Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania.

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

The domain architecture of the protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity.

J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (ß-d-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania JBP1 has two known functional domains: an N-terminal T hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins and a J-DNA-binding domain (JDBD) that resides in the middle of JBP1. Here, we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered structure. We found that this Δ-JDBD domain retains TH activity in vitro but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA and on Δ-JDBD enabled us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein.

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency.

Autotaxin (ATX) is a secreted glycoprotein and the only member of the ectonucleotide pyrophosphatase/phosphodiesterase family that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key responses, such as cell migration, proliferation, and survival, implicating ATX-LPA signaling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, called the "pocket" and the "tunnel," respectively. However, the mechanisms in allosteric modulation of ATX's activity as a lysophospholipase D are unclear. Here, using the physiological LPC substrate, a new fluorescent substrate, and diverse ATX inhibitors, we revisited the kinetics and allosteric regulation of the ATX catalytic cycle, dissecting the different steps and pathways leading to LPC hydrolysis. We found that ATX activity is stimulated by LPA and that LPA activates ATX lysophospholipase D activity by binding to the ATX tunnel. A consolidation of all experimental kinetics data yielded a comprehensive catalytic model supported by molecular modeling simulations and suggested a positive feedback mechanism that is regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend the current understanding of ATX hydrolysis in light of the allosteric regulation by ATX-produced LPA species and have implications for the design and application of both orthosteric and allosteric ATX inhibitors.

The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1.

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (ß-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a 'helical bouquet' with a 'ribbon' helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.

Distant sequence regions of JBP1 contribute to J-DNA binding.

Base-J (ß-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.

Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding.

Base J (ß-D-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and Leishmania, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (~400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 µM(-1) s(-1) and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T's in spatial proximity, resulting in regional introduction of base J into the DNA.

Posttranslational modification of microtubules by the MATCAP detyrosinase.

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Target highlights in CASP9: Experimental target structures for the critical assessment of techniques for protein structure prediction.

One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iß dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (ß-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae.

Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1-SVBP.

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.

Characterization and structure determination of a llama-derived nanobody targeting the J-base binding protein 1.

J-base binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (ß-D-glucosylhydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single-domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance screening identified Nb6 as a strong binder, recognizing JBP1 with a 1:1 stoichiometry and high affinity (Kd = 30 nM). Crystallization trials of JBP1 in complex with Nb6 yielded crystals that diffracted to 1.47 Šresolution. However, the dimensions of the asymmetric unit and molecular replacement with a nanobody structure clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in space group P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64 Å. Ensemble refinement suggests that in the ligand-free state one of the complementarity-determining regions (CDRs) is flexible, while the other two adopt well defined conformations.

Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1.

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

Rational Design of Autotaxin Inhibitors by Structural Evolution of Endogenous Modulators.

Autotaxin produces the bioactive lipid lysophosphatidic acid (LPA) and is a drug target of considerable interest for numerous pathologies. We report the expedient, structure-guided evolution of weak physiological allosteric inhibitors (bile salts) into potent competitive Autotaxin inhibitors that do not interact with the catalytic site. Functional data confirms that our lead compound attenuates LPA mediated signaling in cells and reduces LPA synthesis in vivo, providing a promising natural product derived scaffold for drug discovery.

Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.