Human Cellular Retinol Binding Protein II Forms a Domain;#8208;Swapped Trimer Representing a Novel F
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Ghanbarpour, Alireza↗
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Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9
Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA ( tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo–electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.
Science & Technology - Other Topics↗
Acute deletion of TET enzymes results in aneuploidy in mouse embryonic stem cells through decreased expression of Khdc3
TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.
59 BASIC BIOLOGICAL SCIENCES↗
A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB
Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe 2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe 2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response
TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response.
59 BASIC BIOLOGICAL SCIENCES↗
Adaptive responses of marine diatoms to zinc scarcity and ecological implications
Scarce dissolved surface ocean concentrations of the essential algal micronutrient zinc suggest that Zn may influence the growth of phytoplankton such as diatoms, which are major contributors to marine primary productivity. However, the specific mechanisms by which diatoms acclimate to Zn deficiency are poorly understood. Using global proteomic analysis, we identified two proteins (ZCRP-A/B, Zn/Co Responsive Protein A/B) among four diatom species that became abundant under Zn/Co limitation. Characterization using reverse genetic techniques and homology data suggests putative Zn/Co chaperone and membrane-bound transport complex component roles for ZCRP-A (a COG0523 domain protein) and ZCRP-B, respectively. Metaproteomic detection of ZCRPs along a Pacific Ocean transect revealed increased abundances at the surface (<200 m) where dZn and dCo were scarcest, implying Zn nutritional stress in marine algae is more prevalent than previously recognized. These results demonstrate multiple adaptive responses to Zn scarcity in marine diatoms that are deployed in low Zn regions of the Pacific Ocean.
54 ENVIRONMENTAL SCIENCES↗
Bipartite chromatin recognition by Hop1 from two diverged Holozoa
In meiosis, ploidy reduction is driven by a complex series of DNA breakage and recombination events between homologous chromosomes, orchestrated by meiotic HORMA domain proteins (HORMADs). Meiotic HORMADs possess a central chromatin binding region (CBR) whose architecture varies across eukaryotic groups. Here, we determine high-resolution crystal structures of the meiotic HORMAD CBR from two diverged aquatic Holozoa,Schistosoma mansoniandPatiria miniata, which reveal tightly associated plant homeodomain (PHD) and winged helix-turn-helix (wHTH) domains. We show that PHD–wHTH CBRs bind duplex DNA through their wHTH domains, and identify key residues that disrupt this interaction. Combining experimental and predicted structures, we show that the CBRs’ PHDs likely interact with the tail of histone H3, and may discriminate between unmethylated and trimethylated H3 lysine 4. Finally, we show that Holozoa Hop1 CBRs bind nucleosomes in vitro in a bipartite manner involving both the PHD and wHTH domain. Our data reveal how meiotic HORMADs with PHD–wHTH CBRs can bind chromatin and potentially discriminate between chromatin states to drive meiotic recombination to specific chromosomal regions.
Life Sciences & Biomedicine - Other Topics↗
MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers
The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.
Novosphingobium↗
Reduction of a Heme Cofactor Initiates N -Nitroglycine Degradation by NnlA
Linear nitramines are potentially carcinogenic environmental contaminants. The NnlA enzyme from Variovorax sp. strain JS1663 degrades the nitramine N-nitroglycine (NNG)—a natural product produced by some bacteria—to glyoxylate and nitrite (NO 2 –). Ammonium (NH 4 +) was predicted as the third product of this reaction. A source of nonheme Fe II was shown to be required for initiation of NnlA activity. However, the role of this Fe II for NnlA activity was unclear. This work reveals that NnlA contains a b-type heme cofactor. Reduction of this heme—either by a nonheme iron source or dithionite—is required to initiate NnlA activity. Therefore, Fe II is not an essential substrate for holoenzyme activity. Our data show that reduced NnlA (Fe II -NnlA) catalyzes at least 100 turnovers and does not require O 2 . Finally, NH 4 + was verified as the third product, accounting for the complete nitrogen mass balance. Size exclusion chromatography showed that NnlA is a dimer in solution. Additionally, Fe II -NnlA is oxidized by O 2 and NO 2 – and stably binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with heme-binding PAS domains. Furthermore, a structural homology model of NnlA was generated using the PAS domain from Pseudomonas aeruginosa Aer2 as a template. The structural homology model suggested His 73 is the axial ligand of the NnlA heme. Site-directed mutagenesis of His 73 to alanine decreased the heme occupancy of NnlA and eliminated NNG activity, validating the homology model. We conclude that NnlA forms a homodimeric heme-binding PAS domain protein that requires reduction for initiation of the activity.
59 BASIC BIOLOGICAL SCIENCES↗
Intermediate scattering functions of a rigid body monoclonal antibody protein in solution studied by dissipative particle dynamic simulation
In the past decade, there was increased research interest in studying internal motions of flexible proteins in solution using Neutron Spin Echo (NSE) as NSE can simultaneously probe the dynamics at the length and time scales comparable to protein domain motions. However, the collective intermediate scattering function (ISF) measured by NSE has the contributions from translational, rotational, and internal motions, which are rather complicated to be separated. Widely used NSE theories to interpret experimental data usually assume that the translational and rotational motions of a rigid particle are decoupled and independent to each other. To evaluate the accuracy of this approximation for monoclonal antibody (mAb) proteins in solution, dissipative particle dynamic computer simulation is used here to simulate a rigid-body mAb for up to about 200 ns. The total ISF together with the ISFs due to only the translational and rotational motions as well as their corresponding effective diffusion coefficients is calculated. The aforementioned approximation introduces appreciable errors to the calculated effective diffusion coefficients and the ISFs. For the effective diffusion coefficient, the error introduced by this approximation can be as large as about 10% even though the overall agreement is considered reasonable. Thus, we need to be cautious when interpreting the data with a small signal change. In addition, the accuracy of the calculated ISFs due to the finite computer simulation time is also discussed.
71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
Expansion of the global RNA virome reveals diverse clades of bacteriophages
High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.
59 BASIC BIOLOGICAL SCIENCES↗
Generating Protein Structures for Pathway Discovery Using Deep Learning
Resolving the intricate details of biological phenomena at the molecular level is fundamentally limited by both length- and time scales that can be probed experimentally. Molecular dynamics (MD) simulations at various scales are powerful tools frequently employed to offer valuable biological insights beyond experimental resolution. However, while it is relatively simple to observe long-lived, stable configurations of, for example, proteins, at the required spatial resolution, simulating the more interesting rare transitions between such states often takes orders of magnitude longer than what is feasible even on the largest supercomputers available today. One common aspect of this challenge is pathway discovery, where the start and end states of a scientific phenomenon are known or can be approximated, but the mechanistic details in between are unknown. Here, we propose a representation-learning-based solution that uses interpolation and extrapolation in an abstract representation space to synthesize potential transition states, which are automatically validated using MD simulations. The new simulations of the synthesized transition states are subsequently incorporated into the representation learning, leading to an iterative framework for targeted path sampling. Our approach is demonstrated by recovering the transition of a RAS-RAF protein domain (CRD) from membrane-free to interacting with the membrane using coarse-grain MD simulations.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
XND1 Regulates Secondary Wall Deposition in Xylem Vessels through the Inhibition of VND Functions
Secondary wall deposition in xylem vessels is activated by Vascular-Related NAC Domain proteins (VNDs) that belong to a group of secondary wall NAC (SWN) transcription factors. By contrast, Xylem NAC Domain1 (XND1) negatively regulates secondary wall deposition in xylem vessels when overexpressed. The mechanism by which XND1 exerts its functions remains elusive. We employed the promoter of the fiber-specific Secondary Wall-Associated NAC Domain1 (SND1) gene to ectopically express XND1 in fiber cells to investigate its mechanism of action on secondary wall deposition. Ectopic expression of XND1 in fiber cells severely diminished their secondary wall deposition and drastically reduced the expression of SWN-regulated downstream transcription factors and secondary wall biosynthetic genes but not that of the SWN genes themselves. Transactivation analyses revealed that XND1 specifically inhibited SWN-activated expression of these downstream genes but not their MYB46-activated expression. Both the NAC domain and the C-terminus of XND1 were required for its inhibitory function and its NAC domain interacted with the DNA-binding domains of SWNs. XND1 was shown to be localized in the cytoplasm and the nucleus and its co-expression with VND6 resulted in the cytoplasmic sequestration of VND6. Furthermore, the C-terminus of XND1 was indispensable for the XND1-mediated cytoplasmic retention of VND6 and its fusion to VND6 was able to direct VND6 to the cytoplasm and render it unable to activate the gene expression. Since the XND1 gene is specifically expressed in xylem cells, these results indicate that XND1 acts through inhibiting VND functions to negatively regulate secondary wall deposition in xylem vessels.
Cell Biology↗
A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step
Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell -1 . Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.
59 BASIC BIOLOGICAL SCIENCES↗
Loss of MBD2 affects early T cell development by inhibiting the WNT signaling pathway
Highlights: • MBD2 deficiency resulted in retardation of T cell differentiation at the DN3 stage. • Decreased proliferative capacity and increased apoptosis were detected in Mbd2{sup −/−} DN thymocytes. • MBD2 affects early T cell development by regulating the expression of key genes of WNT pathway. DNA methylation alters the expression of certain genes without any alteration to the DNA sequence and is a dynamic process during normal hematopoietic differentiation. As an epigenetic regulator, methyl-CpG-binding domain protein 2 (MBD2) is an important member of the MBD protein family and is acknowledged as a “reader” of DNA methylation. We used a mouse model to study the effects of MBD2 on the early development of T cells. Here, we found that MBD2 deficiency led to retardation of T cell differentiation at the DN3 stage. Meanwhile, decreased proliferative capacity and increased apoptosis were detected in Mbd2{sup −/−} DN thymocytes. Furthermore, we found the WNT pathway was significantly down-regulated in Mbd2{sup −/−} DN thymocytes: DKK1 (Dickkopf-1) expression was significantly increased, while TCF7 (transcription factor 7) and c-MYC were down-regulated. Thus, these findings established that MBD2 acted as a dominant regulator to imprint DN T cell development via the WNT pathway.
60 APPLIED LIFE SCIENCES↗
Conformational motions and ligand-binding underlying gating and regulation in IP 3 R channel
Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP 3 and Ca 2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP 3 R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP 3 , Ca 2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP 3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP 3 R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.
59 BASIC BIOLOGICAL SCIENCES↗
A plant genetic network for preventing dysbiosis in the phyllosphere
The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Here, the pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.
59 BASIC BIOLOGICAL SCIENCES↗
In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus
The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.
99 GENERAL AND MISCELLANEOUS↗