A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (“tail”) domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (“heads”) that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

ActA is a dimer

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97–126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP’s ability to occlude its DNA binding domain through dimerization. Using a “pull-down” based assay, we find that TBP dimers dissociate slowly (t½ = 6–10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.

A cyclodextrin dimer with a photocleavable linker as a possible carrier for the photosensitizer in photodynamic tumor therapy

A cyclodextrin dimer has been synthesized with two β-cyclodextrins linked by a flexible chain containing a carbon–carbon double bond. This dimer binds and solubilizes a phthalocyanine-based photosensitizer that generates singlet oxygen on irradiation. When the complex is irradiated, the singlet oxygen cleaves the carbon–carbon link, and the cyclodextrins are released, liberating the photosensitizer into the light path. Ideas about how this phenomenon could be used to make photodynamic tumor therapy into a more selective process are described.

Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

d-dimer testing as an adjunct to ultrasonography in patients with clinically suspected deep vein thrombosis: prospective cohort study

Objective To investigate the efficacy of using a rapid plasma d-dimer test as an adjunct to compression ultrasound for diagnosing clinically suspected deep vein thrombosis.

Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2∷kan mutant

Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution. Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli. PriA mutants are Rec− and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression. PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4′,6-diamidino-2-phenylindole-stained log phase cells. Two populations of cells were seen. Eighty four percent appeared like wild type, and 16% of the cells were filamented and had poorly partitioned chromosomes (Par−). To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant. Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning. Mutating either recA or recB virtually eliminated the Par− phenotype. Filamentation in the recB mutant decreased to 3%, but increased to 28% in the recA mutant. The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal. Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK. Mutation of dif showed no change in phenotype whereas ftsK1∷cat was lethal with priA2∷kan. A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication.

The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Human white blood cells contain cyclobutyl pyrimidine dimer photolyase.

Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (supercoiled pET-2, nonsupercoiled bacteriophage lambda, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells.