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Supplementary Table S1. List of methylated histone proteins identified from in vitro HMT assay on calf thymus extracts followed by LC-MS/MS analysis

SMYD3 methyltransferase is nearly undetectable in normal human tissues but highly expressed in several cancers, including breast cancer, although its contributions to pathogenesis in this setting are unclear. Here we report that histone H2A.Z.1 is a substrate of SMYD3 that supports malignancy. SMYD3-mediated dimethylation of H2A.Z.1 at lysine 101 (H2A.Z.1K101me2) increased stability by preventing binding to the removal chaperone ANP32E and facilitating its interaction with histone H3. Moreover, a microarray analysis identified cyclin A1 as a target coregulated by SMYD3 and H2A.Z.1K101me2. The colocalization of SMYD3 and H2A.Z.1K101me2 at the promoter of cyclin A1 activated its expression and G1–S progression. Enforced expression of cyclin A1 in cells containing mutant H2A.Z.1 rescued tumor formation in a mouse model. Our findings suggest that SMYD3-mediated H2A.Z.1K101 dimethylation activates cyclin A1 expression and contributes to driving the proliferation of breast cancer cells. Cancer Res; 76(20); 6043–53. ©2016 AACR.

Fig. S1. Representative mass spectrometry data of methylated H2A.Z. Fig. S2. Methylation activity of SMYD3 on histones in the context of nucleosome and evaluation of the H2A.ZK101me2 antibody. Fig. S3. Dimethylation of H2A.Z is critical for its protein interaction. Fig. S4. Dimethylation of H2A.Z is critical for promoting cell growth. Fig. S5. Genes affected by SMYD3 knockdown and H2A.ZK101Q expression. Fig. S6. Cyclin A1 is one of the downstream targets co-regulated by SMYD3 and H2A.ZK101me2.

Supplementary Materials and Methods, Supplementary Figure Legends and Supplementary Tables

Nature has developed complexity–generating reactions within natural product biosynthetic pathways. However, direct utilization of these pathways to prepare compound libraries remains challenging due to limited substrate scopes, involvement of multiple-step reactions, and moderate robustness of these sophisticated enzymatic transformations. Synthetic chemistry, on the other hand, offers an alternative approach to prepare natural product analogs. However, owing to complex and diverse functional groups appended on the targeted molecules, dedicated design and development of synthetic strategies are typically required. Herein, by leveraging the power of chemo-enzymatic synthesis, we report an approach to bridge the gap between biological and synthetic strategies in the preparation of quinolone alkaloid analogs. Leading by in silico analysis, the predicted substrate analogs were chemically synthesized. The AsqJ-catalyzed asymmetric epoxidation of these substrate analogues was followed by an Lewis Acid-triggered ring contraction to complete the viridicatin formation. We evaluated the robustness of this method in gram-scale reactions. Lastly, through chemoenzymatic cascades, a library of quinolone alkaloids is effectively prepared.

Phosphodiesterase 5A1 (PDE5) is a key target for treating cardiovascular diseases and erectile dysfunction. Here, we report the crystal structure of PDE5 complexed with the sole second generation drug avanafil. Analysis of protein-drug interactions revealed the structural basis of avanafil's superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N(6)-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.

Significance The completion of the tetracyclic core of etoposide, classified by the World Health Organization as an essential medicine, by the Fe/2OG oxygenase deoxypodophyllotoxin synthase follows a hybrid radical-polar pathway not previously seen in other members of this enzyme class. The implication of a substrate-based benzylic carbocation in this mechanism will inform ongoing efforts to create analogs of this important drug with improved or emergent properties and represents a new route for resolution of the initial substrate radical that is common to members of the class. This study adds to our understanding on a growing number of biochemical transformations in which carbocation intermediates are likely to be crucial., Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate–dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS’s catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel–Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mossbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.

Background The Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis. Methods The Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients. Results Slug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130–212 and 33–129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients. Conclusions Ubc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC. Electronic supplementary material The online version of this article (10.1186/s13046-018-0996-8) contains supplementary material, which is available to authorized users.

Figure S2. The activities of Slug mutants. (a) Mutation of individual lysine affects the SUMOylated level of Slug. HEK293T cells were cotransfected with plasmids encoding different 3xFlag-tagged Slug mutants and GFP-tagged SUMO-1. The lysates were used to examine the SUMOylation levels by immunoblotting with anti-Flag antibodies. (b) Different levels of SUMOylation between Slug mutants. HEK293T cells were transfected with expression vectors encoding GFP-tagged SUMO-1 and different 3xFlag-tagged Slug mutants (22 M, all lysines were replaced with arginines; 5 M: lysines at 239, 240, 244, 248, and 258 were replaced with arginines; 6 M: lysines at 188, 239, 240, 244, 248, and 258 were replaced with arginines). These lysates were also examined by immunoblotting with anti-Flag antibodies. The asterisk and arrowhead indicate Slug modified and not modified by SUMO-1, respectively. (c) The transcriptional repression activity of wild-type and mutant Slug proteins. HEK293T cells were cotransfected with the SBS–Gal4–luciferase reporter and Gal4–VP16 activator expression plasmids together with the wild-type or mutant Slug expression plasmid (8 M: lysines at 135, 145, 188, 239, 240, 244, 248, and 258 were replaced with arginines), and the luciferase assay was performed to determine the transcriptional repression activity of Slug. Immunoblotting results are presented alongside the luciferase assay results to demonstrate the expression of the Slug mutant proteins. (d) The DNA-binding activity of wild-type and mutant Slug proteins. The wild-type and mutant Slug proteins used in the EMSA were produced using an in vitro transcription/translation system. The protein expression levels were evaluated by immunoblotting with anti-Slug antibodies (top panel). Phosphor image analysis of the EMSA gel showing 32P-labeled E-box oligonucleotides incubated with in vitro-translated proteins (4 μl) or with Slug antibodies (Ab: antibody, 0.3 μg) (bottom panel). (PDF 152 kb)

Figure S11. Effects of Slug and Slug5M overexpression on cultured cell proliferation. HEK293 cells were driven to express wild-type or mutant Slug using a lentiviral system. Cells were counted at the indicated time points after plating. No significant difference in the cell proliferation rate was found between the different cell lines based on one-way ANOVA. Error bars indicate mean values Âą SEM. (PDF 69 kb)

Figure S8. HDAC1 stronger associated with the E-cadherin promoter in Hop62 cells overexpressing Ubc9 compare with the vector control cells. The normal mouse IgG and HDAC1 antibodies were used to pull down protein-DNA complexes in Hop62 cells with or without Ubc9 overexpression; and the E-cadherin promoter level in the samples was determined by PCR using a gene-specific primer set. Input, an aliquot of each sample was prepared and used as a template for PCR to examine the level of the E-cadherin promoter before immunoprecipitation (IP). (PDF 25 kb)

Figure S5. Structure of the Slug/PIASy/Ubc9/SUMO-1 complex. (a) Schematic showing the regions of Slug that interact with PIASy, Ubc9, and SUMO. Slug is 268 amino acids in length and contains a SNAG repression domain at its N-terminus and five zinc finger (ZnF) domains at its C-terminus. ND means no detection. (b) A 3D structure of Slug/PIASy/Ubc9/SUMO-1 complex was generated using prediction software (orange, Slug; purple, PIASy; green, Ubc9; gray, SUMO-1). A rotated view of this complex is shown in the lower panel. (PDF 127 kb)

Figure S7. Slug recruits corepressors more abundantly than Slug5M. The nuclear fractions of Slug- and Slug5M-overexpressing HEK293 cells were obtained by adding hypotonic buffer to the cells. Subsequently, the samples were subjected to immunoprecipitation using an anti-Slug antibody. The accompanying precipitates were analyzed by immunoblotting using the indicated antibodies (left panel). Lamin B was used as a nuclear marker. The relative densitometry results (the results for Slug were normalized to one) were calculated using two programs (ImageJ and GelPro3.1), and the average values are plotted in the right panel. (PDF 61 kb)

Figure S6. Characterization of Slug and Slug5M protein. (a) The DNA-binding ability of Slug is not altered by the inserted mutations. Equal amounts of in vitro-translated Slug and Slug5M were used in the EMSAs (left panel). Slug and Slug5M bound to the E-box C probes in a dose-dependent manner (+: 0.1 μl; ++: 0.3 μl; +++: 1 μl) (right panel). Anti-Slug antibodies were used to confirm that the shifted bands were formed specifically by Slug and Slug5M. (b) The protein stability of Slug is not altered by the inserted mutations. Protein stability was not significantly different between the wild-type and mutant forms of Slug. Slug- and Slug5M-overexpressing HEK293 cells were treated with cycloheximide (CHX) to prevent further protein synthesis for the indicated periods. The expression of Slug was analyzed by immunoblotting. β-actin was used as the internal control. Relative densitometry results are plotted in the bottom panel. (PDF 68 kb)

Figure S9. SUMOylation affects the expression of Slug-regulated downstream targets. (a) CL1–5 cells were induced to express the wild-type and mutant Slug, respectively, using a lentiviral system. The mRNA expression of the indicated genes was determined via RT-PCR. Gβ-like was used as the internal control. (b) The protein expression of the Slug downstream target, E-cadherin, in HEK293 (left) and CL1–2 (right) over-expressing Slug wild-type or Slug5M cells. The results were analyzed by immunoblotting with the indicated antibodies. β-actin was used as the internal control. (PDF 174 kb)

Figure S4. Direct interaction of Slug with PIAS family members. A pull-down assay was used to determine the physical interaction between Slug and PIAS family members. Recombinant GST and GSTâ Slug proteins were produced from bacteria, and the translated products of HA-tagged PIAS family member genes were obtained using an in vitro transcription/translation system. The production of these proteins was demonstrated by immunoblotting using anti-GST and anti-HA antibodies, respectively. GSTâ Slug was used in the pull-down assay for in vitro interaction with HA-tagged PIAS family members. The GST protein alone was used as a negative control. (PDF 24 kb)

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.

The programmed induction of meiotic DNA double-strand breaks (DSBs) by the evolutionarily conserved SPO-11 protein, which is structurally related to archaeal Topo VIA topoisomerases, triggers meiotic recombination. Identification of several meiosis-specific factors that are required for SPO-11-mediated DSB formation raises the question whether SPO-11 alone can cleave DNA. Here, we have developed procedures to express and purify C. elegans SPO-11 in a soluble, untagged, and monodispersed form. Our biochemical and biophysical analyses demonstrate that SPO-11 is monomeric and binds DNA, double-stranded DNA in particular. Importantly, SPO-11 does not exhibit DNA cleavage activity under a wide range of reaction conditions, suggesting that co-factors are needed for DSB induction activity. Our SPO-11 purification system and the findings reported herein should facilitate future mechanistic studies directed at delineating the mechanism of action of the SPO-11 ensemble in meiotic DSB formation.

Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2β, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2β revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2β, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2β more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2β are practically irreversible. Crystallographic analyses of hTop2β complexed with DNA and etoplatin-N2β demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.

Human cytosolic NADP+-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.

Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme–DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 β-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3′-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs’ structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.

Type IIA topoisomerases catalyze the passage of two DNA duplexes across each other to resolve the entanglements and coiling of cellular DNA. The ability of these enzymes to interact simultaneously but differentially with two DNA segments is central to their DNA-manipulating functions: one duplex DNA is bound and cleaved to produce a transient double-strand break through which another DNA segment can be transported. Recent structural analyses have revealed in atomic detail how type IIA enzymes contact DNA and how the enzyme-DNA interactions may be exploited by drugs to achieve therapeutic purposes. This review summarizes these new findings, with a special focus on the assembly and structural features of the enzymes' composite DNA-binding surfaces.

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways’ end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.

SMYD3 methyltransferase is nearly undetectable in normal human tissues but highly expressed in several cancers, including breast cancer, although its contributions to pathogenesis in this setting are unclear. Here we report that histone H2A.Z.1 is a substrate of SMYD3 that supports malignancy. SMYD3-mediated dimethylation of H2A.Z.1 at lysine 101 (H2A.Z.1K101me2) increased stability by preventing binding to the removal chaperone ANP32E and facilitating its interaction with histone H3. Moreover, a microarray analysis identified cyclin A1 as a target coregulated by SMYD3 and H2A.Z.1K101me2. The colocalization of SMYD3 and H2A.Z.1K101me2 at the promoter of cyclin A1 activated its expression and G1–S progression. Enforced expression of cyclin A1 in cells containing mutant H2A.Z.1 rescued tumor formation in a mouse model. Our findings suggest that SMYD3-mediated H2A.Z.1K101 dimethylation activates cyclin A1 expression and contributes to driving the proliferation of breast cancer cells. Cancer Res; 76(20); 6043–53. ©2016 AACR.

The β-propeller is a highly symmetrical structure with 4-10 repeats of a four-stranded antiparallel β-sheet motif. Although β-propeller proteins with different blade numbers all adopt disc-like shapes, they are involved in a diverse set of functions, and defects in this family of proteins have been associated with human diseases. However, it has remained ambiguous how variations in blade number could alter the function of β-propellers. In addition to the regularly arranged β-propeller topology, a recently discovered β-pinwheel propeller has been found. Here, we review the structural and functional diversity of β-propeller proteins, including β-pinwheels, as well as recent advances in the typical and atypical propeller structures.

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.

DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA. The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrase-specific 'GyrA-box' motif within this domain are essential for this unique (-) supercoiling activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif. This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting a non-planar beta-pinwheel fold composed of six seemingly spirally arranged beta-sheet blades. Interestingly, structural analysis revealed that the non-planar architecture mainly stems from the tilted packing seen between blades 1 and 2, with the packing geometry likely being defined by a conserved and unusual beta-strand-bearing proline. Consequently, the GyrA-box-containing blade 1 is placed at an angled spatial position relative to the other DNA-binding blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface. Mutagenesis studies support that the proline-induced structural twist contributes directly to gyrase's (-) supercoiling activity. To our knowledge, this is the first demonstration that a beta-strand-bearing proline may impact protein function. Potential relevance of beta-strand-bearing proline to disease phenylketonuria is also noted.

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.

The crystal structure of XC1258 from Xanthomonas campestris: A putative procaryotic Nit protein with an arsenic adduct in the active site Ko-Hsin Chin, Ying-Der Tsai, Nei-Li Chan, Kai-Fa Huang, Andrew H.-J. Wang, and Shan-Ho Chou* 1 National Chung Hsing University Biotechnology Center, National Chung-Hsing University, Taichung, 40227, Taiwan, Republic of China 2 Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan, Republic of China 3 Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China 4 Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China

The mer operon confers bacterial resistance to inorganic mercury (Hg(2+)) and organomercurials by encoding proteins involved in sensing, transport and detoxification of these cytotoxic agents. Expression of the mer operon is under tight control by the dual-function transcriptional regulator MerR. The metal-free, apo MerR binds to the mer operator/promoter region as a repressor to block transcription initiation, but is converted into an activator upon Hg(2+)-binding. To understand how MerR interacts with Hg(2+) and how Hg(2+)-binding modulates MerR function, we report here the crystal structures of apo and Hg(2+)-bound MerR from Bacillus megaterium, corresponding respectively to the repressor and activator conformation of MerR. To our knowledge, the apo-MerR structure represents the first visualization of a MerR family member in its intact and inducer-free form. And the Hg(2+)-MerR structure offers the first view of a triligated Hg(2+)-thiolate center in a metalloprotein, confirming that MerR binds Hg(2+) via trigonal planar coordination geometry. Structural comparison revealed the conformational transition of MerR is coupled to the assembly/disassembly of a buried Hg(2+) binding site, thereby providing a structural basis for the Hg(2+)-mediated functional switching of MerR. The pronounced Hg(2+)-induced repositioning of the MerR DNA-binding domains suggests a plausible mechanism for the transcriptional regulation of the mer operon.

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane-associated by interacting with the N-terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N-terminal domain of XpsL (a GspL homolog). While isolated XpsE exhibits very low intrinsic ATPase activity, association with XpsL appears to stimulate ATP hydrolysis. Mutation at a conserved lysine residue in the XpsE Walker A motif causes reduction in its ATPase activity without significantly influencing its interaction with XpsL, congruent with the notion that XpsE–XpsL association precedes ATP hydrolysis. For the first time, functional significance of ATP binding to GspE in type II secretion system is clearly demonstrated. The implications may also be applicable to type IV pilus biogenesis.

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P4(3)2(1)2 is composed of a 90-residue alpha/beta sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsE(N) may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4(1)22, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P4(3)2(1)2 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

Bacteria possess two closely related yet functionally distinct essential type IIA topoisomerases (Topos). DNA gyrase supports replication and transcription with its unique supercoiling activity, whereas Topo IV preferentially relaxes (+) supercoils and is a decatenating enzyme required for chromosome segregation. Here we report the crystal structure of the C-terminal domain of Topo IV ParC subunit (ParC-CTD) from Bacillus stearothermophilus and provide a structure-based explanation for how Topo IV and DNA gyrase execute distinct activities. Although the topological connectivity of ParC-CTD is similar to the recently determined CTD structure of DNA gyrase GyrA subunit (GyrA-CTD), ParC-CTD surprisingly folds as a previously unseen broken form of a six-bladed β-propeller. Propeller breakage is due to the absence of a DNA gyrase-specific GyrA box motif, resulting in the reduction of curvature of the proposed DNA binding region, which explains why ParC-CTD is less efficient than GyrA-CTD in mediating DNA bending, a difference that leads to divergent activities of the two homologous enzymes. Moreover, we found that the topology of the propeller blades observed in ParC-CTD and GyrA-CTD can be achieved from a concerted β-hairpin invasion-induced fold change event of a canonical six-bladed β-propeller; hence, we proposed to name this new fold as “hairpininvaded β-propeller” to highlight the high degree of similarity and a potential evolutionary linkage between them. The possible role of ParC-CTD as a geometry facilitator during various catalytic events and the evolutionary relationships between prokaryotic type IIA Topos have also been discussed according to these new structural insights.

In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.

DNA topoisomerases are nature's tools for resolving the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, formed by a covalent adduct with the enzyme, through which strand passage can occur. The active site tyrosine is responsible for initiating two transesterifications to cleave and then religate the DNA backbone. The cleavage reaction intermediate is exploited by cytotoxic agents, which have important applications as antibiotics and anticancer drugs. The reactions mediated by these enzymes can also be regulated by their binding partners; one example is a DNA helicase capable of modulating the directionality of strand passage, enabling important functions like reannealing denatured DNA and resolving recombination intermediates. In this review, we cover recent advances in mechanistic insights into topoisomerases and their various cellular functions.

The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts., Author Summary Giardia lamblia becomes infective by differentiation into water-resistant cysts. During encystation, cyst wall proteins (CWPs) are highly synthesized and are targeted to the cyst wall. However, little is known about the regulation mechanisms of these genes. DNA topoisomerases can resolve the topological problems and are needed for a variety of key cellular functions, including cell proliferation, cell differentiation and organ development in higher eukaryotes. We found that giardial Topo II was highly expressed during encystation. Topo II is present in Giardia nuclei and is associated with the encystation-induced cwp gene promoters. Topo II has typical DNA cleavage activity of type II topoisomerases. Interestingly, overexpression of Topo II can induce cwp gene expression and cyst formation. Addition of a type II topoisomerase inhibitor, etoposide, significantly decreased the levels of cwp gene expression and cyst formation. Etoposide also has growth inhibition effect on Giardia. Our results suggest that Topo II plays an important role in induction of encystation by up-regulation of the cwp gene expression. Our results provide insights into the function of Topo II in parasite differentiation into cysts and help develop ways to interrupt the parasite life cycle.

The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9 Å resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.75, b = 95.61, c = 183.83 Å.