P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Structural basis for cell type specific DNA binding of C/EBPß: The case of cell cycle inhibitor p15INK4b promoter.

C/EBPß is a key regulator of numerous cellular processes, but it can also contribute to tumorigenesis and viral diseases. It binds to specific DNA sequences (C/EBP sites) and interacts with other transcription factors to control expression of multiple eukaryotic genes in a tissue and cell-type dependent manner. A body of evidence has established that cell-type-specific regulatory information is contained in the local DNA sequence of the binding motif. In human epithelial cells, C/EBPß is an essential cofactor for TGFß signaling in the case of Smad2/3/4 and FoxO-dependent induction of the cell cycle inhibitor, p15INK4b. In the TGFß-responsive region 2 of the p15INK4b promoter, the Smad binding site is flanked by a C/EBP site, CTTAA•GAAAG, which differs from the canonical, palindromic ATTGC•GCAAT motif. The X-ray crystal structure of C/EBPß bound to the p15INK4b promoter fragment shows how GCGC-to-AAGA substitution generates changes in the intermolecular interactions in the protein-DNA interface that enhances C/EBPß binding specificity, limits possible epigenetic regulation of the promoter, and generates a DNA element with a unique pattern of methyl groups in the major groove. Significantly, CT/GA dinucleotides located at the 5'ends of the double stranded element maintain local narrowing of the DNA minor groove width that is necessary for DNA recognition. Our results suggest that C/EBPß would accept all forms of modified cytosine in the context of the CpT site. This contrasts with the effect on the consensus motif, where C/EBPß binding is modestly increased by cytosine methylation, but substantially decreased by hydroxymethylation.

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.

Fever, confusion, acute kidney injury: is this atypical neuroleptic malignant syndrome following polypharmacy with clozapine and risperidone?

OBJECTIVE: Clozapine is the gold-standard antipsychotic medication for treatment-refractory schizophrenia (TRS). However, one potentially lethal side effect of clozapine, as with other antipsychotics, is neuroleptic malignant syndrome (NMS) which could present differently in clozapine therapy. 'Atypical NMS' is a recognised variant of NMS with less rigidity and delayed elevation of creatine kinase; this variant is associated with clozapine. METHOD: A case from the author's clinical practice was reviewed. RESULTS: A 67-year-old man with TRS was treated with clozapine. Unfortunately, his physical condition deteriorated and he presented with atypical NMS, which initially was treated as presumable urinary tract infection. CONCLUSIONS: Atypical NMS is associated with clozapine. This case exposes the potential difficulties in diagnosis, and highlights the importance of considering less common diagnoses in acutely unwell psychiatric patients.

Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300.

Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723-1818 of p300 Taz2 and residues 37-61 of C/EBPℇ. The segment corresponding to the C/EBPℇ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBPℇ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP-p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPß N-terminus, which associates with the C/EBPß-p300 complex.

Differential temperature dependence of tobacco etch virus and rhinovirus 3C proteases.

Because of their stringent sequence specificity, the 3C-like proteases from tobacco etch virus (TEV) and human rhinovirus are often used for the removal of affinity tags. The latter enzyme is rumored to have greater catalytic activity at 4 °C, the temperature at which fusion protein substrates are usually digested. Here we report that experiments with fusion protein and peptide substrates confirm this conjecture. Whereas the catalytic efficiency of rhinovirus 3C protease is approximately the same at its optimum temperature (30 °C) and at 4 °C, TEV protease is 10-fold less active at the latter temperature due primarily to a reduction in k(cat).

Structure of Helicobacter pylori dihydroneopterin aldolase suggests a fragment-based strategy for isozyme-specific inhibitor design.

Dihydroneopterin aldolase (DHNA) is essential for folate biosynthesis in microorganisms. Without a counterpart in mammals, DHNA is an attractive target for antimicrobial agents. Helicobacter pylori infection occurs in human stomach of over 50% of the world population, but first-line therapies for the infection are facing rapidly increasing resistance. Novel antibiotics are urgently needed, toward which structural information on potential targets is critical. We have determined the crystal structure of H. pylori DHNA (HpDHNA) in complex with a pterin molecule (HpDHNA:Pterin) at 1.49-Å resolution. The HpDHNA:Pterin complex forms a tetramer in crystal. The tetramer is also observed in solution by dynamic light scattering and confirmed by small-angle X-ray scattering. To date, all but one reported DHNA structures are octameric complexes. As the only exception, ligand-free Mycobacterium tuberculosis DHNA (apo-MtDHNA) forms a tetramer in crystal, but its active sites are only partially formed. In contrast, the tetrameric HpDHNA:Pterin complex has well-formed active sites. Each active site accommodates one pterin molecule, but the exit of active site is blocked by two amino acid residues exhibiting a contact distance of 5.2 â€‹Å. In contrast, the corresponding contact distance in Staphylococcus aureus DHNA (SaDHNA) is twice the size, ranging from 9.8 to 10.5 â€‹Å, for ligand-free enzyme, the substrate complex, the product complex, and an inhibitor complex. This large contact distance indicates that the active site of SaDHNA is wide open. We propose that this isozyme-specific contact distance (ISCD) is a characteristic feature of DHNA active site. Comparative analysis of HpDHNA and SaDHNA structures suggests a fragment-based strategy for the development of isozyme-specific inhibitors.

Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation.

Coactivators CREB-binding protein and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short alpha helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p53(1-39) phosphorylated at Thr(18) is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser(15) and Thr(18).

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Structure and activity of PPX/GppA homologs from Escherichia coli and Helicobacter pylori.

Rapid adaptation to environmental changes is crucial for bacterial survival. Almost all bacteria possess a conserved stringent response system to prompt transcriptional and metabolic responses toward stress. The adaptive process relies on alarmones, guanosine pentaphosphate (pppGpp), and tetraphosphate (ppGpp), to regulate global gene expression. The ppGpp is more potent than pppGpp in the regulatory activity, and pppGpp phosphohydrolase (GppA) plays a key role in (p)ppGpp homeostasis. Sharing a similar domain structure, GppA is indistinguishable from exopolyphosphatase (PPX), which mediates the metabolism of cellular inorganic polyphosphate. Here, our phylogenetic analysis of PPX/GppA homologs in bacteria shows a wide distribution with several distinct subfamilies, and our structural and functional analysis of Escherichia coli GppA and Helicobacter pylori PPX/GppA reveals unique properties of each homolog. These results explain how each homolog possesses its distinct functionality.

Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation.

The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive posttranslational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogues CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser(15) or Thr(18) increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser(15) and Ser(37) or Thr(18) and Ser(20) further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 posttranslational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation, the change in heat capacity at constant pressure, DeltaC(p), was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of DeltaC(p) for Taz2 binding to the first (-330 cal/(mol.K)) compared to the second site (-234 cal/(mol.K)) suggests that the importance of nonpolar and polar interactions differs between the two sites.

Overproduction, purification and structure determination of human dual-specificity phosphatase 14.

Dual-specificity phosphatases (DUSPs) are enzymes that participate in the regulation of biological processes such as cell growth, differentiation, transcription and metabolism. A number of DUSPs are able to dephosphorylate phosphorylated serine, threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs) and thus are also classified as MAPK phosphatases (MKPs). As an increasing number of DUSPs are being identified and characterized, there is a growing need to understand their biological activities at the molecular level. There is also significant interest in identifying DUSPs that could be potential targets for drugs that modulate MAPK-dependent signaling and immune responses, which have been implicated in a variety of maladies including cancer, infectious diseases and inflammatory disorders. Here, the overproduction, purification and crystal structure at 1.88 A resolution of human dual-specificity phosphatase 14, DUSP14 (MKP6), are reported. This structural information should accelerate the study of DUSP14 at the molecular level and may also accelerate the discovery and development of novel therapeutic agents.

Structure of the Taz2 domain of p300: insights into ligand binding.

CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813-1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed.

Expression and purification of soluble His(6)-tagged TEV protease.

This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease.

Successful clozapine rechallenge in a patient with suspected drug induced lupus.

Clozapine is the most effective treatment for patients with refractory schizophrenia. Clozapine is also associated with serious and potentially lethal side effects including drug induced lupus (DIL). There have been four previous published case reports describing clozapine inducing a lupus-like syndrome including one previous case where a clozapine rechallenge was attempted without success. This case report describes a successful clozapine rechallenge in a patient with suspected DIL.

Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.

Energy-dependent Lon proteases play a key role in cellular regulation by degrading short-lived regulatory proteins and misfolded proteins in the cell. The structure of the catalytically inactive S679A mutant of Escherichia coli LonA protease (EcLon) has been determined by cryo-EM at the resolution of 3.5 Å. EcLonA without a bound substrate adopts a hexameric open-spiral quaternary structure that might represent the resting state of the enzyme. Upon interaction with substrate the open-spiral hexamer undergoes a major conformational change resulting in a compact, closed-circle hexamer as in the recent structure of a complex of Yersinia pestis LonA with a protein substrate. This major change is accomplished by the rigid-body rearrangement of the individual domains within the protomers of the complex around the hinge points in the interdomain linkers. Comparison of substrate-free and substrate-bound Lon structures allows to mark the location of putative pivotal points involved in such conformational changes.

Targeting Protein-Protein Interactions of Tyrosine Phosphatases with Microarrayed Fragment Libraries Displayed on Phosphopeptide Substrate Scaffolds.

Chemical library screening approaches that focus exclusively on catalytic events may overlook unique effects of protein-protein interactions that can be exploited for development of specific inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide motifs comprise minimal recognition elements that determine the substrate specificity of protein tyrosine phosphatases (PTPases). We incorporated aminooxy-containing amino acid residues into a 7-residue epidermal growth factor receptor (EGFR) derived phosphotyrosine-containing peptide and subjected the peptides to solution-phase oxime diversification by reacting with aldehyde-bearing druglike functionalities. The pTyr residue remained unmodified. The resulting derivatized peptide library was printed in microarrays on nitrocellulose-coated glass surfaces for assessment of PTPase catalytic activity or on gold monolayers for analysis of kinetic interactions by surface plasmon resonance (SPR). Focusing on amino acid positions and chemical features, we first analyzed dephosphorylation of the peptide pTyr residues within the microarrayed library by the human dual-specificity phosphatases (DUSP) DUSP14 and DUSP22, as well as by PTPases from poxviruses (VH1) and Yersinia pestis (YopH). In order to identify the highest affinity oxime motifs, the binding interactions of the most active derivatized phosphopeptides were examined by SPR using noncatalytic PTPase mutants. On the basis of high-affinity oxime fragments identified by the two-step catalytic and SPR-based microarray screens, low-molecular-weight nonphosphate-containing peptides were designed to inhibit PTP catalysis at low micromolar concentrations.

Crystal structure of the human dual specificity phosphatase 1 catalytic domain.

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38-alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.

A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag.

A generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His6-MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His6-tobacco etch virus protease to separate the His6-MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique.