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The protein Cereblon, part of an ubiquitin E3 ligase complex, is the target for anticancer thalidomide analogs. The crystal structure of human Cereblon-DDB1 with bound lenalidomide reveals how the drug affects E3 substrate recruitment. The Cul4–Rbx1–DDB1–Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

G protein–coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins 1 . Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains 2 . Downregulation of mth increases the life span of Drosophila melanogaster 3 ; inhibitors of Mth signaling should therefore enhance longevity. We used mRNA display selection 4 , 5 to identify high-affinity ( K d = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane domains. Flies constitutively expressing a Mth antagonist peptide have a robust life span extension, which suggests that the peptides inhibit Mth signaling in vivo . Our work thus provides new life span–extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N . fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N . fowleri . The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N . fowleri research. In 2017, 178 N . fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Abstract Proteases can play essential roles in severe human pathology, ranging from degenerative and inflammatory illnesses to infectious diseases, with some, such as Taspase1, involved in growth and progression of tumors at primary and metastatic sites. Taspase1 is a N-terminal nucleophile (Ntn)-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse xenograft models of glioblastoma, thus this protein has the potential to become a novel anticancer drug target. It belongs to the family of Ntn-hydrolases, a unique family of proteins synthesized as enzymatically inactive proenzymes that become activated upon cleavage of the peptide bond on the N-terminal side of a threonine residue, which then becomes the catalytic site nucleophile. The activation process simultaneously changes the conformation of a long domain at the C-terminus of the alpha-subunit for which no full-length structural information exists and its function is poorly understood. Here we present a novel cloning strategy to generate a fully active, circularly permuted form of Taspase1 to determine the crystallographic structure of catalytically active human Taspase1 to 3.04Å. We discovered that this region forms a long helical domain and is indispensable for the catalytic activity of Taspase1. Together, our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases and suggests that this long domain could be a novel target for the design of inhibitors with the potential to be developed into anticancer therapeutics. Competing Interest Statement The authors have declared no competing interest.

ABSTRACT Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. N. fowleri genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen. Competing Interest Statement The authors have declared no competing interest. Footnotes * Revised to include ORCIDs of authors.

G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins1. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains2. Down-regulation of mth increases the lifespan of Drosophila melanogaster3— inhibitors of Mth signaling would thus be expected to enhance longevity. We used mRNA display selection4,5 to identify high affinity (KD = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane (TM) domains. Flies constitutively expressing a Mth antagonist peptide exhibit a robust lifespan extension, suggesting that the peptides inhibit Mth signaling in vivo. Our work thus provides novel lifespan-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.

Protein drug design is a viable approach to developing potent therapeutics. We investigated a variety of compounds that were designed to be isozyme specific ligands of nitric oxide synthase (NOS), specifically binding within the highly conserved active site located in the heme domain of NOS protein. Using X-ray crystallography, site directed mutagenesis, enzyme kinetics, and computational methods we discovered new features to explore within the active site. Escherichia coli were used in the production of recombinant bovine endothelial nitric oxide synthase (eNOS) proteins. Mutants (nNOS D597N, D597N/M336V, D597N/M336V/Y706A, and eNOS N368D, N368D/V106M, Y477A) were generated using quik-change site directed mutagenesis. The soluble holo proteins were co-expressed with calmodulin and purified in multiple steps generating protein for enzyme kinetics. The holo protein was also trypsinized, generating a truncated heme containing domain for crystallization. The structures of the constitutive NOSs (cNOSs), nNOS and eNOS, complexed with ligands (both mutants and wild-type) were used in MM-PBSA computational analysis to better understand ligand interactions. Four series of NOS ligands; aminopyridine pyrrolidine diamine, aminopyridine pyrrolidine ethoxy amine, double headed aminopyridine, and aminopyridine pyrrolidine ethoxy amine derived were complexed with cNOSs and the structures were observed crystallographically. Both the aminopyridine pyrrolidine diamine and aminopyridine pyrrolidine ethoxy amine ligands were determined to be potent selective inhibitors of nNOS, but bound eNOS in the same way, including a newly observed flipped orientation. Key residue differences and residue movement between the cNOSs contributed to this selectivity within the active site. A new zinc binding site was discovered within the nNOS active site when complexed with non selective double headed aminopyridine ligands. The aminopyridine pyrrolidine ethoxy amine derived ligands appear to exhibit the same features as its predecessors, but also appears to be more bioavailable and slightly more potent. Further analysis is needed to appreciate this series of ligands. Some of the ligands were analyzed enzymatically, to determine isoform selectivity and inhibitor potency by competitive binding with the substrate L-Arginine. Ligands were analyzed computationally to aid our understanding of the relationship within the active site. Additionally, nNOS and eNOS mutants complexed with ligands were utilized to confirm our conclusions both crystallographically and enzymatically.