Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B(12), 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.

Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.

SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators.

Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.

Transforming growth factor-β activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.