.5, b = 115.8, c = 179.3 Å, β = 112.3°. The structure was refined to a resolution of 2.9 Å, with R
cryst and R
free values of 0.250 and 0.286, respectively.
vascular adhesion protein 1; VAP-1; SSAO; copper amine oxidases; topaquinone; TPQ
A fragment-based screen against human immunodeficiency virus type 1 (HIV) integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF) binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.
The expression, purification and crystallization of S. aureus 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, an essential enzyme from the folate-biosynthesis pathway, is reported.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg2+-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 Å, α = γ = 90.0, β = 100.2°. The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V
M) of 2.04 Å3 Da−1 and an estimated solvent content of 39.6%.
HPPK; folate pathway; Staphylococcus aureus
Development of an ontology for the description of crystallization experiments and results is proposed.
When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world’s crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.
crystallization screening data; crystallization ontology
The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, Kd, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and 15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including 15N-1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.
Part of the latest SAMPL challenge was to predict how a small fragment library of 500 commercially available compounds would bind to a protein target. In order to assess the modellers’ work, a reasonably comprehensive set of data was collected using a number of techniques. These included surface plasmon resonance, isothermal titration calorimetry, protein crystallization and protein crystallography. Using these techniques we could determine the kinetics of fragment binding, the energy of binding, how this affects the ability of the target to crystallize, and when the fragment did bind, the pose or orientation of binding. Both the final data set and all of the raw images have been made available to the community for scrutiny and further work. This overview sets out to give the parameters of the experiments done and what might be done differently for future studies.
X-ray crystallography; Surface plasmon resonance; Isothermal titration calorimetry; Modelling; Fragment screening
Complexes of both hIGF-II and hIGF-IIE with a Fab have been crystallized and investigated by X-ray analysis.
Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab–IGF-II complexes (M64-F02–IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 Å resolution and belonged to space group P212121, with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 Å. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P212121, with unit-cell parameters a = 50.7, b = 107, c = 111.5 Å, and also diffracted X-rays to 2.2 Å resolution.
human insulin-like growth factor; antibodies
This paper describes how four general optimization techniques, growth-rate modulation, fine screening, seeding and additive screening, have been adapted for automation in a medium-throughput crystallization service facility.
The use of crystallization robots for initial screening in macromolecular crystallization is well established. This paper describes how four general optimization techniques, growth-rate modulation, fine screening, seeding and additive screening, have been adapted for automation in a medium-throughput crystallization service facility. The use of automation for more challenging optimization experiments is discussed, as is a novel way of using both the Mosquito and the Phoenix nano-dispensing robots during the setup of a single crystallization plate. This dual-dispenser technique plays to the strengths of both machines.
crystallization; automation; Phoenito experiments
We have identified a virus, B/Perth/211/2001, with a spontaneous mutation, D197E in the neuraminidase (NA), which confers cross-resistance to all NA inhibitors. We analyzed enzyme properties of the D197 and E197 NAs and compared these to a D197N NA, known to arise after oseltamivir treatment. Zanamivir and peramivir bound slowly to the wild type NA, but binding of oseltamivir was more rapid. The D197E/N mutations resulted in faster binding of all three inhibitors. Analysis of the crystal structures of D197 and E197 NAs with and without inhibitors showed that the D197E mutation compromised the interaction of neighboring R150 with the N-acetyl group, common to the substrate sialic acid and all NA inhibitors. Although rotation of the E275 in the NA active site occurs upon binding peramivir in both the D197 and E197 NAs, this does not occur upon binding oseltamivir in the E197 NA. Lack of the E275 rotation would also account for the loss of slow binding and the partial resistance of influenza B wild type NAs to oseltamivir.
Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.
Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F420H2 to catalyse the reduction of the α,β-unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F420H2 dependent reductase (FDR-A and -B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5′-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F420H2 binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro-reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.
The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the ‘Toblerone’ fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser–Lys catalytic dyads. A single catalytic dyad (Ser85–Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure–function relationship.