Conserved Serine Residue Research Articles

Loss-of-Function Mutation in tet2 in Zebrafish Leads to Early MDS like Phenotype

Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia.Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent.We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant.These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss.Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype.Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation.This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Expression of novel nitrate reductase genes in the harmful alga, Chattonella subsalsa

Eukaryotic nitrate reductase (NR) catalyzes the first step in nitrate assimilation and is regulated transcriptionally in response to external cues and intracellular metabolic status. NRs are also regulated post-translationally in plants by phosphorylation and binding of 14-3-3 proteins at conserved serine residues. 14-3-3 binding motifs have not previously been identified in algal NRs. A novel NR (NR2-2/2HbN) with a 2/2 hemoglobin domain was recently described in the alga Chattonella subsalsa. Here, a second NR (NR3) in C. subsalsa is described with a 14-3-3 binding motif but lacking the Heme-Fe domain found in other NRs. Transcriptional regulation of both NRs was examined in C. subsalsa, revealing differential gene expression over a diel light cycle, but not under constant light. NR2 transcripts increased with a decrease in temperature, while NR3 remained unchanged. NR2 and NR3 transcript levels were not inhibited by growth on ammonium, suggesting constitutive expression of these genes. Results indicate that Chattonella responds to environmental conditions and intracellular metabolic status by differentially regulating NR transcription, with potential for post-translational regulation of NR3. A survey of algal NRs also revealed the presence of 14-3-3 binding motifs in other algal species, indicating the need for future research on regulation of algal NRs.

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From Arabidopsis thaliana

The β-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

3147 - Loss-of-Function Mutation in TET2 in Zebrafish Leads to Early MDS Like Phenotype

Abstract Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia. Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent. We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant. These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss. Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype. Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation. This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apo-structure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded FO domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS175N) prevents FO-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors (aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20–30 Å) from the original mutation. The possibility to compensate through long-range effects the aS175N mutation is an interesting observation that holds promise for the development of therapeutic molecules.

IPD3 and IPD3L Function Redundantly in Rhizobial and Mycorrhizal Symbioses.

Legume plants form symbiotic associations with either nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi, which are regulated by a set of common symbiotic signaling pathway genes. Central to the signaling pathway is the activation of the DMI3/IPD3 protein complex by Ca2+ oscillations, and the initiation of nodule organogenesis and mycorrhizal symbiosis. DMI3 is essential for rhizobial infection and nodule organogenesis; however, ipd3 mutants have been shown to be impaired only in infection thread formation but not in root nodule organogenesis in Medicago truncatula. We identified an IPD3-like (IPD3L) gene in the M. truncatula genome. A single ipd3l mutant exhibits a normal root nodule phenotype. The ipd3l/ipd3-2 double mutant is completely unable to initiate infection threads and nodule primordia. IPD3L can functionally replace IPD3 when expressed under the control of the IPD3 promoter, indicating functional redundancy between these two transcriptional regulators. We constructed a version of IPD3 that was phosphomimetic with respect to two conserved serine residues (IPD3-2D). This was sufficient to trigger root nodule organogenesis, but the increased multisite phosphorylation of IPD3 (IPD3-8D) led to low transcriptional activity, suggesting that the phosphorylation levels of IPD3 fine-tune its transcriptional activity in the root nodule symbiosis. Intriguingly, the phosphomimetic version of IPD3 triggers spontaneous root-like nodules on the roots of dmi3-1 and dmi2-1 (DMI2 is an LRR-containing receptor-like kinase gene which is required for Ca2+ spiking), but not on the roots of wild-type or ipd3l ipd3-2 plants. In addition, fully developed arbuscules were formed in the ipd3l ipd3-2 mutants but not the ccamk/dmi3-1 mutants. Collectively, our data indicate that, in addition to IPD3 and IPD3L, another new genetic component or other new phosphorylation sites of IPD3 function downstream of DMI3 in rhizobial and mycorrhizal symbioses.

Dampened STING-Dependent Interferon Activation in Bats

Compared with terrestrial mammals, bats have a longer lifespan and greater capacity to co-exist with a variety of viruses. In addition to cytosolic DNA generated by these viral infections, the metabolic demands of flight cause DNA damage and the release of self-DNA into the cytoplasm. However, whether bats have an altered DNA sensing/defense system to balance high cytosolic DNA levels remains an open question. We demonstrate that bats have a dampened interferon response due to the replacement of the highly conserved serine residue (S358) in STING, an essential adaptor protein in multiple DNA sensing pathways. Reversing this mutation by introducing S358 restored STING functionality, resulting in interferon activation and virus inhibition. Combined with previous reports on bat-specific changes of other DNA sensors such as TLR9, IFI16, and AIM2, our findings shed light on bat adaptation to flight, their long lifespan, and their unique capacity to serve as a virus reservoir.

GATA6 phosphorylation by Erk1/2 propels exit from pluripotency and commitment to primitive endoderm

The transcription factor GATA6 and the Fgf/Ras/MAPK signaling pathway are essential for the development of the primitive endoderm (PrE), one of the two lineages derived from the pluripotent inner cell mass (ICM) of mammalian blastocysts. A mutant mouse line in which Gata6-coding exons are replaced with H2BGFP (histone H2B Green Fluorescence Protein fusion protein) was developed to monitor Gata6 promoter activity. In the Gata6-H2BGFP heterozygous blastocysts, the ICM cells that initially had uniform GFP fluorescence signal at E3.5 diverged into two populations by the 64-cell stage, either as the GFP-high PrE or the GFP-low epiblasts (Epi). However in the GATA6-null blastocysts, the originally moderate GFP expression subsided in all ICM cells, indicating that the GATA6 protein is required to maintain its own promoter activity during PrE linage commitment. In embryonic stem cells, expressed GATA6 was shown to bind and activate the Gata6 promoter in PrE differentiation. Mutations of a conserved serine residue (S264) for Erk1/2 phosphorylation in GATA6 protein drastically impacted its ability to activate its own promoter. We conclude that phosphorylation of GATA6 by Erk1/2 compels exit from pluripotent state, and the phosphorylation propels a GATA6 positive feedback regulatory circuit to compel PrE differentiation. Our findings resolve the longstanding question on the dual requirements of GATA6 and Ras/MAPK pathway for PrE commitment of the pluripotent ICM.

Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response

As sessile organisms, plants must adapt to variations in the environment. Environmental stress triggers various responses, including growth inhibition, mediated by the plant hormone abscisic acid (ABA). The mechanisms that integrate stress responses with growth are poorly understood. Here, we discovered that the Target of Rapamycin (TOR) kinase phosphorylates PYL ABA receptors at a conserved serine residue to prevent activation of the stress response in unstressed plants. This phosphorylationdisrupts PYL association with ABA and with PP2C phosphatase effectors, leading to inactivationof SnRK2 kinases. Under stress, ABA-activated SnRK2s phosphorylate Raptor, a component of the TOR complex, triggering TOR complex dissociationand inhibition. Thus, TOR signaling represses ABA signaling and stress responses in unstressed conditions, whereas ABA signaling represses TOR signaling and growth during times of stress. Plants utilize this conserved phospho-regulatory feedback mechanism to optimize the balance of growth and stress responses.

Trametinib suppresses HIV-1 replication by interfering with the disassembly of human immunodeficiency virus type 1 capsid core

Our previous study showed that the phosphorylation of a highly conserved serine residue, Ser16 in the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is promoted by virion-incorporated extracellular signal-regulated kinase 2 (ERK2) and required for proper peptidyl-prolyl isomerase (Pin1)-mediated uncoating. Interestingly, western blot analysis demonstrated that phosphorylated/activated mitogen-activated protein kinase kinase 1/2 (MEK1/2), the upstream activator of ERK2, as well as ERK2 are incorporated into virions. Here, we show that the MEK1/2 selective allosteric inhibitor Trametinib reduces HIV-1 infectivity via the decrease in virion-incorporated ERK2 phosphorylation. The treatment of chronic HIV-1-infected T-cell line, CEM/LAV-1 cells with Trametinib results in a decrease in ERK2 phosphorylation in the virions. The viruses have relatively low infectivity and impaired reverse transcription. Cell-based fate-of-capsid uncoating assay showed that the reduction in infectivity was caused by a functional impairment of the uncoating process. Furthermore, the viruses from Trametinib-treated CEM/LAV-1 cells also showed decreased reverse transcription efficiency and attenuated multiple rounds of replication in human peripheral blood mononuclear cells (PBMCs). Taken together, these findings suggest that Trametinib suppresses HIV-1 replication by abrogating the proper disassembly of CA core.

Regulatory Mechanisms of a Highly Pectinolytic Mutant of Penicillium occitanis and Functional Analysis of a Candidate Gene in the Plant Pathogen Fusarium oxysporum.

Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin) or repressing conditions (glucose) was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum, a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

Crocodylian nuclear factor kappa B.

We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element.

Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts.

Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK.

Nosiheptide (NOS) is a highly modified thiopeptide antibiotic that displays formidable in vitro activity against a variety of Gram-positive bacteria. In addition to a central hydroxypyridine ring, NOS contains several other modifications, including multiple thiazole rings, dehydro-amino acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety is required for NOS efficacy and is synthesized from l-tryptophan in a series of reactions that have not been fully elucidated. Herein, we describe the role of NosJ, the product of an unannotated gene in the biosynthetic operon for NOS, as an acyl carrier protein that delivers 3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI as the enzyme responsible for catalyzing the ATP-dependent activation of MIA and MIA’s attachment to the phosphopantetheine moiety of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray crystal structure of NosK, solved to 2.3 Å resolution, reveals that the protein is an α/β-fold hydrolase. Ser102 interacts with Glu210 and His234 to form a catalytic triad located at the bottom of an open cleft that is large enough to accommodate the thiopeptide framework.

Flaviviridae viruses use a common molecular mechanism to escape nucleoside analogue inhibitors

The RNA-dependent RNA polymerases of Flaviviridae viruses are crucial for replication. The Flaviviridae polymerase is organized into structural motifs (A-G), with motifs F, A, C and E containing interrogating, priming and catalytic substrate-interacting sites. Modified nucleoside analogues act as antiviral drugs by targeting Flaviviridae polymerases and integrating into the synthesized product causing premature termination. A threonine mutation of a conserved serine residue in motif B of Flaviviridae polymerases renders resistance to 2′-C-methylated nucleoside analogues. The mechanism how this single mutation causes Flaviviridae viruses to escape nucleoside analogues is not yet known. Given the pivotal position of the serine residue in motif B that supports motif F, we hypothesized the threonine mutation causes alterations in nucleoside exploration within the entry tunnel. Implementing a stochastic molecular software showed the all-atom 2′-C-methylated analogue reaction within the active sites of wild type and serine-threonine mutant polymerases from Hepacivirus and Flavivirus. Compared with the wild type, the serine-threonine mutant polymerases caused a significant decrease of analogue contacts with conserved interrogating residues in motif F and a displacement of metal ion cofactors. The simulations significantly showed that during the analogue exploration of the active site the hydrophobic methyl group in the serine-threonine mutant repels water-mediated hydrogen bonds with the 2′-C-methylated analogue, causing a concentration of water-mediated bonds at the substrate-interacting sites. Collectively, the data are an insight into a molecular escape mechanism by Flaviviridae viruses from 2′-C-methylated nucleoside analogue inhibitors.

Crystal structure of MBP-PigG fusion protein and the essential function of PigG in the prodigiosin biosynthetic pathway in Serratia marcescens FS14

Prodigiosin, a tripyrrole red pigment is synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway; MBC (4-methoxy-2,2′-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. PigI, PigG and PigA have been shown to be involved in the first steps of MBC biosynthesis (proline incorporation). The crystal structure of PigG was resolved to elucidate its function and mechanism. PigG, an acyl carrier protein (ACP), features the ACP architecture:, a helical bundle fold containing three major helices and a minor distorted helix together with a conserved “S” motif. An in-frame deletion mutation of the pigG gene abolished the synthesis of prodigiosin in Serratia marcescens FS14. The production of prodigiosin was fully restored by complementation of intact pigG; however the S36A mutant was not able to restore function in the in-frame deletion pigG mutant, indicating that PigG and the conserved serine residue (S36) of PigG are essential for the synthesis of prodigiosin.

Molecular Dynamics Simulations for Understanding IR Spectra and the Distribution of Environments Around the Phosphopantetheine Arm of Acyl Carrier Proteins

Natural product biosynthesis in polyketide synthases (PKSs) and fatty acid synthases (FASs) is heavily reliant on both specificity and selectivity of protein-protein interactions. Interactions are mediated by the acyl carrier protein (ACP), which utilizes a 4′-phosphopantateine (Ppant) arm attached to a conserved serine residue to shuttle carbon intermediates between partner enzymes. Recent studies have shown that site-specific vibrational spectroscopy can identify the local environment of the arm through cyanylation of the terminal sulfur of the 4′Ppant arm and introduction of a unique SCN residue. We aim to use molecular dynamics simulations in GROMACS to simulate IR spectra from atom-level physical interactions and better elucidate the dynamics and structural distribution of the Ppant arm. A strong understanding of the behavior of the ACP's Ppant arm will provide both a reliable and generalizable method for confirming experimental IR data, and inform bioengineering efforts by further characterizing ACP's interactions with PKSs and FASs that give rise to complex natural products.

Identification and Crystallization of Penicillin-Binding Protein/β-Lactamase Homolog (Rp46) from Ruegeria Pomeroyi

In spite of the enormous biological and clinical significance of penicillin-binding protein (PBP)/β-lactamase (βL), few of their many homologs (PBP)/βLs homologs) have been studied crystallographically, and have known functions. Herein, X-ray crystallographic study of a PBP/βL homolog (Rp46) from Ruegeria pomeroyi is described. Multiple sequence alignments indicate that Rp46 has a conserved serine residue within the S70-X-X-K73 motif (Motif I), acting as the catalytic nucleophile. Moreover, an invariant tyrosine residue (Tyr185) and a Trp365-X-Gly motif (Motif III) were also identified. The recombinant Rp46 protein was expressed in Escherichia coli and purified to homogeneity judging from the SDS-PAGE analysis. Rp46 was crystallized using a solution consisting of 20% (w/v) PEG 3000, 0.1 M Tris-HCl, pH 7.0, 0.2 M calcium acetate, and the X-ray diffraction data were collected to a resolution of 1.90 Å with an Rmerge of 7.4%. The crystals of Rp46 belong to the space group I422, with unit cell parameters a = b = 141.26 Å, and c = 119.75. The structure determination and biochemical characterization are in progress. (Synopsis: A penicillin-binding protein/β-lactamase homolog (Rp46) from Ruegeria pomeroyi was identified and crystallized in the space group I4, and the diffraction data were collected to a resolution of 1.90 Å.)