The impact of the C-terminal domain on the interaction of human DNA topoisomerase II α and β with DNA

Background

Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction.

Methodology/Principal Findings

We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes.

Conclusions/Significance

The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme''s K_D for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn.     

Sea anemone collagen: Further evidence for the existence of only oneα-chain type

Summary Collagen from an inferior invertebrate, namely from the sea anemoneActinia equina L., appears to consist of three identical subunits (-chains). New evidence for this situation is reported on the basis of preparative-scale experiments.No fractionation of sea anemone collagen was obtained by CM-cellulose chromatography which is the method of choice for the isolation of vertebrate collagen subunits. On the other hand, the-component (mol. wt., 95.000) could be isolated by gel chromatography. It was homogenous when investigated by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulphate) and by ion exchange chromatography on CM-cellulose. The amino acid composition of the-component was identical with that of unfractionated collagen.Divergent evolution of the collagen molecule appears likely since vertebrate collagen molecules are made up of different-chain types.     

The C-terminal αI domain linker as a critical structural element in the conformational activation of αI integrins

The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2) in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/β intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.     

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2 months of life with an approximate 20% penetrance (~ 35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9 nm versus exon 2-deleted: 88.2 nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2 nm, SPARC-null/exon 2-deleted: 40.8 nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.     

The gene coding for the α-chain of human propionyl-CoA carboxylase maps to chromosome band 13q32

Summary The human gene encoding the -polypeptide of propionyl-CoA carboxylase (PCC) has hitherto been localized to the distal half of the long arm of chromosome 13, segment 13q22q34. We studied the enzyme activities of mitochondrial carboxylases in cell cultures obtained from patients with different deletions of chromosome 13. By setting the PCC activity in normal diploid cell cultures (control group) at 100%, cell cultures with trisomy 13 showed 150% activity. In contrast, one of four patients with partial monosomy 13 had an enzyme activity of only 50%. Thus, by comparative deletion mapping, combined with studies of the gene-dosage effect, we have been able to assign the PCCA gene locus to chromosome band 13q32.     

Crystal structure of the C-terminal domain of human DNA primase large subunit: Implications for the mechanism of the primase—polymerase α switch

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes'' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.Key words: DNA primase, prim1, prim2, replication, 4Fe-4S cluster, crystal structure, DNA polymerase α     

Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine

Background

NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far.

Methodology/Principal Findings

We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K_D = 148±9.54 nM).

Conclusion/Significance

Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Three unrelated individuals with perinatally lethal osteogenesis imperfecta resulting from identical Gly502Ser substitutions in the α2-chain of type I collagen

In general, osteogenesis imperfecta is caused by heterozygous mutations in either of the genes encoding the 1 or 2 chains of type I collagen (COL1A1 and COL1A2, respectively). Usually, these mutations are unique to the affected individual or individuals within a family. In this study, single-strand conformation polymorphism mapping analysis has been coupled with sequence analysis to identify a single base mutation in the 2(I) gene of type I collagen; this mutation is identical in three unrelated individuals with perinatal lethal osteogenesis imperfecta. The heterozygous G to A transition at a CpG dinucleotide results in a Gly502Ser substitution in the 2 chain of type I collagen.     

The cleavage site preference of the porcine pepsin on the N-terminal α1 chain of bovine type I collagen: a focal analysis with mass spectrometry

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH₂ was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH₂ (13?16) were readily identified, pQLSK(MOCAc)GY (1?6) and DEKSTGISK(Dnp)P-NH₂ (7?16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.     

The role of the acidic domain of α-synuclein in amyloid fibril formation: a molecular dynamics study

The detailed mechanism of the pathology of α-synuclein in the Parkinson’s disease has not been clearly elucidated. Recent studies suggested a possible chaperone-like role of the acidic C-terminal region of α-synuclein in the formation of amyloid fibrils. It was also previously demonstrated that the α-synuclein amyloid fibril formation is accelerated by mutations of proline residues to alanine in the acidic region. We performed replica exchange molecular dynamics simulations of the acidic and nonamyloid component (NAC) domains of the wild type and proline-to-alanine mutants of α-synuclein under various conditions. Our results showed that structural changes induced by a change in pH or an introduction of mutations lead to a reduction in mutual contacts between the NAC and acidic regions. Our data suggest that the highly charged acidic region of α-synuclein may act as an intramolecular chaperone by protecting the hydrophobic domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins may provide novel insights into the mechanism of amyloid formation.     

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε–subunit's C-terminal domain are mutually exclusive

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F₁ complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for F_OF₁ from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF₁, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F₁ with the membrane-embedded F_O complex modulate inhibition by the εCTD. Here, we study membranes containing EcF_OF₁ with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).     

In vitro α1-3 or α1-4 fucosylation of type I and II oligosaccharides with secreted forms of recombinant human fucosyltransferases III and VI

Transgalactosylation of chitobiose and chitotriose employing -galactosidase from bovine testes yielded mixtures with 1-3 linked galactose (type I) and 1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human 1, 3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewisa structure, whereas approximately 10% fucosylation of the type II isomers to the Lewisx oligosaccharides was observed in long-term incubations.Employing large amounts of human 1, 3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewisx product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely 1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a 1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an 1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewisa oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.