Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum.

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex. In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.

Structural features of a minimal intact methyltransferase of a type I restriction-modification system.

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.

BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

Crystal structures of UDP-N-acetylmuramic acid L-alanine ligase (MurC) from Mycobacterium bovis with and without UDP-N-acetylglucosamine.

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

Crystal structure of human interleukin-2 in complex with TCB2, a new antibody-drug candidate with antitumor activity.

Immunotherapy via interleukin-2 (IL-2) mediated activation of anti-tumor immune response is a promising approach for cancer treatment. The multi-potent cytokine, IL-2 has a central role in immune cell activation and homeostasis. Since IL-2 preferentially activates immunosuppressive T regulatory cells by IL-2Rα dependent manner, blocking IL-2:IL-2Rα interaction is a key to amplify the IL-2 activity in effector T cells toward anti-tumor response. Anti-IL-2 monoclonal antibodies are good candidates to control the IL-2:IL-2Rα interaction. In a previous study, we developed a new IL-2Rα mimetic antibody, TCB2, and showed that the human IL-2(hIL-2):TCB2 complex can stimulate T effector cells specifically and elicit potent anti-cancer immunotherapeutic effect, especially when administered in combination with immune checkpoint inhibitors. To understand the molecular mechanism, we determined the crystal structure of TCB2-Fab in a complex with hIL-2 at 2.5 Å resolution. Our structural analysis reveals that TCB2 binds to the central area of the hIL-2Rα binding region on hIL-2, and binding angle and epitope are different from previously known hIL-2Rα mimicking antibody NARA1 which recognizes the top part of hIL-2. TCB2 binding to hIL-2 also induces an allosteric effect that increases the affinity for the hetero-dimeric hIL-2 receptor, IL-2R(ß + Î³), on effector T cells.

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex.

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.

A short guide to histone deacetylases including recent progress on class II enzymes.

The interaction between histones and DNA is important for eukaryotic gene expression. A loose interaction caused, for example, by the neutralization of a positive charge on the histone surface by acetylation, induces a less compact chromatin structure, resulting in feasible accessibility of RNA polymerase and increased gene expression. In contrast, the formation of a tight chromatin structure due to the deacetylation of histone lysine residues on the surface by histone deacetylases enforces the interaction between the histones and DNA, which minimizes the chance of RNA polymerases contacting DNA, resulting in decreased gene expression. Therefore, the balance of the acetylation of histones mediated by histone acetylases (HATs) and histone deacetylases (HDACs) is an issue of transcription that has long been studied in relation to posttranslational modification. In this review, current knowledge of HDACs is briefly described with an emphasis on recent progress in research on HDACs, especially on class IIa HDACs.

Sample delivery using viscous media, a syringe and a syringe pump for serial crystallography.

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X-ray beam position in a stable manner. Using this system, the room-temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Šand 1.75 Å, respectively, were determined. This straightforward syringe pump-based sample delivery system can be utilized in SX.

Characterization of a Dimeric Arginase From Zymomonas mobilis ZM4.

Many organisms have genes to protect themselves from toxic conditions such as high ethanol and/or ammonia concentrations. When a high ethanol condition is induced to Zymomonas mobilis ZM4, a representative ethanologenic organism, this bacterium overexpresses several genes to overcome this ethanol stress. Among them, we characterized a gene product annotated as an arginase (zmARG) from Z. mobilis ZM4. Even though all of the arginase-determining sequence motifs are not strictly conserved in zmARG, this enzyme converts L-arginine to urea and L-ornithine in the presence of a divalent manganese ion. The revealed high-resolution crystal structure of zmARG shows that it has a typical globular α/ß arginase fold with a protruded C-terminal helix. Two zinc ions reside in the active site, where one metal ion is penta-coordinated and the other has six ligands, discerning this zmARG from the reported arginases with two hexa-liganded metal ions. zmARG forms a dimeric structure in solution as well as in the crystalline state. The dimeric assembly of zmARG is formed mainly by interaction formed between the C-terminal α-helix of one molecule and the α/ß hydrolase fold of another molecule. The presented findings demonstrate the first reported dimeric arginase formed by the C-terminal tail and has two metal ions coordinated by different number of ligands.

Structural basis of the specific interaction of SMRT corepressor with histone deacetylase 4.

Modification of chromatin and related transcription factors by histone deacetylases (HDACs) is one of the major strategies for controlling gene expression in eukaryotes. The HDAC domains of class IIa HDACs repress the respective target genes by interacting with the C-terminal region of the silencing mediator for retinoid and thyroid receptor (SMRT) repression domain 3 (SRD3c). However, latent catalytic activity suggests that their roles as deacetylases in gene regulation are unclear. Here, we found that two conserved GSI-containing motifs of SRD3c are critical for HDAC4 binding. Two SMRT peptides including these motifs commonly form a ß-hairpin structure in the cleft and block the catalytic entry site of HDAC4. They interact mainly with class IIa HDAC-specific residues of HDAC4 in a closed conformation. Structure-guided mutagenesis confirmed critical interactions between the SMRT peptides and HDAC4 and -5 as well as the contribution of the Arg1369 residue in the first motif for optimal binding to the two HDACs. These results indicate that SMRT binding does not activate the cryptic deacetylase activity of HDAC4 and explain how class IIa HDACs and the SMRT-HDAC3 complex are coordinated during gene regulation.

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apo-structure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through ß-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA.

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central ß-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the ß-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5'-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Trehalose-6-phosphate phosphatase as a broad-spectrum therapeutic target against eukaryotic and prokaryotic pathogens.

As opposed to organism-based drug screening approaches, protein-based strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors and thus afford a precise targeting. Capitalising on the increasing number of genome and transcriptome datasets, novel targets in pathogens for therapeutic intervention can be identified in a more rational manner when compared with conventional organism-based methodologies. Trehalose-6-phosphate phosphatases (TPPs) are structurally and functionally conserved enzymes of the trehalose biosynthesis pathway which play a critical role for pathogen survival, in particular, in parasites. The absence of these enzymes and trehalose biosynthesis from mammalian hosts has recently given rise to increasing interest in TPPs as novel therapeutic targets for drugs and vaccines. Here, we summarise some key aspects of the current state of research towards novel therapeutics targeting, in particular, nematode TPPs.

Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation.

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study with Xenopus laevis egg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif of Xenopus Cep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous to Xenopus p-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis.

Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

Cloning, crystallization and preliminary X-ray diffraction analysis of an intact DNA methyltransferase of a type I restriction-modification enzyme from Vibrio vulnificus.

Independently of the restriction (HsdR) subunit, the specificity (HsdS) and methylation (HsdM) subunits interact with each other, and function as a methyltransferase in type I restriction-modification systems. A single gene that combines the HsdS and HsdM subunits in Vibrio vulnificus YJ016 was expressed and purified. A crystal suitable for X-ray diffraction was obtained from 25%(w/v) polyethylene glycol monomethylether 5000, 0.1 M HEPES pH 8.0, 0.2 M ammonium sulfate at 291 K by hanging-drop vapour diffusion. Diffraction data were collected to a resolution of 2.31 Šusing synchrotron radiation. The crystal belonged to the primitive monoclinic space group P21, with unit-cell parameters a = 93.25, b = 133.04, c = 121.49 Å, ß = 109.7°. With four molecules in the asymmetric unit, the crystal volume per unit protein weight was 2.61 Å(3) Da(-1), corresponding to a solvent content of 53%.

Crystal structure of cbbF from Zymomonas mobilis and its functional implication.

A phosphate group at the C1-atom of inositol-monophosphate (IMP) and fructose-1,6-bisphosphate (FBP) is hydrolyzed by a phosphatase IMPase and FBPase in a metal-dependent way, respectively. The two enzymes are almost indiscernible from each other because of their highly similar sequences and structures. Metal ions are bound to residues on the ß1- and ß2-strands and one mobile loop. However, FBP has another phosphate and FBPases exist as a higher oligomeric state, which may discriminate FBPases from IMPases. There are three genes annotated as FBPases in Zymomonas mobilis, termed also cbbF (ZmcbbF). The revealed crystal structure of one ZmcbbF shows a globular structure formed by five stacked layers. Twenty-five residues in the middle of the sequence form an α-helix and a ß-strand, which occupy one side of the catalytic site. A non-polar Leu residue among them is protruded to the active site, pointing out unfavorable access of a bulky charged group to this side. In vitro assays have shown its dimeric form in solution. Interestingly, two ß-strands of ß1 and ß2 are disordered in the ZmcbbF structure. These data indicate that ZmcbbF might structurally belong to IMPase, and imply that its active site would be reorganized in a yet unreported way.

Peptide-based inhibitors of Plk1 polo-box domain containing mono-anionic phosphothreonine esters and their pivaloyloxymethyl prodrugs.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.