PubMed Health⌕ Search

Biomedical subjects

S J Lippard

Publications and source records attributed to S J Lippard.

At least 37 records · Page 2Linked to original sources
PubMed2001

Decomposition of alkyl-substituted urea molecules at a hydroxide-bridged dinickel center.

The interactions between N-methylurea, N,N'-dimethylurea, N,N-dimethylurea, tetramethylurea, and thiourea and the hydroxide-bridged dinickel complex [Ni(2)(mu-OH)(mu-H2O)(bdptz)(H2O2](OTs)(3) were investigated. Structural characterization of [Ni(2)(mu-OH)(mu-H2O)(bdptz)(Me-urea)(CH3CN)](ClO4)(3) (1) and [Ni(2)(mu-OH)(mu-H2O)(bdptz)(thiourea)(CH3CN)](ClO4)(3) (2) provided insight into the interactions of the substrates with the dinickel center. In 1, the methylurea molecule coordinates to the dinickel complex through its carbonyl oxygen atom. Complex 2 has a similar geometry, with the thiourea molecule bound to a nickel ion through its sulfur atom. When the urea substrates are heated in the presence of the hydroxide-bridged dinickel complex, N-methylurea and N,N-dimethylurea react to form methylammonium cyanate and dimethylammonium cyanate, respectively. After long reaction times, thiourea reacts similarly, producing ammonium thiocyanate. The other substrates are unreactive. These results indicate that the dinickel complex promotes the elimination of alkylamines from urea substrates to form cyanate but cannot effect the direct hydrolysis of such substrates.

Alkanes↗
PubMed2001

Crystal structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) demonstrating geometrical variability at the dinuclear iron active site.

The oxidation of methane to methanol is performed at carboxylate-bridged dinuclear iron centers in the soluble methane monooxygenase hydroxylase (MMOH). Previous structural studies of MMOH, and the related R2 subunit of ribonucleotide reductase, have demonstrated the occurrence of carboxylate shifts involving glutamate residues that ligate the catalytic iron atoms. These shifts are thought to have important mechanistic implications. Recent kinetic and theoretical studies have also emphasized the importance of hydrogen bonding and pH effects at the active site. We report here crystal structures of MMOH from Methylococcus capsulatus (Bath) in the diiron(II), diiron(III), and mixed-valent Fe(II)Fe(III) oxidation states, and at pH values of 6.2, 7.0, and 8.5. These structures were investigated in an effort to delineate the range of possible motions at the MMOH active site and to identify hydrogen-bonding interactions that may be important in understanding catalysis by the enzyme. Our results present the first view of the diiron center in the mixed-valent state, and they indicate an increased lability for ferrous ions in the enzyme. Alternate conformations of Asn214 near the active site according to redox state and a distortion in one of the alpha-helices adjacent to the metal center in the diiron(II) state have also been identified. These changes alter the surface of the protein in the vicinity of the catalytic core and may have implications for small-molecule accessibility to the active site and for protein component interactions in the methane monooxygenase system. Collectively, these results help to explain previous spectroscopic observations and provide new insight into catalysis by the enzyme.

Binding Sites↗
PubMed2001

Cisplatin: from DNA damage to cancer chemotherapy.

Cisplatin [cis-DDP, cis-diamminedichloroplatinum(II)] is a potent anticancer drug that has been used successfully to treat tumors of the head, neck, lungs, and genitourinary tract. The biological activity of cisplatin was discovered serendipitously more than 30 years ago, and since that time research efforts have focused on elucidating its mechanism of action. The present review provides a historical perspective of our attempts to understand this complex phenomenon and the results of recent work that guides our current activities in this field. Continued efforts to understand the mechanism of genotoxicity of cisplatin are expected to lead to the discovery of new drugs and combinations for the improvement of cancer chemotherapy.

Amino Acid Sequence↗
PubMed2000

Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA.

Cisplatin exerts its anticancer activity by forming covalent adducts with DNA. High-mobility group (HMG)-domain proteins recognize the major 1,2-intrastrand cisplatin-DNA cross-links and can mediate cisplatin cytotoxicity. The crystal structure of HMG1 domain A bound to cisplatin-modified DNA, further analyzed here, reveals intercalation of a key Phe37 residue. Other published structures of HMG domains bound to DNA, including NHP6A and HMG-D, similarly indicate amino acid side chains intercalating into linear DNA to form a bend. To delineate the importance of such side chain intercalations and further to explore the binding modes of different HMG domains toward prebent DNA structures, site-directed mutagenesis was used to generate HMG1 domain A and domain B mutants. The affinities of these mutant proteins for cisplatin-modified DNA were determined in gel electrophoresis mobility shift assays. The results indicate that intercalating residues at positions 16 or 37 can both contribute to the binding affinity. The data further reveal that the length of the loop between helices I and II is not critical for binding affinity. Footprinting analyses indicate that the position of the intercalating residue dictates the binding mode of the domain toward platinated DNA. Both congruent and offset positioning of the HMG domain with respect to the locus of the cisplatin-induced bend in the DNA were encountered. Packing interactions in the crystal structure suggest how full-length HMG1 might bind to DNA by contacting more than one duplex simultaneously. Taken together, these results demonstrate that cisplatin modification of DNA provides an energetically favorable, prebent target for HMG domains, which bind to these targets through one or more side chain and favorable hydrophobic surface interactions.

Amino Acid Sequence↗
PubMed2000

Synthesis and characterization of several dicopper(I) complexes and a spin-delocalized dicopper(I,II) mixed-valence complex using a 1,8-naphthyridine-based dinucleating ligand.

The synthesis of dicopper(I) complexes [Cu2(BBAN)(MeCN)2](OTf)2 (1), [Cu2(BBAN)(py)2](OTf)2 (2), [Cu2(BBAN)(1-Me-BzIm)2](OTf)2 (3), [Cu2(BBAN)(1-Me-Im)2](OTf)2 (4), and [Cu2(BBAN)(mu-O2CCPh3)](OTf) (5), where BBAN = 2,7-bis((dibenzylamino)methyl)-1,8-naphthyridine, py = pyridine, 1-Me-Im = 1-methylimidazole, and 1-Me-BzIm = 1-methylbenzimidazole, are described. Short copper-copper distances ranging from 2.6151(6) to 2.7325(5) A were observed in the solid-state structures of these complexes depending on the terminal ligands used. The cyclic voltammogram of compound 5 dissolved in THF exhibited a reversible redox wave at E1/2 = -25 mV vs Cp2Fe+/Cp2Fe. When complex 5 was treated with 1 equiv of silver(I) triflate, a mixed-valence dicopper(I,II) complex [Cu2(BBAN)(mu-O2CCPh3)(OTf)](OTf) (6) was prepared. A short copper-copper distance of 2.4493(14) A observed from the solid-state structure indicates the presence of a copper-copper interaction. Variable-temperature EPR studies showed that complex 6 has a fully delocalized electronic structure in frozen 2-methyltetrahydrofuran solution down to liquid helium temperature. The presence of anionic ligands seems to be an important factor to stabilize the mixed-valence dicopper(I,II) state. Compounds 1-4 with neutral nitrogen-donor terminal ligands cannot be oxidized to the mixed-valence analogues either chemically or electrochemically.

Journal Article↗
PubMed2000

Oxygen kinetic isotope effects in soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH(3)CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH(2)CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 +/- 0.0007 and 1.0167 +/- 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.

Kinetics↗
PubMed2000

Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent.

Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.

Base Sequence↗
PubMed2000

HMG-domain protein recognition of cisplatin 1,2-intrastrand d(GpG) cross-links in purine-rich sequence contexts.

HMG-domain proteins bind strongly to bent DNA structures, including cruciform and cisplatin-modified duplexes. Such protein-platinated DNA complexes, formed where the DNA is modified by the active cis but not the inactive trans isomer of diamminedichloroplatinum(II), are implicated in the cytotoxic mechanism of the drug. A series of oligonucleotide duplexes with deoxyguanosine nucleosides flanking a cis-[Pt(NH(3))(2)¿d(GpG)-N7(1),-N7(2)¿] cross-link have been synthesized. These probes were used to determine the flanking sequence dependence of the affinity of the individual HMG domains of HMG1 toward cisplatin-modified DNA. Nine related sequences, where N(1) and N(2) are not dG and GG is the 1,2-intrastrand cisplatin adduct in N(1)GGN(2), were previously investigated [Dunham, S. U., and Lippard, S. J. (1997) Biochemistry 36, 11428-11436]. Three of the seven remaining possible sequences for which N(1) and/or N(2) was dG were prepared here by using normal deoxyguanosine, but the rest, where N(1) is dG and N(2) is dA, dC, T, or dG, could not be isolated in pure form. These sequences were accessed by using the synthetic bases 7-deazaadenine and 7-deazaguanine, which lack the nucleophilic N7 atom in the purine ring. Deaza nucleotides accurately mimic the properties of the natural bases, allowing the interaction of the HMG-domain proteins with cisplatin-modified DNA to be examined. These experiments reveal that the flexibility of A.T versus G.C flanking base pairs, rather than base-specific contacts, determines HMG1domA protein selectivity. This conclusion was supported by use of mutant HMG1domA and HMG1domB proteins, which exhibit identical flanking sequence selectivity. The methods and results obtained here not only improve our understanding of how proteins might mediate cisplatin genotoxicity but also should apply more generally in the investigation of how other proteins interact with damaged DNA.

Adenine↗
PubMed2000

Aminotroponiminates as ligands for potential metal-based nitric oxide sensors.

A family of new fluorescently labeled ligands, HRDATI, was prepared to develop transition-metal-based NO sensing strategies. The ligands are composed of aminotroponiminates (ATIs) with a dansyl fluorophore on one of the imine nitrogen atoms and an alkyl substituent, either i-Pr (8), t-Bu (9), or Bz (10), on the other. Bis(chelate) Co2+ ([Co(i-PrDATI)2] (12), [Co(t-BuDATI)2] (14), [Co(BzDATI)2] (15)) and Zn2+ ([Zn(i-PrDATI)2] (13)) complexes were prepared and characterized by X-ray crystallography. The bis(ATI) complex [Co(i-Pr2ATI)2] (11) was also prepared and its X-ray crystal structure determined. Cyclic voltammetry reveals reversible redox waves at -2.57 and -0.045 V (vs Cp2Fe/Cp2Fe+) in THF for the Co2+/Co+ and Co3+/Co2+ couples, respectively, of 11. Only a Co2+/Co+ wave at -2.09 V is observed for 12. When excited at 350 nm, the HRDATI ligands and the diamagnetic Zn2+ complex 13 fluoresce around 500 nm, whereas the paramagnetic Co2+ complexes quench the fluorescence. These air-stable cobalt compounds react with nitric oxide to dissociate a DATI ligand and form neutral dinitrosyl complexes, [Co(NO)2(RDATI)]. The release of the fluorophore-containing ligand is accompanied by an increase in fluorescence intensity, thus providing a strategy for fluorescent NO sensing. Linking two DATI moieties via a tetramethylene chain affords the ligand H2DATI-4 (18). The Co2+ complex [Co(DATI-4)] (19) reacts more readily with NO than the bis(DATI) compounds and also displays an increase in fluorescence intensity upon NO binding.

Journal Article↗
PubMed2000

Synthesis of PDK, a novel porphyrin-linked dicarboxylate ligand.

A novel trinucleating ligand system H4(PDK), for porphyrin-based diamine bis(Kemp's triacid)imide, was constructed by using two molecules of Kemp's triacid and one molecule of porphyrin as building blocks. The dimeso-substituted octaalkylporphyrin unit, carrying bromomethyl groups at the ortho positions of two trans-positioned meso-phenyl groups, was synthesized from (3,3'-diethyl-4,4'-dimethyl-2,2'-dipyrrolyl)methane and (alpha-bromo-o-tolualdehyde. The structures of both of the two atropisomers (alpha,alpha- and alpha,beta-) of bromoporphyrin, H2(alpha,alpha-BP) and H2(alpha,beta-BP), were determined by X-ray crystallography, with the alpha,beta-isomer as the free base form and the alpha,alpha-isomer as the zinc complex. Both the alpha,alpha- and alpha,beta-isomers of the bromomethyl porphyrins were converted to their aminomethyl derivatives, H2(alpha,alpha-AP) and H2(alpha,beta-AP), through the corresponding imidoporphyrin intermediates, H2(alpha,alpha-IP) and H2(alpha,beta-IP), by the Gabriel synthesis. The alpha,alpha- and alpha,beta-aminoporphyrins were coupled with Kemp's triacid anhydride-chloride to form H4(alpha,alpha-PDK) and H4(alpha,beta-PDK), respectively. Unlike the alpha,beta-isomer, the structure of which was determined by X-ray crystallography for the zinc complex, H4(alpha,alpha-PDK) has a remarkable and complicated solvent-dependent 1H NMR spectrum that suggests the existence of hydrogen bonding interactions between two convergent, tethered Kemp's triacid units, as predicted in a modeling study. The convergent feature is essential for the target ligand H4(alpha,alpha-PDK) to form trinuclear metal complexes with a bis(carboxylato) dimetallic unit sitting above a metalloporphyrin ring.

Dicarboxylic Acids↗
PubMed2000

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA.

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.

Amino Acid Sequence↗
PubMed2000

Reactivity of mu-hydroxodizinc(II) centers in enzymatic catalysis through model studies.

The stable dinuclear complex [Zn2(BPAM)(mu-OH)(mu-O2PPh2)](ClO4)2, where BPAN = 2,7-bis[2-(2-pyridylethyl)-aminomethyl]-1,8-naphthyridine, was chosen as a model to investigate the reactivity of (mu-hydroxo)dizinc(II) centers in metallohydrolases. Two reactions, the hydrolysis of phosphodiesters and the hydrolysis of beta-lactams, were studied. These two processes are catalyzed in vivo by zinc(II)-containing enzymes: P1 nucleases and beta-lactamases, respectively. The former catalyzes the hydrolysis of single-stranded DNA and RNA. beta-Lactamases, expressed in many types of pathogenic bacteria, are responsible for the hydrolytic degradation of beta-lactam antibiotic drugs. In the first step of phosphodiester hydrolysis promoted by the dinuclear model complex, the substrate replaces the bridging diphenylphosphinate. The bridging hydroxide serves as a general base to deprotonate water, which acts as a nucleophile in the ensuing hydrolysis. The dinuclear model complex is only 1.8 times more reactive in hydrolyzing phosphodiesters than a mononuclear analogue, Zn(bpta)(OTf)2, where bpta = N,N-bis(2-pyridylmethyl)-tert-butylamine. Hydrolysis of nitrocefin, a beta-lactam antibiotic analogue, catalyzed by [Zn2(BPAN)(mu-OH)(mu-O2PPh2)](ClO4)2 involves monodentate coordination of the substrate via its carboxylate group, followed by nucleophilic attack of the zinc(II)-bound terminal hydroxide at the beta-lactam carbonyl carbon atom. Collapse of the tetrahedral intermediate results in product formation. Mononuclear complexes Zn(cyclen)-(NO3)2 and Zn(bpta)(NO3)2, where cyclen = 1,4,7,10-tetraazacyclododecane, are as reactive in the beta-lactam hydrolysis as the dinuclear complex. Kinetic and mechanistic studies of the phosphodiester and beta-lactam hydrolyses indicate that the bridging hydroxide in [Zn2(BPAN)(mu-OH)(mu-O2PPh2)](ClO4)2 is not very reactive, despite its low pKa value. This low reactivity presumably arises from the two factors. First, the briding hydroxide and coordinated substrate in [Zn2(BPAN)(mu-OH)(substrate)]2+ are not aligned properly to favor nucleophilic attack. Second, the nucleophilicity of the bridging hydroxide is diminished because it is simultaneously bound to the two zinc(II) ions.

Catalysis↗
PubMed2000

Enhanced binding of the TATA-binding protein to TATA boxes containing flanking cisplatin 1,2-cross-links.

The TATA-binding protein (TBP) is essential for transcription initiation in eukaryotes. TBP recognizes and binds to the minor groove of a consensus sequence, TATAAA, known as the TATA box or TATA element. DNA binding is affected largely by hydrophobic contacts and through the intercalation of two sets of adjacent phenylalanine residues. The resultant duplex is sharply kinked, bending toward the major groove. Inspired by prior structural information showing intercalation of a phenylalanine side chain of a high mobility group (HMG) domain into the site of a cisplatin 1, 2-intrastrand d(GpG) cross-link, a series of DNA probes was prepared with one or two such adducts flanking the TATA box positions at or near the sites of TBP intercalation. The platinum adducts bend the DNA toward the major groove and result in as much as a 175-fold increase in binding affinity of the TBP over the unmodified target sequence. Kinetic studies indicate that the enhanced binding to the modified TATA box is predominantly a consequence of a >30-fold slower dissociation rate of the protein-platinated DNA complex. This work demonstrates that it is feasible to design rationally and to synthesize an enhanced affinity-binding site for a sequence-specific DNA-binding protein by appropriate chemical modification of flanking sequences. It also has implications for the mechanism of action of cisplatin.

Binding, Competitive↗
PubMed2000

Steroid hormones induce HMG1 overexpression and sensitize breast cancer cells to cisplatin and carboplatin.

Cisplatin is an anticancer drug that has enjoyed remarkable success against testicular tumors, but dose limiting side-effects have limited its application against a broader range of cancers. Previous studies have shown that high-mobility group (HMG) domain proteins such as HMG1 sensitize cells to cisplatin by shielding its major DNA adducts from nucleotide excision repair. Estrogen treatment increases HMG1 mRNA levels in breast cancer MCF-7 cells. Herein, we describe that treatment of human cancer cells having steroid hormone receptors with the appropriate hormone, estrogen and/or progesterone, significantly increases the potency of cisplatin and its analogue carboplatin by causing the overexpression of HMG1. These findings suggest that the proper combination of these drugs, which are already approved by the Food and Drug Administration, could have potential benefit in treating tumors such as ovarian or breast that carry the hormone receptors.

Adenocarcinoma↗
PubMed2000

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA.

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.

Base Pairing↗