Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Defining subdomains of the K domain important for protein–protein interactions of plant MADS proteins

The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.     

Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.     

Arm-domain interactions can provide high binding cooperativity

Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology. A well-studied example of this type of protein-protein interaction occurs between the yeast homeodomain proteins, MAT alpha2 and MAT a1, which form a high-affinity heterodimer on DNA. The carboxyl-terminal arm extending from MAT alpha2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1. Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change. This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase. The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B

Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B. Folding was reversible in secondary and tertiary structures as well as in activity. Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two-state mechanism without thermodynamically stable intermediates. Consistent with the two-state model, one dimensional (1D) NMR spectra and gel-filtration chromatography analysis did not show any evidence for a folded monomeric intermediate. Interestingly enough, Cys 81 located at the dimeric interface was modified by DTNB before unfolding. This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation. The refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step. Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step. During the refolding process, negative ellipticity at 225 nm increased very fast within 80 msec to account for >80% of the total amplitude. This result suggests that the protein folds into a monomer containing most of the alpha-helical structures before dimerization. Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.     

Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M

We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g <--> e' (i, i' +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T(m) = 28 degrees C) or nanomolar (T(m) = 40 degrees C) range, respectively, but heterodimerize with a T(m) >90 degrees C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 microM and 6 degrees C but that heterodimerize with a T(m) = 74 degrees C or K(d(37)) = 1.1 x 10(-11) M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T(m) = 73 degrees C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.     

Effects of ovariectomy and estrogen treatment on learning and hippocampal neurotransmitters in mice

This study examined the effects of long-term estrogen treatment (sc 17 beta-estradiol minipellets) on learning in C57BL/6J female and male mice using a position discrimination task in the T-maze and a win-stay task (1/8 arms baited) in the radial arm maze (RAM). In addition, hippocampal monoamines and ChAT activity were measured at the end of the study and correlated to task performance. Female sham-operated (gonadally intact) and ovariectomized (OVX) mice were treated with estrogen either for 7 or 40 days before the behavioral tests and intact male mice for 7 days before the behavioral tests. In sham-operated mice the 40-day estrogen treatment improved RAM performance and in OVX mice both the 7- and 40-day estrogen treatments improved the performance in both maze tasks. The estrogen treatment also improved RAM performance in males. The hippocampal ChAT, NA, 5-HIAA, and DOPAC levels were decreased in OVX mice. Furthermore, the effects of estrogen treatment on the levels of hippocampal 5-HT and its metabolite 5-HIAA were different in sham-operated than in OVX mice. We could find no correlation between cognitive measures and neurochemical variables. This study gives new information about the effects of estrogen on learning and hippocampal neurotransmitters in mice.