The PIAS-like Coactivator Zmiz1 Is a Direct and Selective Cofactor of Notch1 in T Cell Development and Leukemia.

Pan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors might also promote cancer because NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction might combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

Differential dynamics of cullin deneddylation via COP9 signalosome subunit 5 interaction.

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation-deneddylation conversion.

The differentially expressed gene signatures of the Cullin 3-RING ubiquitin ligases in neuroendocrine cancer.

Cullin-RING ubiquitin E3 ligase (CRLs) composed of four components including cullin scaffolds, adaptors, substrate receptors, and RING proteins mediates the ubiquitination of approximately 20% of cellular proteins that are involved in numerous biological processes. While CRLs deregulation contributes to the pathogenesis of many diseases, including cancer, how CRLs deregulation occurs is yet to be fully investigated. Here, we demonstrate that components of CRL3 and its transcriptional regulators are possible prognosis marker of neuroendocrine (NE) cancer. Analysis of Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal revealed that expression of CRL3 scaffold Cullin 3 (CUL3) highly correlates with NE signature, and CUL3 silencing inhibited NE cancer proliferation. Moreover, subset of 151 BTB (Bric-a-brac, Tramtrack, Broad complex) domain-containing proteins that have dual roles as substrate receptors and adaptor subunits in CRL3, as well as the expression of transcription factors (TFs) that control the transcription of BTB genes were upregulated in NE cancer. Analysis using published ChIP-sequencing data in small cell lung cancer (SCLC), including NE or non-NE SCLC verified that gene promoter of candidates which show high correlation with NE signature enriched H3K27Ac. These observations suggest that CRL3 is a master regulator of NE cancer and knowledge of specifically regulated CRL3 genes in NE cancer may accelerate new therapeutic approaches.

Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit-Activating Enzyme CahJ.

Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.

NADP+-dependent cytosolic isocitrate dehydrogenase provides NADPH in the presence of cadmium due to the moderate chelating effect of glutathione.

Cadmium (Cd2+) is toxic to living organisms because it causes the malfunction of essential proteins and induces oxidative stress. NADP+-dependent cytosolic isocitrate dehydrogenase (IDH) provides reducing energy to counteract oxidative stress via oxidative decarboxylation of isocitrate. Intriguingly, the effects of Cd2+ on the activity of IDH are both positive and negative, and to understand the molecular basis, we determined the crystal structure of NADP+-dependent cytosolic IDH in the presence of Cd2+. The structure includes two Cd2+ ions, one coordinated by active site residues and another near a cysteine residue. Cd2+ presumably inactivates IDH due to its high affinity for thiols, leading to a covalent enzyme modification. However, Cd2+ also activates IDH by providing a divalent cation required for catalytic activity. Inactivation of IDH by Cd2+ is less effective when the enzyme is activated with Cd2+ than Mg2+. Although reducing agents cannot restore activity following inactivation by Cd2+, they can maintain IDH activity by chelating Cd2+. Glutathione, a cellular sulphydryl reductant, has a moderate affinity for Cd2+, allowing IDH to be activated with residual Cd2+, unlike dithiothreitol, which has a much higher affinity. In the presence of Cd2+-consuming cellular antioxidants, cells must continually supply reductants to protect against oxidative stress. The ability of IDH to utilise Cd2+ to generate NADPH could allow cells to protect themselves against Cd2+.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

Two Loops Undergoing Concerted Dynamics Regulate the Activity of the ASH1L Histone Methyltransferase.

ASH1L (absent, small, or homeotic-like 1) is a histone methyltransferase (HMTase) involved in gene activation that is overexpressed in multiple forms of cancer. Previous studies of ASH1L's catalytic SET domain identified an autoinhibitory loop that blocks access of histone substrate to the enzyme active site. Here, we used both nuclear magnetic resonance and X-ray crystallography to identify conformational dynamics in the ASH1L autoinhibitory loop. Using site-directed mutagenesis, we found that point mutations in the autoinhibitory loop that perturb the structure of the SET domain result in decreased enzyme activity, indicating that the autoinhibitory loop is not a simple gate to the active site but is rather a key feature critical to ASH1L function. We also identified a second loop in the SET-I subdomain of ASH1L that experiences conformational dynamics, and we trapped two different conformations of this loop using crystallographic studies. Mutation of the SET-I loop led to a large decrease in ASH1L enzymatic activity in addition to a significant conformational change in the SET-I loop, demonstrating the importance of the structure and dynamics of the SET-I loop to ASH1L function. Furthermore, we found that three C-terminal chromatin-interacting domains greatly enhance ASH1L enzymatic activity and that ASH1L requires native nucleosome substrate for robust activity. Our study illuminates the role of concerted conformational dynamics in ASH1L function and identifies structural features important for ASH1L enzymatic activity.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus.

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Substrate binding mechanism of a type I extradiol dioxygenase.

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a ß-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and ß-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.

Development of potent dimeric inhibitors of GAS41 YEATS domain.

GAS41 is an emerging oncogene overexpressed and implicated in multiple cancers, including non-small cell lung cancer (NSCLC). GAS41 is a dimeric protein that contains the YEATS domain, which is involved in the recognition of lysine-acylated histones. Here, we report the development of GAS41 YEATS inhibitors by employing a fragment-based screening approach. These inhibitors bind to GAS41 YEATS domain in a channel constituting a recognition site for acylated lysine on histone proteins. To enhance inhibitory activity, we developed a dimeric analog with nanomolar activity that blocks interactions of GAS41 with acetylated histone H3. Our lead compound engages GAS41 in cells, blocks proliferation of NSCLC cells, and modulates expression of GAS41-dependent genes, validating on-target mechanism of action. This study demonstrates that disruption of GAS41 protein-protein interactions may represent an attractive approach to target lung cancer cells. This work exemplifies the use of bivalent inhibitors as a general strategy to block challenging protein-protein interactions.

Structural basis for substrate binding to human pyridoxal 5'-phosphate phosphatase/chronophin by a conformational change.

Human pyridoxal 5'-phosphate phosphatase (PLPP), also known as a chronophin, is a phosphatase belonging to subfamily II of the HAD phosphatases, characterized by a large cap domain. As a member of the subfamily, its cap-open conformation is expected for substrate binding. We determined apo and PLP-bound PLPP/chronophin structures showing a cap-closed conformation. The active site, in which a PLP molecule was found, is too small to accommodate a phospho-cofilin peptide, the substrate of chronophin. A conformational change to a cap-open conformation may be required for substrate binding. The core and cap domains are joined through linker peptide hinges that change conformation to open the active site. The crystal structures reveal that a disulphide bond between the cap and core domains restricts the hinge motion. The enzyme displays PLP dephosphorylation activity in the cap-closed conformation with the disulphide bond and even in the crystal state, in which repositioning of the cap and core domains is restricted. Structural analysis suggests that a small substrate such as PLP can bind to the active site through a small movement of a local motif. However, a change to the cap-open conformation is required for binding of larger substrates such as phosphopeptides to the active site.

A Review of the Microbial Production of Bioactive Natural Products and Biologics.

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

O2- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis.

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.

Crystallization and preliminary crystallographic analysis of the second GAF domain of DevS from Mycobacterium smegmatis.

Mycobacterium tuberculosis is known to transform into the nonreplicating persistence state under the influence of hypoxia or nitric oxide. DevS-DevR is a two-component regulatory system that mediates the genetic response for the transformation. DevS is a histidine kinase that contains two GAF domains for sensing hypoxia or nitric oxide. The second GAF from M. smegmatis DevS was crystallized using the sitting-drop vapour-diffusion method in the presence of sodium citrate and 2-propanol as precipitants. X-ray diffraction data were collected from crystals containing selenomethionine to a maximum resolution of 2.0 A on a synchrotron beamline. The crystals belong to the hexagonal space group P6(1). The asymmetric unit contains one molecule, corresponding to a packing density of 2.5 A(3) Da(-1). The selenium substructure was determined by the single anomalous dispersion method and structure refinement is in progress.