Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin.

Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of ß-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

Novel Susceptibility Genes Drive Familial Non-Medullary Thyroid Cancer in a Large Consanguineous Kindred.

Familial non-medullary thyroid cancer (FNMTC) is a well-differentiated thyroid cancer (DTC) of follicular cell origin in two or more first-degree relatives. Patients typically demonstrate an autosomal dominant inheritance pattern with incomplete penetrance. While known genes and chromosomal loci account for some FNMTC, the molecular basis for most FNMTC remains elusive. To identify the variation(s) causing FNMTC in an extended consanguineous family consisting of 16 papillary thyroid carcinoma (PTC) cases, we performed whole exome sequence (WES) analysis of six family patients. We demonstrated an association of ARHGEF28, FBXW10, and SLC47A1 genes with FNMTC. The variations in these genes may affect the structures of their encoded proteins and, thus, their function. The most promising causative gene is ARHGEF28, which has high expression in the thyroid, and its protein-protein interactions (PPIs) suggest predisposition of PTC through ARHGEF28-SQSTM1-TP53 or ARHGEF28-PTCSC2-FOXE1-TP53 associations. Using DNA from a patient's thyroid malignant tissue, we analyzed the possible cooperation of somatic variations with these genes. We revealed two somatic heterozygote variations in XRCC1 and HRAS genes known to implicate thyroid cancer. Thus, the predisposition by the germline variations and a second hit by somatic variations could lead to the progression to PTC.

Asgard ESCRT-III and VPS4 reveal conserved chromatin binding properties of the ESCRT machinery.

The archaeal Asgard superphylum currently stands as the most promising prokaryotic candidate, from which eukaryotic cells emerged. This unique superphylum encodes for eukaryotic signature proteins (ESP) that could shed light on the origin of eukaryotes, but the properties and function of these proteins is largely unresolved. Here, we set to understand the function of an Asgard archaeal protein family, namely the ESCRT machinery, that is conserved across all domains of life and executes basic cellular eukaryotic functions, including membrane constriction during cell division. We find that ESCRT proteins encoded in Loki archaea, express in mammalian and yeast cells, and that the Loki ESCRT-III protein, CHMP4-7, resides in the eukaryotic nucleus in both organisms. Moreover, Loki ESCRT-III proteins associated with chromatin, recruited their AAA-ATPase VPS4 counterpart to organize in discrete foci in the mammalian nucleus, and directly bind DNA. The human ESCRT-III protein, CHMP1B, exhibited similar nuclear properties and recruited both human and Asgard VPS4s to nuclear foci, indicating interspecies interactions. Mutation analysis revealed a role for the N terminal region of ESCRT-III in mediating these phenotypes in both human and Asgard ESCRTs. These findings suggest that ESCRT proteins hold chromatin binding properties that were highly preserved through the billion years of evolution separating Asgard archaea and humans. The conserved chromatin binding properties of the ESCRT membrane remodeling machinery, reported here, may have important implications for the origin of eukaryogenesis.

Evolution of homo-oligomerization of methionine S-adenosyltransferases is replete with structure-function constrains.

Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S-adenosyltransferases (MATs)-dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer-dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate-induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.

Current view of iron biomineralization in magnetotactic bacteria.

Biomineralization is the process of mineral formation by living organisms. One notable example of these organisms is magnetotactic bacteria (MTB). MTB are Gram-negative bacteria that can biomineralize iron into magnetic nanoparticles. This ability allows these aquatic microorganisms to orient themselves according to the geomagnetic field. The biomineralization process takes place in a specialized sub-cellular membranous organelle, the magnetosome. The magnetosome contains a defined set of magnetosome-associated proteins (MAPs) that controls the biomineralization environment, including iron concentration, redox, and pH. Magnetite formation is subjected to a tight regulation within the magnetosome that affects the nanoparticle nucleation, size, and shape, leading to well-defined magnetic properties. The formed magnetite nanoparticles have unique characteristics of a stable, single magnetic domain with narrow size distribution and high crystalline structures, which turned MTB into the subject of interest in multidisciplinary research. This graphical review provides a current overview of iron biomineralization in magnetotactic bacteria, focusing on Alphaproteobacteria. To better understand this complex mechanism, we present the four main steps and the main MAPs participating in the process of magnetosome formation.

SAMase of Bacteriophage T3 Inactivates Escherichia coli's Methionine S-Adenosyltransferase by Forming Heteropolymers.

S-Adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host Escherichia coli cells, thereby inactivating a crucial metabolite involved in a plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection, and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy. It allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle cryoelectron microscopy (cryo-EM) and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM but also by interacting with and efficiently inhibiting the host's methionine S-adenosyltransferase (MAT), the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency, high-amplitude active-site-deforming modes. The amplification of uncorrelated motions between active-site residues weakens MAT's substrate binding affinity, providing a possible explanation for the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools. IMPORTANCE Self-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances, filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy: silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes is a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.

Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We consistently found that recombinant purified L8 directly binds MTs and L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that the largely unstructured L8 facilitates flexible anchoring of Cin8 to the MTs. This flexible anchoring enables the direct observation of bi-directional motility in motility assays. Remarkably, although L8-deleted Cin8 variants exhibit a strong minus-end directed bias at the single-molecule level, they also exhibit plus-end directed motility in an MT-gliding assay. Thus, L8-induced flexible MT anchoring is required for bi-directional motility of single Cin8 molecules but is not necessary for context-dependent directionality reversal of Cin8 in an MT-gliding assay.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.

Folding of an Intrinsically Disordered Iron-Binding Peptide in Response to Sedimentation Revealed by Cryo-EM.

Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.

A common genetic variant in zinc transporter ZnT2 (Thr288Ser) is present in women with low milk volume and alters lysosome function and cell energetics.

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.

From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

The interdimeric interface controls function and stability of Ureaplasma urealiticum methionine S-adenosyltransferase.

Methionine S-adenosyltransferases (MATs) are predominantly homotetramers, comprised of dimers of dimers. The larger, highly conserved intradimeric interface harbors two active sites, making the dimer the obligatory functional unit. However, functionality of the smaller, more diverged, and recently evolved interdimeric interface is largely unknown. Here, we show that the interdimeric interface of Ureaplasmaurealiticum MAT has evolved to control the catalytic activity and structural integrity of the homotetramer in response to product accumulation. When all four active sites are occupied with the product, S-adenosylmethionine (SAM), binding of four additional SAM molecules to the interdimeric interface prompts a ∼45° shift in the dimer orientation and a concomitant ∼60% increase in the interface area. This rearrangement inhibits the enzymatic activity by locking the flexible active site loops in a closed state and renders the tetramer resistant to proteolytic degradation. Our findings suggest that the interdimeric interface of MATs is subject to rapid evolutionary changes that tailor the molecular properties of the entire homotetramer to the specific needs of the organism.

Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29-121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical 'tip motif'. A flexible region preceding the BteA helical bundle contains the characteristic ß-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P2-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P2 analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P2 and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P2-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1-287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.

Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine.

The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression of STAT3 with the Elk receptor tyrosine kinase, we were able to characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

A Colorimetric Method for Measuring Iron Content in Plants.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

Directed evolution of SIRT6 for improved deacylation and glucose homeostasis maintenance.

Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

Structural Basis for p53 Lys120-Acetylation-Dependent DNA-Binding Mode.

Normal cellular homeostasis depends on tight regulation of gene expression, which requires the modulation of transcription factors' DNA-binding specificity. That said, the mechanisms that allow transcription factors to distinguish between closely related response elements following different cellular signals are not fully understood. In the tumor suppressor protein p53, acetylation of loop L1 residue Lys120 within the DNA-binding domain has been shown to promote the transcription of proapoptotic genes such as bax. Here, we report the crystal structures of Lys120-acetylated p53 DNA-binding domain in complex with a consensus response element and with the natural BAX response element. Our structural analyses reveal that Lys120 acetylation expands the conformational space of loop L1 in the DNA-bound state. Loop L1 flexibility is known to increase p53's DNA-binding specificity, and Lys120-acetylation-dependent conformational changes in loop L1 enable the formation of sequence-dependent DNA-binding modes for p53. Furthermore, binding to the natural BAX response element is accompanied by global conformational changes, deformation of the DNA helical structure, and formation of an asymmetric tetrameric complex. Based on these findings, we suggest a model for p53's Lys120 acetylation-dependent DNA-binding mode.

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.