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A click-based oxime protocol to extend small molecule microarray-derived TDP1 inhibitory platform to project into the DNA and TOP1 peptide substrate-binding channels.

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

C/EBPβ 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/EBPβ is an essential cofactor for TGFβ signaling in the case of Smad2/3/4 and FoxO-dependent induction of the cell cycle inhibitor, p15INK4b. In the TGFβ-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/EBPβ 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/EBPβ 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/EBPβ 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/EBPβ binding is modestly increased by cytosine methylation, but substantially decreased by hydroxymethylation.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3′- and 5′-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3′-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors., Using small molecule microarray TDP1 inhibitors have been identified that bind in a trivalent mode.

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., Graphical abstract In Brief The structure of the E. coli LonA protease was determined by cryo-EM. The unliganded hexameric complex adopts an open spiral quaternary structure. Upon interaction with substrate it likely undergoes a rigid-body conformational change resulting in a compact, closed-circle hexamer as reported in the Yersinia pestis Lon structure.Image 1, Highlights • E. coli LonA(S679A) protease adopts an open spiral quaternary structure. • This conformation might represent the resting state of the complex. • Upon substrate binding the open spiral will close into a circular hexamer.

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 A 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.

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.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

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.

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.

4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in complex with pNPP. The structure illuminates the molecular basis for substrate binding and may also facilitate the structure-assisted development of DUSP22 inhibitors.

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.

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/EBP∊. The segment corresponding to the C/EBP∊ 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/EBP∊ 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/EBPβ N-terminus, which associates with the C/EBPβ–p300 complex.

We have developed competitive and direct binding methods to examine small-molecule inhibitors of protein tyrosine phosphatase activity. Focusing on the Yersinia pestis outer protein H, a potent bacterial protein tyrosine phosphatase, we describe how an understanding of the kinetic interactions involving Yersinia pestis outer protein H, peptide substrates, and small-molecule inhibitors of protein tyrosine phosphatase activity can be beneficial for inhibitor screening, and we further translate these results into a microarray assay for high-throughput screening.

Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed.

The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.

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.

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.

Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here, we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

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.

Yersinia pestis, the causative agent of the plague, employs a type III secretion system (T3SS) to secrete and translocate virulence factors into to the cytoplasm of mammalian host cells. One of the secreted virulence factors is YopR. Little is known about the function of YopR other than that it is secreted into the extracellular milieu during the early stages of infection and that it contributes to virulence. Hoping to gain some insight into the function of YopR, we determined the crystal structure of its protease-resistant core domain, which consists of residues 38-149 out of 165 amino acids. The core domain is composed of five alpha-helices that display unexpected structural similarity with one domain of YopN, a central regulator of type III secretion in Y. pestis. This finding raises the possibility that YopR may play a role in the regulation of type III secretion.

VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave alpha-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 A resolution. The shape of the molecule resembles the letter "V," with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as "chaperones" to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 A resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

Summary RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family represented by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the family. Previously, we have shown that RNase III uses two catalytic sites to create the 2-nucleotide (nt) 3′ overhangs in its products. Here, we present three crystal structures of RNase III in complex with double-stranded RNA, demonstrating how Mg2+ is essential for the formation of a catalytically competent protein–RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modelled a protein–substrate complex and a protein–reaction intermediate (transition state) complex on the basis of the crystal structures. Together, the crystal structures and the models suggest a stepwise mechanism for RNase III to execute the phosphoryl transfer reaction.

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9–5.8 mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1–α2–α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity.

Cell contact by the plague bacterium Yersinia pestis initiates the injection of several virulence factors that target biochemical pathways critical for host clearance of bacteria. Despite this impairment of innate immunity, it is unclear whether antigen recognition by T cells is equally affected. We present evidence that human cytolytic T cells respond to Y. pestis virulence proteins presented by infected monocytes and dendritic cells. These T cell antigens consisted of a panel of proteins encoded by pCD1, a 70-kDa plasmid that harbors virulence factors and transport proteins of the cell contact-dependent, type III secretion system. Infected cells retained the ability to process and present tetanus toxoid to T cells, which indicates that responses to unrelated antigens were also maintained. Our results indicate that T cell immunity remains functional during Y. pestis infection, which thus suggests the potential benefits of therapeutic vaccination and strategies that emphasize the inclusion of cytotoxic T lymphocyte responses.

The proto-oncogenic properties of the POK family of transcriptional repressors BCL6, PLZF, and LRF have been well established. These proteins utilize their amino-terminal POZ domains for multimerization and the recruitment of co-repressors. Because LRF represses the production of the tumor suppressor p19(Arf) (ARF), it is regarded as an attractive therapeutic target for the treatment of many types of cancer. The crystal structure of the LRF POZ domain reveals a high degree of structural conservation with the corresponding domains of BCL6 and PLZF. However, striking differences between the electrostatic properties of the BCL6 and LRF POZ domains suggest that if, like BCL6, LRF interacts with the co-repressor SMRT, it almost certainly uses a different mechanism to do so. These differences may also explain why LRF interacts with BCL6 but not with PLZF. Finally, the conservation of crystal packing contacts suggests the probable location of the interface that mediates LRF/BCL6 complex formation.

Smallpox, a highly contagious infectious disease caused by the variola major virus, has an overall mortality rate of about 30%. Because there currently is no specific treatment for smallpox, and the only prevention is vaccination, there is an urgent need for the development of effective antiviral drugs. The dual specificity protein phosphatase encoded by the smallpox virus (H1) is essential for the production of infectious viral particles, making it a promising molecular target for antiviral therapeutics. Here, we report the molecular cloning, overproduction, purification, and initial biochemical characterization of H1 phosphatase, thereby paving the way for the discovery of small molecule inhibitors.

SummaryMembers of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3′ overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.

Summary Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III•dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.

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).

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.

Binary complexes formed by components of the Yersinia pestis type III secretion system were investigated by surface plasmon resonance (SPR) and matrix-assisted laser desorption time-of-flight mass spectrometry. Pairwise interactions between 15 recombinant Yersinia outer proteins (Yops), regulators, and chaperones were first identified by SPR. Mass spectrometry confirmed over 80% of the protein-protein interactions suggested by SPR, and new binding partners were further characterized. The Yop secretion protein (Ysc) M2 of Yersinia enterocolitica and LcrQ of Y. pestis, formerly described as ligands only for the specific Yop chaperone (Syc) H, formed stable complexes with SycE. Additional previously unreported complexes of YscE with the translocation regulator protein TyeA and the thermal regulator protein YmoA and multiple potential protein contacts by YscE, YopK, YopH, and LcrH were also identified. Because only stably folded proteins were examined, the interactions we identified are likely to occur either before or after transfer through the injectosome to mammalian host cells and may have relevance to understanding disease processes initiated by the plague bacterium.

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.

Yersinia pestis, the causative agent of bubonic plague, secretes a eukaryotic-like protein tyrosine phosphatase (PTPase) termed Yersinia outer protein H (YopH) that is essential for virulence. We have determined, for the first time, the crystal structure of the YopH PTPase domain in complex with a nonhydrolyzable substrate analogue, the hexapeptide mimetic Ac-DADE-F(2)Pmp-L-NH(2). As anticipated, the mode of ligand binding in the active site is similar to the way in which the corresponding phosphohexapeptide binds to the structurally homologous human PTP1B. Unexpectedly, however, the crystal structure also revealed a second substrate-binding site in YopH that is not present in PTP1B. The mode of binding and structural conformation of the hexapeptide analogue is quite different in the two sites. Although the biological function of the second substrate-binding site remains to be investigated, the structure of a substrate analogue in the active site of Y. pestis YopH opens the door for the structure-based design and optimization of therapeutic countermeasures to combat this potential agent of bioterrorism.

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.

UNLABELLED Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble et al. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F. tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis. DATABASE The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

Background: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg 2+ -dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. Results: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn 2+ . The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg 2+ ion, which provides the first glimpse of RNase III in action. Conclusions: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn 2+ binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.