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

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 signalling in cells, and reduces LPA synthesis in vivo, providing a promising natural product derived scaffold for drug discovery

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

15 pags, 9 figsOne 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 Iβ 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 (β-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. © 2011 Wiley-Liss, Inc.Grant sponsor: Spanish Ministry of Education and Science; Grant number: BFU2008-01588; Grant sponsor: European Commission; Grant number: NMP4-CT-2006-033256; Grant sponsor: Spanish Ministry of Education and Science (José Castillejo fellowship); Grant sponsor: Xunta de Galicia (Angeles Alvariño fellowship); Grant sponsor: National Institutes of Health; Grant numbers: K22-CA124517 (D.E.C.); R01-GM090161 (C.K.) GM074942; GM094585; Grant sponsor: U. S. Department of Energy, Office of Biological and Environmental Research; Grant number: DE-AC02-06CH11357 (to A.J.); Grant sponsor: Foundation for Polish Science (to K.M.); Grant sponsor: NSF; Grant number: DBI 0829586

Target highlights in CASP14 : Analysis of models by structure providers

Abstract The biological and functional significance of selected 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 modelled 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. This article is protected by copyright. All rights reserved.Peer reviewe

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

Distant sequence regions of JBP1 contribute to J-DNA binding

Base-J (β-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.</p

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

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