Crystallographic insights into a cobalt (III) sepulchrate based alternative cofactor system of P450 BM3 monooxygenase.

P450 BM3 is a multi-domain heme-containing soluble bacterial monooxygenase. P450 BM3 and variants are known to oxidize structurally diverse substrates. Crystal structures of individual domains of P450 BM3 are available. However, the spatial organization of the full-length protein is unknown. In this study, crystal structures of the P450 BM3 M7 heme domain variant with and without cobalt (III) sepulchrate are reported. Cobalt (III) sepulchrate acts as an electron shuttle in an alternative cofactor system employing zinc dust as the electron source. The crystal structure shows a binding site for the mediator cobalt (III) sepulchrate at the entrance of the substrate access channel. The mediator occupies an unusual position which is far from the active site and distinct from the binding of the natural redox partner (FAD/NADPH binding domain).

Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ß strands stacked between α helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

A Crystallin Fold in the Interleukin-4-inducing Principle of Schistosoma mansoni Eggs (IPSE/α-1) Mediates IgE Binding for Antigen-independent Basophil Activation.

The IL-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1), the major secretory product of eggs from the parasitic worm S. mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/α-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEΔNLS, a monomeric IPSE/α-1 mutant, revealed that IPSE/α-1 is a new member of the ßγ-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEΔNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/α-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/α-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism.

Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.

Cloning, expression, purification and preliminary X-ray analysis of EstN2, a novel archaeal α/ß-hydrolase from Candidatus Nitrososphaera gargensis.

EstN2 is a novel α/ß-hydrolase originating from the ammonia-oxidizing thaumarchaeon Candidatus Nitrososphaera gargensis. The genome of the organism was sequenced and genes conferring putative lipolytic activity were amplified and cloned into Escherichia coli as a heterologous host. Through function-based screening, esterase and lipase activity was detected. A recombinant enzyme designated EstN2 was successfully expressed, purified and crystallized. The crystals belonged to space group I2, with one molecule per asymmetric unit, and diffracted X-rays to 1.5 Šresolution.

Cloning, overexpression, purification and preliminary X-ray analysis of the protein kinase domain of enhanced disease resistance 1 (EDR1) from Arabidopsis thaliana.

Enhanced disease resistance 1 is a member of the Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK) family that negatively regulates disease resistance, ethylene-induced senescence and programmed cell death in response to both abiotic and biotic stresses. A catalytically inactive form of the EDR1 kinase domain was successfully cloned, expressed, purified and crystallized. Crystallization was conducted in the presence of the ATP analogue AMP-PNP. The crystals belonged to space group P3221 and contained two molecules in the asymmetric unit. The crystals diffracted X-rays to 2.55 Šresolution.

Automation in biological crystallization.

Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given.

Crystallographic identification of an unexpected protein complex in silkworm haemolymph.

The first crystal structure of a complex formed by two storage proteins, SP2 and SP3, isolated from their natural source, mulberry silkworm (Bombyx mori L.) haemolymph, has been determined. The structure was solved by molecular replacement using arylphorin, a protein rich in aromatic amino-acid residues, from oak silkworm as the initial model. The quality of the electron-density maps obtained from the X-ray diffraction experiment allowed the authors to detect that the investigated crystal structure was composed of two different arylphorins: SP2 and SP3. This discovery was confirmed by N-terminal sequencing. SP2 has been extensively studied previously, whereas only a few reports on SP3 are available. However, to date no structural studies have been reported for these proteins. These studies revealed that SP2 and SP3 exist in the silkworm body as a heterohexamer formed by one SP2 trimer and one SP3 trimer. The overall fold, consisting of three haemocyanin-like subdomains, of SP2 and SP3 is similar. Both proteins contain a conserved N-glycosylation motif in their structures.

Cloning, overexpression, purification and preliminary X-ray analysis of the catalytic domain of the ethylene receptor ETR1 from Arabidopsis thaliana.

Ethylene is a gaseous plant hormone which controls many aspects of plant growth and development. It is perceived by membrane-bound receptors with a similarity to bacterial two-component systems. The catalytic and ATP-binding domain of the histidine kinase domain of ETR1 from Arabidopsis thaliana has been cloned, overexpressed and crystallized. The protein was crystallized together with various nucleotides. Crystals obtained in the presence of ADP belonged to space group I222 or I2(1)2(1)2(1) with one molecule per asymmetric unit. They diffracted X-ray radiation to beyond 1.85 Šresolution.

Cloning, expression, purification and preliminary X-ray analysis of the dimerization domain of ethylene response sensor 1 (ERS1) from Arabidopsis thaliana.

Ethylene signalling is initiated by a group of membrane-bound receptors with similarity to two-component systems. ERS1 belongs, together with ETR1, to subfamily 1, which plays a predominant role in ethylene signalling. The dimerization domain of ERS1 was crystallized in space groups C222(1) and P2(1)2(1)2, with two and four molecules per asymmetric unit, respectively. The crystals diffracted X-ray radiation to 1.9 Šresolution.

Two crystal structures of Bombyx mori lipoprotein 3 - structural characterization of a new 30-kDa lipoprotein family member.

The 30-kDa family of lipoproteins from insect hemolymph has been the focus of a number of studies over the last few years. Recently, four crystal structures of Bombyx mori lipoprotein 7 have been determined. Here we report two crystal structures of another member of the 30-kDa lipoprotein family, Bombyx mori lipoprotein 3 (Bmlp3). The protein was isolated from its natural source, mulberry silkworm hemolymph. It crystallized in two different crystal forms, Bmlp3-p21 (space group P21) and Bmlp3-c2 (space group C2). The crystal structures were solved by molecular replacement using the coordinates of Bmlp7 as a starting model. The crystals of Bmlp3-p21 diffracted X-rays to 2.4 Å resolution and of Bmlp3-c2 to 2.1 Å resolution. Bmlp3 has an overall fold characteristic of 30-kDa lipoproteins, with a VHS-type N-terminal domain and ß-trefoil C-terminal domain. Structural comparison of Bmlp3 and Bmlp7 shows that the loops present in the C-terminal domain are flexible and participate in dimer formation. Additionally, new putative binding sites of Bmlp3 have been analyzed in detail and the electrostatic potential of the protein surface at physiological pH 7.4 conditions has been calculated. The results of these calculations are the starting point for an explanation of the recently reported cell-penetrating properties of the 30-kDa lipoproteins.

P450 BM3 crystal structures reveal the role of the charged surface residue Lys/Arg184 in inversion of enantioselective styrene epoxidation.

Solved crystal structures of P450 BM3 variants in complex with styrene provide on the molecular level a first explanation of how a positively charged surface residue inverts the enantiopreference of styrene epoxidation. The obtained insights into productive and non-productive styrene binding modes deepened our understanding of enantioselective epoxidation with P450 BM3.

Optimization of protein buffer cocktails using Thermofluor.

The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing.

The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

High-resolution structure of Bombyx mori lipoprotein 7: crystallographic determination of the identity of the protein and its potential role in detoxification.

Three crystal structures of a lipoprotein (Bmlp7) of unknown function, a member of the 30 kDa lipoprotein family from mulberry silkworm (Bombyx mori L.) haemolymph, have been determined. The 1.33 Å resolution structure is an excellent example of how a precise crystallographic study can contribute to protein identification. The correct sequence of this haemolymph-isolated protein was assigned thanks to superb-quality electron-density maps. Two unexpected cadmium cations were found in this crystal structure [Bmlp7-I(Cd)] and their presence may be connected to a detoxification mechanism in this insect. For a comparison of the metal-binding sites, the crystal structure of a platinum complex (Bmlp7-Pt) was also solved at 1.94 Å resolution. The third (2.50 Å resolution) structure, of the native protein harvested in a different season (Bmlp7-II), corresponds to a different polymorph with an altered pattern of intermolecular interactions and with a total absence of cadmium ions and highlights the possible involvement of Bmlp7 in the response to environmental pollution. The N-terminal domain of Bmlp7 has a fold resembling a clockwise spiral created by six helices and can be classified as a VHS domain. The C-terminal domain is folded as a ß-trefoil. The biological function of Bmlp7 is unknown, but its structural homology to sugar-binding proteins suggests that, in analogy to other 30 kDa haemolymph lipoproteins, it could play a role as an anti-apoptotic factor or function in the immune response of the insect to fungal infections.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of succinyl-diaminopimelate desuccinylase (Rv1202, DapE) from Mycobacterium tuberculosis.

Succinyl-diaminopimelate desuccinylase from Mycobacterium tuberculosis (DapE, Rv1202) has been cloned, heterologously expressed in Escherichia coli and purified using standard chromatographic techniques. Diffraction-quality crystals were obtained at acidic pH from ammonium sulfate and PEG and diffraction data were collected from two crystals to resolutions of 2.40 and 2.58 Å, respectively. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 79.7, b = 76.0, c = 82.9 Å, ß = 119°. The most probable content of the asymmetric unit was two molecules of DapE, which would correspond to a solvent content of 56%. Both examined crystals turned out to be pseudo-merohedrally twinned, with twin operator -h, -k, h + l and twin fractions of approximately 0.46 and 0.16, respectively.

Cloning, expression, purification and preliminary X-ray analysis of a putative metagenome-derived lipase.

LipS is a novel thermostable putative lipase that was isolated from a metagenomic library using functional screening methods. The corresponding gene shows high similarity to that encoding a putative but uncharacterized esterase from Symbiobacterium thermophilum IAM14863 (99% nucleotide-sequence similarity). Two different constructs of the recombinant lipase were crystallized. Crystals belonging to space group P4(2)2(1)2 diffracted X-ray radiation to 2.8 Šresolution and crystals belonging to space group P4 diffracted to 2.0 Šresolution. The most probable content of their asymmetric units were two molecules (P4(2)2(1)2) and four or five molecules (P4), respectively.

Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk.

Ethylene controls many aspects of plant growth and development. Signaling by the gaseous phytohormone is initiated by disulfide-linked membrane-bound receptors, and the formation of heteromeric receptor clusters contributes to the broad range of ethylene responsiveness. In Arabidopsis thaliana, the TCS-like ethylene receptors interact with the cytosolic serine/threonine kinase constitutive triple response 1 (CTR1), a proposed mitogen-activated protein kinase kinase kinase. In the absence of the hormone, the receptor and therefore CTR1 are active. Hence, ethylene acts as an inverse agonist of its signaling pathway. The three-dimensional structures of the active, triphosphorylated and the unphosphorylated, inactive kinase domain of CTR1 in complex with staurosporine illustrate the conformational rearrangements that form the basis of activity regulation. Additionally, in analytical ultracentrifugation experiments, active kinase domains form back-to-back dimers, while inactive and activation loop variants are monomers. Together with a front-to-front activation interface, the active protein kinase dimers thereby engage in interactions that promote CTR1-mediated cross talk between ethylene receptor clusters. This model provides a structural foundation for the observed high sensitivity of plants to ethylene.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulatory domain of aspartokinase (Rv3709c) from Mycobacterium tuberculosis.

The regulatory domain of Mycobacterium tuberculosis aspartokinase (Mtb-AK, Mtb-Ask, Rv3709c) has been cloned, heterologously expressed in Escherichia coli and purified using standard chromatographic techniques. Screening for initial crystallization conditions using the regulatory domain (AK-ß) in the presence of the potential feedback inhibitor threonine identified four conditions which yielded crystals suitable for X-ray diffraction analysis. From these four conditions five different crystal forms of Mtb-AK-ß resulted, three of which belonged to the orthorhombic system, one to the tetragonal system and one to the monoclinic system. The highest resolution (1.6 Å) was observed for a crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=53.70, b=63.43, c=108.85 Šand two molecules per asymmetric unit.

Cloning, expression, purification and preliminary X-ray analysis of the protein kinase domain of constitutive triple response 1 (CTR1) from Arabidopsis thaliana.

Ethylene, a gaseous plant hormone, is perceived by a group of membrane-bound receptors. Constitutive triple response 1 (CTR1) from Arabidopsis thaliana directly interacts with ethylene receptors and thus links signal reception to the intracellular signalling pathway. The C-terminal protein kinase domain of CTR1 has been crystallized in its wild-type form and as a kinase-dead mutant. The wild-type crystals diffracted X-ray radiation to 3 Šresolution and the crystals of the kinase-dead mutant diffacted to 2.5 Šresolution. The crystals belonged to space groups P4(1)2(1)2 and P4(2)2(1)2, respectively, with two molecules per asymmetric unit in both cases.