Zinc-binding Region Research Articles

Partial lipodystrophy, severe dyslipidaemia and insulin resistant diabetes as early signs of Werner syndrome

Werner syndrome is a premature ageing disorder caused by biallelic variants in the WRN gene. WRN encodes a dual DNA helicase/exonuclease enzyme. Molecular diagnosis is commonly only made at a late disease stage in the third or fourth decade, when cardinal features have become apparent. We describe a 28 year-old woman who presented with early onset diabetes associated with partial lipodystrophy, severe dyslipidaemia and rapidly progressive liver fibrosis related to non-alcoholic steatohepatitis in the absence of progeroid features. Werner syndrome was diagnosed by trio exome analysis, which revealed compound heterozygous WRN mutations: the known variant c.1290_1293del (p.Asn430Lysfs*7) and the novel intronic splice site variant c.2732+5G>A. cDNA analysis demonstrated this to lead to in-frame skipping of exon 22, predicted to delete most of the zinc binding region of the helicase domain. We suggest that including the WRN gene in genetic analysis of early onset diabetes, lipodystrophy or dyslipidaemia would allow for the opportunity to diagnose some cases of Werner syndrome long before clinical criteria are met, thereby allowing early implementation of important primary prevention interventions.

Mutational Impact on "in-Between-Ring" (IBR) Domain of PARKIN on Protein Stability and Function.

Mutations in parkin, which is encoded by the PARK2 gene, are associated with a rare form of Parkinson's disease called autosomal recessive juvenile parkinsonism (ARJP). Parkin is a member of RBR family of E3 ubiquitin ligase. Parkin contains a RING1-In-Between-Ring (IBR)-RING2 motif. The IBR domain is located at the C-terminal end of the parkin protein. Two zinc-binding sites are present in the IBR domain which shows zinc ion-dependent folding and maintains the orientation and geometry of RING domains. So, mutation in a zinc-binding region can be responsible for improper folding of parkin protein, which eventually affects the protein structure and function. Abnormalities in parkin protein increase the aggregation of mis-folded proteins in the brain cell. As a consequence, cellular toxicity occurs. The IBR domain also interacts with UbcH7 and UbcH8 proteins belonging to E2 protein family and facilitates synphilin-1, Sept5, and SIM2 protein ubiquitination. It is reported that missense mutation in parkin protein are responsible for autosomal recessive juvenile Parkinson disease. In this work, we first collected the missense mutations in the IBR domain from literature and sequence databases. Then, using various computational tools, we predicted their pathogenicity and involvements in causing possible changes in various protein properties. Evolutionary conservation of amino acids, solvent accessible surface areas, the physico-chemical properties, and changes of protein structure were analyzed. We, for the first time, analyzed the effects of these mutations in parkin to decipher the plausible molecular mechanism of Parkinson's disease.

Non-native fold of the putative VPS39 zinc finger domain.

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

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.

Novel mutations in the RING-finger domain of BRCA1 gene in clinically diagnosed breast cancer patients.

In the present study, we investigated the frequency of BRCA1 gene mutations in 30 breast cancer (BC) patients of independent family history and 30 healthy control subjects. The immunohistochemistry (IHC) of BCpatients showed duct cell carcinoma and distinct expression of the human epidermal growth factor receptor2 (HER2). The genomic DNA was extracted from the BC patients and control subjects, the BRCA1 gene was PCR amplifiedand sequenced. The sequence analysis revealed that BRCA1 gene mutations were detected in 5/30 (16.6%) unrelated patients. One novel deleterious c.53delT mutation was detected in 3/30 (10%) unrelated patients leading to p.Met18Serfs*5 frame shift mutation in exon 2. Two patients 2/30 (6%) had novel c.297_301delinsCTCAA mutation in exon 5 leading to p.Leu99_Tyr101delinsPheSerAsn. Interestingly, the qRT-PCR analysis showed high expression of BRCA1 gene in all these patients having mutations compared with control subjects. Further, in silico analysis revealed loss of zinc-binding region ofthe RING-finger domain in BRCA1 structure due to these mutations, variable number of helices, helix-helix interactions, β-turns, and γ-turns were identified in the secondary structure, resulted in the formation of non-functional protein which is unable to activate BRCA1-associated genome surveillance complex (BASC) leading to uncontrolled cell proliferation. Moreover, the molecular dynamics (MD) simulations of mutated BRCA1 protein demonstrated extensive variations in the domain and non-domain regions compared with the wild-type structure as indicated by RMSD values. All these results conclusively explain that the c.53delT mutation may be the probable founder of deleterious mutation in this ethnic group.

Low Frequency of MKRN3 and DLK1 Variants in Chinese Children with Central Precocious Puberty.

Background Central precocious puberty (CPP) is defined by gonadotropin-dependent development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. MKRN3 and DLK1 are two genes, disease-causing variants of which have recently been discovered to cause idiopathic CPP. Methods We screened 173 Chinese patients (9 males and 164 females; 9 familial and 164 sporadic) with ICPP and 43 patients (9 males and 34 females; 3 familial and 40 sporadic) with early puberty for variants in MKRN3. We also screened 19 patients with ICPP and early puberty for variants of DLK1 (17 males and 2 females; 5 familial and 14 sporadic). Results We identified four novel missense variants of MKRN3, c.1138G > A (p.Glu380Lys), c.1420T > A (p.Leu474Met), c.673C > G (p.Leu225Val), and c.1071C > G (p.Ile357Met) in two sporadic cases and three familial cases. According to ACMG standards, two MKRN3 variant (p.Glu380Lys and p.Ile357Met) are likely pathogenic, and two others are of uncertain significance. We also performed bioinformatic analysis to evaluate the impact of variants on MKRN3 protein structures, which showed that Ile357Met locates at the zinc-binding region (C3HC4 RING finger motif), while Glu380Lys is spatially extremely close to the C3HC4 RING finger, MKRN-specific Cys-His domain, and the third C3H1 zinc-finger motif region. Per Glu380Lys, Glu with negative charges has been changed into Lys with positive charges, which may affect the hydrogen bond formation between amino acids and the stability of the local structure, thus affecting the binding of zinc iron to MKRN3 protein. Besides, we did not identify any variants of DLK1 gene in our patients. Conclusions In this study, we report four novel MKRN3 variants in patients with ICPP. Moreover, we did not find any variants of DLK1 gene. Variants of MKRN3 are relatively uncommon in Chinese ICPP patients.

Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.IMPORTANCECucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH.

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein–protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity.

Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

Quantitative Fluorescence Microscopy Reveals Fibroblast Growth Factor Receptor 5 Signaling Complex Formation

Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinase receptors that modulate many cellular processes including adhesion and survival. FGFR5 is a non-canonical family member that lacks a kinase domain and is presumed to be a negative regulator of signaling. However, we previously showed that overexpression of FGFR5 results in activation of downstream signaling targets, thereby challenging our understanding of this receptor. In particular, it was unclear whether FGFR5 and FGFR1 interact on the plasma membrane to form a signaling complex. We previously used quantitative fluorescence microscopy, including number and brightness analysis (N&B) and homo-forster resonance energy transfer (homoFRET), to study the dynamics and the stoichiometry of the FGFR1 signaling complex. We therefore created a library of fluorescent protein tagged FGFR1 and FGFR5 constructs to conduct similar studies of receptor membrane aggregation. FGFR5 has a unique cytoplasmic C-terminus that includes a histidine-rich zinc binding region as well as a putative SH2-binding motif. HomoFRET and anisotropy imaging revealed that FGFR5 forms homo-dimers while a C-terminal deficient isoform (FGFR5ΔC) does not, suggesting that the C-terminus drives the interaction. We validated these findings using N&B analysis of Venus-tagged FGFR5ΔC observing no change in relative brightness in the presence or absence of non-Venus tagged FGFR5ΔC (i.e. “dark receptor”). Finally, we measured changes in anisotropy of Cerulean-tagged FGFR5 when co-expressed with an excess dark FGFR1 (i.e. mCherry-tagged FGFR1). We observed an increase in FGFR5 anisotropy indicative of dimer to monomer transition, suggesting that FGFR5 also forms heterodimers with FGFR1. Currently, we are investigating the effect of FGFs and scaffolding proteins on modulating the FGFR5-FGFR1 heterocomplex. These studies will allow us to better understand the regulatory mechanisms that govern FGFR-signaling and the downstream cellular processes.

MALDI-TOF MS and CD spectral analysis for identification and structure prediction of a purified, novel, organic solvent stable, fibrinolytic metalloprotease from Bacillus cereus B80.

The ability to predict protein function from structure is becoming increasingly important; hence, elucidation and determination of protein structure become the major steps in proteomics. The present study was undertaken for identification of metalloprotease produced by Bacillus cereus B80 and recognition of characteristics that can be industrially exploited. The enzyme was purified in three steps combining precipitation and chromatographic methods resulting in 33.5% recovery with 13.1-fold purification of enzyme which was detected as a single band with a molecular mass of 26 kDa approximately in SDS-PAGE and zymogram. The MALDI-TOF MS showed that the enzyme exhibited 70–93% similarity with zinc metalloproteases from various strains Bacillus sp. specifically from Bacillus cereus group. The sequence alignment revealed the presence of zinc-binding region VVVHEMCHMV in the most conserved C terminus region. Secondary structure of the enzyme was obtained by CD spectra and I-TASSER. The enzyme kinetics revealed a Michaelis constant (K m) of 0.140 μmol/ml and V max of 2.11 μmol/min. The application studies showed that the enzyme was able to hydrolyze various proteins with highest affinity towards casein followed by BSA and gelatin. The enzyme exhibited strong fibrinolytic, collagenolytic, and gelatinolytic properties and stability in various organic solvents.

Towards understanding the molecular recognition process in prokaryotic zinc-finger domain.

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.

Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc-binding sites in the C-terminal domain.

Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N-terminal catalytic domain and a 30 kDa C-terminal zinc-binding region (ZD domain) which has three adjacent tetra-cysteine zinc-binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc-binding motifs in ZD domain and both the first and second zinc-binding motifs are required for iron binding in TopA. The site-directed mutagenesis studies further reveal that while the mutation of the third zinc-binding motif has very little effect on TopA's activity, mutation of the first two zinc-binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc-binding motifs in TopA are crucial for its function. The DNA-binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single-stranded (ss) DNA-binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.

Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD

YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.

Zinc supplementation protects human endostatin Fc fusion against proteolytic degradation during cell culture

Endostatin is a potent anti-angiogenesis compound with efficacy in treating solid tumors and other diseases. However, its clinical application has been hampered by the susceptibility to proteolytic degradation during cell culture production. Here we describe a simple and effective strategy for stabilizing a CHO cell-derived human endostatin Fc fusion. Mass spectrometry analysis of the prominent clipped species revealed that the cleavage sites are located at the N-terminal zinc binding region, which is known to be critical for the structural stability of the molecule. Accordingly, we tested the effect of zinc supplementation on stabilizing the molecule and found that micromolar concentrations of zinc chloride significantly reduced the level of clipping. The protective effect appeared to be mediated via direct interaction between zinc and endostatin, as zinc protects purified endostatin spiked into conditioned medium. Interestingly, copper which is known to have high affinity to endostatin, also prevents degradation. The method provides a robust process for manufacturing Fc-endostatin.

Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex

The anaphase promoting complex (APC) is the crucial ubiquitin ligase targeting the regulatory machinery of the cell cycle. Emi1, a major modulator of APC activity, is thought to act competitively as a pseudosubstrate. We show that the modulation of APC activity is more subtle: Emi1 inhibits ubiquitylation at both substrate binding and separately at the step of ubiquitin transfer to APC-bound substrates. The zinc-binding region of Emi1 allows multiple monoubiquitylation of substrates, but preferentially suppresses the ubiquitin chain elongation by UBCH10. Furthermore, the C-terminal tail of Emi1 antagonizes chain elongation by Ube2S, via competitively preventing its binding to APC cullin subunit through electrostatic interaction. Combinatorially, Emi1 effectively stabilizes APC substrates by suppressing ubiquitin chain extension. Deubiquitylating enzymes can then convert inhibited substrates to their basal state. Chain elongation may be a particularly sensitive step for controlling degradation and this study provides the first kinetic evidence for how it is inhibited.