Zinc-binding Domain Research Articles

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Zinc-dependent multimerization of mutant calreticulin is required for MPL binding and MPN pathogenesis

Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.

Studies on Vibrio mimicus derived collagenase variants providing insights into critical role(s) played by the FAXWXXT motifs in its collagen-binding domain

Vibrio mimicus collagenase (VMC), a Class II Vibrio metalloprotease, contains an HEXXH motif in a zinc-binding catalytic domain, and two FAXWXXT motifs in its C-terminal domain, which is its collagen binding domain (CBD). To understand the functional role of the individual CBD motifs in the activity of VMC, if any, we created and characterized a series of VMC variants: i) VMA, with 51 amino acids deleted from the C-terminal end of full-length VMC; ii) VMT1, a form of VMA mutated in the first CBD motif; iii) VMT2, a form of VMA mutated in the second CBD motif; iv) DM, a form of VMA with both CBD motifs mutated; v) CT, a truncated form of VMA, lacking the entire CBD region; and vi) CBD, a construct containing the collagen binding domain alone. The activity of each variant was assessed by multiple means, in relation to VMA. We report that VMT1 and VMT2 show 1.6-fold and 10-fold reduced activity, respectively. The reduced activity of VMT2 correlates with reduced binding to insoluble collagen as well as an inability to cause structural perturbation of collagen. VMC appears to cause unwinding and structural alteration of the collagen triple helix prior to hydrolysis of the substrate (using both motifs for collagen binding), like Clostridium collagenases. In the absence of a known structure for VMC, our findings suggest that Vibrio collagenase, functions like Clostridium collagenases, although the two show very little sequence similarity. Also, VMC shows reduced activity with respect to Clostridium collagenases, making it an ideal enzyme for therapeutic applications.

Zinc Metalloproteins in Epigenetics and Their Crosstalk.

More than half a century ago, zinc was established as an essential micronutrient for normal human physiology. In silico data suggest that about 10% of the human proteome potentially binds zinc. Many proteins with zinc-binding domains (ZBDs) are involved in epigenetic modifications such as DNA methylation and histone modifications, which regulate transcription in physiological and pathological conditions. Zinc metalloproteins in epigenetics are mainly zinc metalloenzymes and zinc finger proteins (ZFPs), which are classified into writers, erasers, readers, editors, and feeders. Altogether, these classes of proteins engage in crosstalk that fundamentally maintains the epigenome’s modus operandi. Changes in the expression or function of these proteins induced by zinc deficiency or loss of function mutations in their ZBDs may lead to aberrant epigenetic reprogramming, which may worsen the risk of non-communicable chronic diseases. This review attempts to address zinc’s role and its proteins in natural epigenetic programming and artificial reprogramming and briefly discusses how the ZBDs in these proteins interact with the chromatin.

The zinc-binding domain of mammalian prolyl-tRNA synthetase is indispensable for catalytic activity and organism viability

SummaryAminoacyl-tRNA synthetases (AARS) participate in decoding the genome by catalyzing conjugation of amino acids to their cognate tRNAs. During evolution, biochemical and environmental conditions markedly influenced the sequence and structure of the 20 AARSs, revealing adaptations dictating canonical and orthogonal activities. Here, we investigate the function of the appended Zn2+-binding domain (ZBD) in the bifunctional AARS, glutamyl-prolyl-tRNA synthetase (GluProRS). We developed GluProRS mutant mice by CRISPR-Cas9 with a deletion of 29 C-terminal amino acids, including two of four Zn2+-coordinating cysteines. Homozygous ZBD mutant mice die before embryonic day 12.5, but heterozygous mice are healthy. ZBD disruption profoundly reduces GluProRS canonical function by dual mechanisms: it induces rapid proteasomal degradation of the protein and inhibits ProRS aminoacylation activity, likely by sub-optimal positioning of ATP in the spatially adjacent catalytic domain. Collectively, our studies reveal the ZBD as a critical determinant of ProRS activity and GluProRS stability in vitro and in vivo.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2’-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

The Role of APOBECs in Viral Replication.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) proteins are a diverse and evolutionarily conserved family of cytidine deaminases that provide a variety of functions from tissue-specific gene expression and immunoglobulin diversity to control of viruses and retrotransposons. APOBEC family expansion has been documented among mammalian species, suggesting a powerful selection for their activity. Enzymes with a duplicated zinc-binding domain often have catalytically active and inactive domains, yet both have antiviral function. Although APOBEC antiviral function was discovered through hypermutation of HIV-1 genomes lacking an active Vif protein, much evidence indicates that APOBECs also inhibit virus replication through mechanisms other than mutagenesis. Multiple steps of the viral replication cycle may be affected, although nucleic acid replication is a primary target. Packaging of APOBECs into virions was first noted with HIV-1, yet is not a prerequisite for viral inhibition. APOBEC antagonism may occur in viral producer and recipient cells. Signatures of APOBEC activity include G-to-A and C-to-T mutations in a particular sequence context. The importance of APOBEC activity for viral inhibition is reflected in the identification of numerous viral factors, including HIV-1 Vif, which are dedicated to antagonism of these deaminases. Such viral antagonists often are only partially successful, leading to APOBEC selection for viral variants that enhance replication or avoid immune elimination.

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/β linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like β-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

Snapshots of ADP-ribose bound to Getah virus macro domain reveal an intriguing choreography

Alphaviruses are (re-)emerging arboviruses of public health concern. The nsP3 gene product is one of the key players during viral replication. NsP3 comprises three domains: a macro domain, a zinc-binding domain and a hypervariable region. The macro domain is essential at both early and late stages of the replication cycle through ADP-ribose (ADPr) binding and de-ADP-ribosylation of host proteins. However, both its specific role and the precise molecular mechanism of de-ADP-ribosylation across specific viral families remains to be elucidated. Here we investigate by X-ray crystallography the mechanism of ADPr reactivity in the active site of Getah virus macro domain, which displays a peculiar substitution of one of the conserved residues in the catalytic loop. ADPr adopts distinct poses including a covalent bond between the C′′1 of the ADPr and a conserved Togaviridae-specific cysteine. These different poses observed for ADPr may represent snapshots of the de-ADP-ribosylation mechanism, highlighting residues to be further characterised.

Structural basis for transcription inhibition by E. coli SspA.

Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

Characterization of Single Gene Deletion Mutants Affecting Alternative Oxidase Production in Neurospora crassa: Role of the yvh1 Gene.

The Neurospora crassa AOD1 protein is a mitochondrial alternative oxidase that passes electrons directly from ubiquinol to oxygen. The enzyme is encoded by the nuclear aod-1 gene and is produced when the standard electron transport chain is inhibited. We previously identified eleven strains in the N. crassa single gene deletion library that were severely deficient in their ability to produce AOD1 when grown in the presence of chloramphenicol, an inhibitor of mitochondrial translation that is known to induce the enzyme. Three mutants affected previously characterized genes. In this report we examined the remaining mutants and found that the deficiency of AOD1 was due to secondary mutations in all but two of the strains. One of the authentic mutants contained a deletion of the yvh1 gene and was found to have a deficiency of aod-1 transcripts. The YVH1 protein localized to the nucleus and a post mitochondrial pellet from the cytoplasm. A zinc binding domain in the protein was required for rescue of the AOD1 deficiency. In other organisms YVH1 is required for ribosome assembly and mutants have multiple phenotypes. Lack of YVH1 in N. crassa likely also affects ribosome assembly leading to phenotypes that include altered regulation of AOD1 production.

Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

The M2-1 protein of human respiratory syncytial virus (HRSV) is a transcription anti-terminator that regulates the processivity of the HRSV RNA-dependent RNA polymerase (RdRP). Here, we report a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residue Phe23 is indispensable for M2-1 to recognize the base of RNA. We also captured spontaneous binding of RNA (SH7) to M2-1 in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both experiments and simulations revealed that the interactions of RNA with two separate domains of M2-1, the zinc-binding domain (ZBD) and the core domain (CD), are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1.

Crystal structure of Arabidopsis thaliana cytidine deaminase

Cytidine deaminase (CDA) catalyzes the (deoxy)cytidine deamination to (deoxy)uridine, which involves in the catabolic and salvage pathways of pyrimidine nucleotides in plants. CDA serves as a prototype of the cytidine deaminase superfamily that contains a number of RNA editing enzymes. Arabidopsis thaliana has only one functional CDA, AtCDA1. We solved the crystal structures of AtCDA1, which is a dimeric zinc-containing enzyme and each protomer consists of an N-terminal zinc-binding catalytic domain and a C-terminal non-catalytic domain. Both domains adopt a typical α/β/α sandwich fold. In vitro biochemical assays showed that the ribose moiety of cytidine is required for ligand binding, and structural analyses revealed a conserved catalytic mechanism is adopted by AtCDA1.

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.

665 Associations of BIRC2/3/5 copy number gains with clinicopathological features of acral melanomas in Taiwan

Anti-apoptotic molecules can protect melanoma cells from apoptosis, thus enhancing their aggression. One of the factors responsible for the difficulties in engaging the apoptotic cascade efficiently in melanoma is the up-regulation of conserved inhibitor of apoptosis proteins (IAPs). All IAPs share up to three conserved zinc-binding baculoviral IAP repeats domains. The best described members are cIAP1, cIAP2, X-linked IAP, and surviving, which are encoded by Baculovirus inhibitor of apoptosis repeat containing genes (BIRC2, BIRC3, BIRC4, and BIRC5). These proteins are associated with chemoresistance and poor outcome in many cancer types. Acral melanoma is the most common subtype of melanoma in Asians. Our previous report found that frequencies of NRAS/KRAS mutations, altered cell cycle regulation, BIRC2/3/5 copy number gain, and amplification of receptor tyrosine kinase genes were significantly enriched in acral melanomas. In addition, BIRC2/3 copy number gain was only detected in the acral melanomas, and acral melanomas had significantly more BIRC2/3/5 copy number gain than the cutaneous melanomas. Most of BIRC2/3/5 copy number gains were detected in acral melanomas developed in stress-bearing areas. Moreover, the melanomas with BIRC2/3/5 gain were associated with ulceration and greater Breslow thickness. Our further analysis found that cell cycle alteration, BIRC2/3/5 copy number gain, and lymph node status were significant prognostic factors in 45 acral melanoma in a univariate analysis. In a multivariate analysis, altered cell cycle and lymph node status remained the most crucial independent prognostic factors in acral melanomas. We identified BIRC2/3/5 might be a critical determinant of survival in the acral melanoma patients.

New insights into two yeast BDHs from the PDH subfamily as aldehyde reductases in context of detoxification of lignocellulosic aldehyde inhibitors.

At least 24 aldehyde reductases from Saccharomyces cerevisiae have been characterized and most function in in situ detoxification of lignocellulosic aldehyde inhibitors, but none is classified into the polyol dehydrogenase (PDH) subfamily of the medium-chain dehydrogenase/reductase (MDR) superfamily. This study confirmed that two (2R,3R)-2,3-butanediol dehydrogenases (BDHs) from industrial (denoted Y)/laboratory (denoted B) strains of S. cerevisiae, Bdh1p(Y)/Bdh1p(B) and Bdh2p(Y)/Bdh2p(B), were members of the PDH subfamily with an NAD(P)H binding domain and a catalytic zinc binding domain, and exhibited reductive activities towards lignocellulosic aldehyde inhibitors, such as acetaldehyde, glycolaldehyde, and furfural. Especially, the highest enzyme activity towards acetaldehyde by Bdh2p(Y) was 117.95U/mg with cofactor nicotinamide adenine dinucleotide reduced (NADH). Based on the comparative kinetic property analysis, Bdh2p(Y)/Bdh2p(B) possessed higher specific activity, substrate affinity, and catalytic efficiency towards glycolaldehyde than Bdh1p(Y)/Bdh1p(B). This was speculated to be related to their 49% sequence differences and five nonsynonymous substitutions (Ser41Thr, Glu173Gln, Ile270Leu, Ile316Met, and Gly317Cys) occurred in their conserved NAD(P)H binding domains. Compared with BDHs from a laboratory strain, Bdh1p(Y) and Bdh2p(Y) from an industrial strain displayed five nonsynonymous mutations (Thr12, Asn61, Glu168, Val222, and Ala235) and three nonsynonymous mutations (Ala34, Ile96, and Ala369), respectively. From a first analysis with selected aldehydes, their reductase activities were different from BDHs of laboratory strain, and their catalytic efficiency was higher towards glycolaldehyde and lower towards acetaldehyde. Comparative investigation of kinetic properties of BDHs from S. cerevisiae as aldehyde reductases provides a guideline for their practical applications in in situ detoxification of aldehyde inhibitors during lignocellulose bioconversion.Key Points• Two yeast BDHs have enzyme activities for reduction of aldehydes.• Overexpression of BDHs slightly improves yeast tolerance to acetaldehyde and glycolaldehyde.• Bdh1p and Bdh2p differ in enzyme kinetic properties.• BDHs from strains with different genetic backgrounds differ in enzyme kinetic properties.

Structural Insight of the Full-Length Ros Protein: A Prototype of the Prokaryotic Zinc-Finger Family

Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56–142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-‘like’ gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1–55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.

Structural basis of bacterial σ28 -mediated transcription reveals roles of the RNA polymerase zinc-binding domain.

In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28 -dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E.coli σ28 -dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28 -RNAP recognizes promoter DNA through strong interactions with the -10 element, but weak contacts with the -35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β' zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further invitro and invivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP-promoter complex by compensating for the weak interaction between σ4 and -35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/-35 element interactions.

Abstract C001: TP53 mutations reprogram fatty acid metabolism and tumor microenvironment in African American patients with non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide, with a 5-year survival rate of ~18% (1-3). Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancer cases. Great strides have been made with the development of more advanced diagnosis, prognosis, and treatment strategies. Despite improvements, African American patients (AAs) continue to present with more advanced stages and later-stage metastatic tumors of lung cancer at diagnosis. The severe types of lung cancer can reduce the 5-year survival rate to only 4% (4). The disparities may partially be due to the socioeconomic disadvantages of AAs in receiving cancer services (5). Etiologically, cigarette smoking and exposure to secondhand smoking are leading preventable causes of lung cancer and premature death in the United States (US) (5). Notably, menthol cigarettes, which have been particularly marketed to communities with high AA population (7), can cause deeper inhalation, increased absorption of tobacco toxicants, a higher nicotine dependence and reduced cessation success (6-8). Some genetic alterations have been suggested to contribute to the disparities of breast cancer (9), but genetic factors have not been well studied in lung cancer disparities. Utilizing the genomic data from the Precision Oncology Initiative (POI) cohort at the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC), our initial analysis has shown that TP53 has a significantly higher mutation rate in AA patients, which was validated with the TCGA cohort (10). Furthermore, codons including Cysteine 176 (C176) and C242 mutated more frequently in AA than CA patients. Considering the four amino acids in the zinc-binding region (C176, Histidine 179 [H179], C238, and C242), AA patients have a higher mutation rate than CA patients within both our POI (15% v.s. 5%, P=5E-4) and the TCGA (14% vs. 6%, P<5E-4) cohorts. NSCLC patients with TP53 mutations experienced a poorer survival outcome, and, importantly, mutant AA had shorter overall survival than their CA counterparts. Utilizing RNA-Seq data of NSCLC patients from the TCGA cohort and Gene Set Enrichment Analysis (GSEA), we also identified increased transcriptional activation of fatty acid metabolism and reactive oxygen species (ROS) among TP53 mutation carriers; both pathways/processes are related to an immunosuppressive tumor microenvironment (TME). More importantly, significantly increased expression of these two gene sets was observed in AA cases with TP53 mutations than in their CA counterparts. The immunosuppressive TME in TP53-mutant (vs. wild-type) and mutant AA (vs. CA) patients were further validated by analyzing the immune cell components in each patient using computation algorithm CIBERSORT (11). These analyses implicate that mutated TP53 differentially contributes to cancer disparities in the AA population through metabolism and immune suppression, and mutations in the zinc-binding domain of p53 protein are specifically crucial. Citation Format: Liang Liu, Farideh Mehraein-Ghomi, Elizabeth Forbes, Umit Topaloglu, W. Jeffrey Petty, Stefan Grant, Jimmy Ruiz, Kristie L. Foley, Karen Winkfield, Boris Pasche, Wei Zhang. TP53 mutations reprogram fatty acid metabolism and tumor microenvironment in African American patients with non-small cell lung cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C001.