Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics.

Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.semaphorin ͉ Tuftsin ͉ VEGF N europilins (Nrps) are essential cell surface receptors with central roles in both angiogenesis and axon guidance (1-3). During angiogenesis, Nrp directly binds VEGF and functions as a coreceptor for VEGF along with VEGF receptor (VEGFR)-2, one of the three VEGFR tyrosine kinases (3, 4). During neural development, Nrp directly binds semaphorin and functions as a semaphorin coreceptor with members of the plexin family (5). Additionally, interactions with both neural adhesion protein L1 and Nrp-interacting protein (NIP), have been shown to be involved in a variety of other cellular processes (6-8).Nrp plays a stimulatory role in angiogenesis, a process critical for growth of solid tumors (reviewed in refs. 4 and 9-11). Nrp expression is observed in tumor vasculature, and overexpression promotes tumorigenesis in vivo for a variety of solid tumors including pituitary, prostate, breast, and colon cancers (12-15). In contrast, a soluble splice form containing only part of the extracellular domain of Nrp inhibits tumorigenesis (16) as do a number of peptides that block VEGF binding to Nrp (17,18). Recent evidence has also demonstrated a role for Nrp in hematological malignancies. Nrp overexpression is observed in both multiple myeloma and acute myeloid leukemia and, in the latter case, is associated with significantly reduced survival (19,20). Strategies to inhibit Nrp activity are thus being developed as potential antitumor therapies (reviewed in ref. 11).Higher eukaryotes possess two Nrp homologs, Nrp-1 and Nrp-2, which share 44% amino acid sequence identity (1). Nrp extracellular regions are composed of two complement binding CUB domains (a1 and a2) followed by two coagulation factor domains (b1 and b2), a MAM (meprin, A5, ) domain (c1), a single membrane-spanning region, and a short cytoplasmic tail (Fig. 1A) (21, 22). The a1 and a2 domains of Nrp are essential for binding to the core seven-bladed Sema domain of semaphorin as well as contributing to interactions with VEGF (23, 24). The coagulation factor domains b1 and b2 contain the high-affinity binding site for the basic heparin binding domain (HBD) of VEGF165 as well a...

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the crossbrace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the Ubox. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to premRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.Ubiquitin (Ub) targeting of proteins for degradation by the proteosome involves polyubiquination of substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2) and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition and enzymology, reflecting their regulatory role in substrate recognition 1 . HECT and RING finger domain-containing proteins constitute two classes of E3 ligases. HECT domains bind Ub through a thioester bond and transfer Ub directly to substrate. RING finger E3s facilitate the transfer of Ub from the E2 to the substrate, rather than binding Ub directly.Correspondence should be addressed to K.L.G. kathy.gould@mcmail.vanderbilt.edu or W.J.C. walter.chazin@vanderbilt.edu. 3 These authors contributed equally to this work. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptA third class of E3 ubiquitin ligases has been recently identified 2,3 . This class of proteins contains a U-box motif, first identified in Saccharomyces cerevisiae Ufd2p. Ufd2p promotes the elongation of poly-ubiquitin chains in a U-box-dependent manner (recently termed an 'E4' activity) 4 . An alignment of U-boxes and RING motifs indicated that U-boxes lack the strictly conserved histidine and cysteine Zn 2+ -chelating residues found in RING fingers, but they share a similar pattern of hydrophobic and polar amino acids (Fig. 1a), raising the possibility that they have similar folds 5 .S. cerevisiae Prp19p is an essential pre-mRNA splicing factor that contains an N-terminal Ubox 6,7 . Interestingly, a mutation within the Prp19p U-box that results in the substitution of an isoleucine for a conserved valine (prp19-1) leads to pre-mRNA splicing defects and the disruption of several key protein-protein interactions within the spliceosome 8,9 . The profound physiological consequences that result from th...

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The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6β1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6β1, establishing an autocrine loop. NRP2 can be targeted in vivo to retard tumour initiation. These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6β1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs.