Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex.

The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.     

Synthesis, crystal structure, cytotoxic activity and DNA-binding properties of the copper (II) and zinc (II) complexes with 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene

A new ligand L, 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene, and its two metal complexes, [Cu(L)3](ClO4)2 (1) and [Zn(L)3](ClO4)2(H2O)2 (2), have been synthesized and characterized. The crystal structure of complex 1 was determined by single crystal X-ray diffraction, which crystallized in monoclinic, space group P2(1)/n with unit cell parameters, a = 12.710(4) angstroms, b = 12.135(3) angstroms, c = 33.450(9) angstroms, beta = 93.281(5) degrees and Z = 4. The Cu atom was six-coordinated to N(1), N(2), N(4), N(5), N(7) and N(8) from three L ligands and formed a slightly distorted octahedral geometry. Complexes 1 and 2, and ligand L were subjected to biological tests in vitro using three different cancer cell lines (HL-60, BGC-823 and MDA-MB-435). Complex 1 showed significant cytotoxic activity against three cancer cell lines. The interactions of complexes 1 and 2, and ligand L with calf thymus DNA were then investigated by thermal denaturation, viscosity measurements and spectrophotometric methods. The experimental results indicated that complexes 1 and 2 bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of complexes 1 and 2, and ligand L with DNA were 1.8 x 10(4), 5.4 x 10(3) and 2.76 x 10(3) M(-1), respectively.     

A theoretical investigation of the intercalative binding of 7-H pyrido[4.3C]carbazole chromophore into a d(CpG)2 minihelix

Theoretical computations are performed of the intercalative binding to a model d(CpG)2 minihelix of 7-H pyrido[4.3C]carbazole, the precursor of the antitumor bisintercalating drug ditercalinium. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structures) of Miller and co-workers. The ligand-nucleotide interactions and the nucleotide conformational energies are computed with the SIBFA procedures (sum of interactions between fragments ab initio computed), which use formulas of empirical origin that reproduce ab initio SCF (self-consistent field) computations. Among the candidate intercalation sites most favored energetically, one has a pattern of conformational angles related to the one determined crystallographically by Sobell et al. in a series of x-ray structural studies of small intercalator-dinucleotide monophosphate complexes. Optimal values of the unwinding angle, found in the range of -12 degrees to -14 degrees, are consistent with available experimental data on DNA.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cationic 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin fully intercalates at 5'-CG-3' steps of duplex DNA in solution

The interaction of 5,10,15, 20-tetrakis(N-methylpyridinium-4-yl)porphyrin (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5'-CG-3' step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site.     

DNA-binding and photoactivated enantiospecific cleavage of chiral polypyridyl ruthenium(II) complexes

A series of enantiomeric polypyridyl ruthenium(II) complexes Delta- and Lambda-[Ru(bpy)2CNOIP](PF6)2 (Delta-1 and Lambda-1; BPY=2,2'-bipyridine, CNOIP=2-(2-chloro-5-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline), Delta- and Lambda-[Ru(bpy)2HPIP](PF6)2 (Delta-2 and Lambda-2; HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline), Delta- and Lambda-[Ru(bpy)2DPPZ](PF6)2 (Delta-3 and Lambda-3; DPPZ=dipyrido[3,2:a-2',3':c]-phenazine), Delta- and Lambda-[Ru(bpy)2TAPTP](PF6)2 (Delta-4 and Lambda-4; TAPTP=4,5,9,18-tetraazaphenanthreno[9,10-b] triphenylene) have been synthesized. Binding of these chiral complexes to calf thymus DNA has been studied by spectroscopic methods, viscosity, and equilibrium dialysis. The experimental results indicated that all the enantiomers of these complexes bound to DNA through an intercalative mode, but the binding affinity of each chiral complex to DNA was different due to the different shape and planarity of the intercalative ligand. After binding to DNA, the luminescence property of complex 1 was distinctly different from complexes 2 to 4. Upon irradiation at 302 nm, complexes 2-4 were found to promote the cleavage of plasmid pBR 322 DNA from supercoiled form I to nicked form II, and obvious enantioselectively was observed on DNA cleavage for the enantiomers of complexes 2 and 4. The mechanisms for DNA cleavage by these enantiomeric complexes were also proposed.     

Conformational variations of the cis-syn cyclobutane-type photodimer in DNA and RNA.

The recent NMR study of a cis-syn photodimer B-DNA 10mer-duplex (Taylor et al., Biochemistry 29, 8858 (1990)) showed the cyclobutane (CB) ring with a puckered-twist in a right-handed sense (CB+). This is opposite to that of the crystal structure of cis-syn d-TpT(cyano-ethyl)(d-T[p]T-CE) which has a left-handed puckered-twist (CB-)(Hruska et al., Biopolymers 25, 1399 (1986)). 2D-NOESY experiments were performed on cis-syn d-T[p]T and cis-syn U[p]U at 25 and 35 degrees C, respectively, to investigate the puckering mode of the cyclobutane ring of isolated cis-syn photodimers of the DNA and RNA types. The DNA photodimers showed interconversion of the puckered-twist of the cyclobutane ring between CB- and CB+ and interconversion of the glycosidic angle between syn and anti in both nucleoside residues. Interestingly, in the RNA photodimer only the CB- puckering mode with syn conformation of the glycosidic angle of the U[p]- was observed. These different dynamical behaviors of the photodimer in DNA and RNA might portend differential conformational effects on their corresponding normal nucleic acid regions. In addition these results indicate differences in the cyclobutane ring conformation of the cis-syn d-T[p]T, not only in solution and crystalline states, but also when the dimer is isolated and in duplex forms.     

Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae.

A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.     

Synthesis, characterization and DNA-binding of novel chiral complexes delta- and lambda-[Ru(bpy)2L]2+ (L = o-mopip and p-mopip)

Novel chiral Ru(II) complexes [Ru(bpy)2L]2+ (bpy = 2,2-bipyridine; L: o-mopip = 2-(2-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline, p-mopip = 2-(4-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline) containing -OCH3 at different positions on the phenyl ring have been synthesized and characterized. The DNA-binding and DNA-photocleavage properties of the complexes were investigated. The theoretical calculations for these complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that: both these two isomer complexes can bind to DNA in an intercalative mode; the DNA-binding affinity of [Ru(bpy)2(p-mopip)] 2 is greater than that of [Ru(bpy)2(o-mopip)] 1; moreover, the DNA-binding affinities of enantiomers delta-1 and delta-2 are all greater than those of lambda-1 and lambda-2, respectively. In addition, a very interesting finding is experimentally obtained, i.e. under a low [DNA]/[Ru] ratio, the emission intensities of delta-1 and lambda-1 are all weaker than those of delta-2 and lambda-2, however, upon a high [DNA]/[Ru] ratio, the emission intensities of both delta-1 and lambda-1 are stronger than those of delta-2 and lambda-2. Such a difference of the emission spectra can be interpreted by the electric effect of substituent on the intercalative ligand. The difference in DNA-binding affinities of these two isomeric complexes can also be reasonably explained by the DFT calculations.     

Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase

Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21, 6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the substitution positions of Br group in intercalative ligand on the DNA-binding behaviors of Ru(II) polypyridyl complexes

Two new polypyridyl ligands containing substituent Br at different positions in the phenyl ring, PBIP [PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline], OBIP [OBIP=2-(2-bromophenyl)imidazo[4,5-f]1,10-phenanthroline] and their Ru(II) complexes, [Ru(phen)2PBIP]2+ 1, [Ru(phen)2OBIP]2+ 2 (phen=1,10-phenanthroline), have been synthesized and characterized. The binding strength of the two complexes to calf thymus DNA (CT DNA) was investigated with spectrophotometric methods, viscosity measurements, as well as equilibrium dialysis and circular dichroism spectroscopy. The theoretical calculations for these two complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that the Br group substituting H at different positions of the phenyl ring in the intercalated ligand has significant effects on the spectral properties and the DNA-binding behaviors of Ru(II) complexes. Both the complexes can bind to CT DNA in intercalative mode and interact with CT DNA enantioselectively. Moreover, complex 1 can bind to CT DNA more strongly than complex 2, and complex 2 can become a much better candidate as an enantioselective binder to CT DNA than complex 1. The theoretical calculations show that both intercalative ligands, PBIP and OBIP, in these two complexes are essentially planar, and the obtained electronic structures of the complexes can be used to explain reasonably some of their experimental regularities or trends. Such experimental and theoretical information will be useful in design of novel probes of nucleic acid structures.     

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.     

A comparative study of the interaction of two structurally analogue ruthenium(II) complexes with DNA

The binding of the ruthenium(II) complexes of [Ru(bpy)2(CAIP)]Cl2 and [Ru(bpy)2(HCIP)]Cl2 (where bpy=2,2'-bipyridine, CAIP=4-carboxyl-imidado[4,5-f][1,10]-phenanthroline, HCIP=3-hydroxyl-4-carboxyl-imidado[4,5-f][1,10]-phenanthroline) to calf thymus DNA (ct-DNA) has been investigated with UV-visible and emission spectroscopy, steady-state emission quenching, and viscosity measurements. The experimental results indicate that the two complexes bind to ct-DNA through an intercalative mode and [Ru(bpy)2(HCIP)]2+ intercalates into DNA more deeply than [Ru(bpy)2(CAIP)]2+ does.     

DNA binding and cleavage properties of certain tetrammine ruthenium(II) complexes of modified 1,10-phenanthrolines--effect of hydrogen-bonding on DNA-binding affinity

A series of ruthenium(II) mixed ligand complexes of the type [Ru(NH(3))(4)(L)](2+), where L=imidazo[4,5-f][1,10]phenanthroline (ip), 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip), 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (hpip), 4,7-diphenyl-1,10-phenanthroline (dip), naphtha[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione (qdppz), 5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-H:2',3'-f]phenazine (hqdppz), have been isolated and characterized. The interaction of these complexes with calf thymus DNA (CT DNA) has been explored by using absorption, emission, and circular dichroic spectral methods, thermal denaturation studies and viscometry. All these studies suggest the involvement of the modified phenanthroline 'face' rather than the ammonia 'face' of the complexes in DNA binding. An intercalative mode of DNA binding, which involves the insertion of the modified phenanthroline ligands in between the base pairs, is suggested. The results from absorption spectral titration and circular dichroism (CD), thermal denaturation and viscosity experiments indicate that the qdppz and hqdppz complexes (K(b) approximately 10(6) and Delta T(m)=11-13 degrees C) bind more avidly than the ip, pip and hpip complexes (K(b) approximately 10(5), Delta T(m)=6-8 degrees C). Intramolecular hydrogen bonding in the hpip and hqdppz complexes increases the surface area of the intercalating diimines and enhances the DNA binding affinity substantially. The ammonia co-ligands of the complexes are possibly involved in hydrogen bonding with the intrastrand nucleobases to favour intercalation of the extended aromatic ligands. Circular dichroism spectral studies reveal that all the complexes effect certain structural changes on DNA duplex; [Ru(NH(3))(4)(ip)](2+) induces a B to A transition while [Ru(NH(3))(4)(qdppz)](2+) a B to Psi conformational change on CT DNA. Cleavage efficiency of the complexes were determined using pBR322 supercoiled plasmid DNA. All the complexes, except hqdppz complex, promote the cleavage of supercoiled plasmid (form I) to relaxed circular form (form II).     

DNA-binding and photocleavage studies of cobalt(III) mixed-polypyridyl complexes containing 2-(2-chloro-5-nitrophenyl)imidazo [4,5-f][1,10]phenanthroline

The ligand 2-(2-chloro-5-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline(CNOIP) and its complexes [Co(bpy)(2)(CNOIP)](3+) (1) and [Co(phen)(2)(CNOIP)](3+) (2) (bpy=2,2'-bipyridine; phen=1,10-phenanthroline) have been synthesized and characterized. Binding of the two complexes with calf thymus DNA has been investigated by spectroscopic methods, cyclic voltammetry, viscosity, and electrophoresis measurements. The experimental results indicate that both complexes bind to DNA through an intercalative mode. In comparison with their parent complexes containing PIP ligand (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline), the introduction of NO(2) and Cl groups to the PIP ligand decreased the binding affinity of complexes 1 and 2 to CT DNA. Both complexes have also been found to promote the photocleavage of plasmid pBR 322 DNA, the hydroxyl radical (OH*) is suggested to be the reactive species responsible for the cleavage.     

Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates

The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.     

Synthesis, DNA-binding and photocleavage studies of cobalt(III) mixed-polypyridyl complexes: [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+)

Two novel cobalt(III) mixed-polypyridyl complexes [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+) (phen=1,10-phenanthroline, dpta=dipyrido-[3,2-a;2',3'-c]- thien-[3,4-c]azine, amtp=3-amino-1,2,4-triazino[5,6-f]1,10-phenanthroline) have been synthesized and characterized. The interaction of these complexes with calf thymus DNA was investigated by spectroscopic, cyclic voltammetry, and viscosity measurements. Results suggest that the two complexes bind to DNA via an intercalative mode. Moreover, these Co(III) complexes have been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365nm. The mechanism studies reveal that hydroxyl radical (OH()) is likely to be the reactive species responsible for the cleavage of plasmid DNA by [Co(phen)(2)(dpta)](3+) and superoxide anion radical (O(2)(-)) acts as the key role in the cleavage reaction of plasmid DNA by [Co(phen)(2)(amtp)](3+).     

Visualization of drug--nucleic acid interactions at atomic resolution. VI. Structure of two drug--dinucleoside monophosphate crystalline complexes, ellipticine--5-iodocytidylyy (3'-5') guanosine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium--5-iodocytidylyl (3'-5') guanosine

Ellipticine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine. These crystals are isomorphous: ellipticine-iodoCpG² crystals are monoclinic, space group P2₁ with $\text{a = 13.88}\text{A}\text{?}\text{, b = 19.11}\text{A}\text{?}\text{, c = 21.42}\text{A}\text{?}$, β = 105.4; TMP-iodoCpG crystals are monoclinic, space group P2₁, with $\text{a = 13.99}\text{A}\text{?}\text{, b = 19.12}\text{A}\text{?}\text{, c = 21.31}\text{A}\text{?}$, β = 104.9 °. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares.The asymmetric unit in the ellipticine-iodoCpG structure contains two ellipticine molecules, two iodoCpG molecules, 20 water molecules and 2 methanol molecules, a total of 144 atoms, whereas, in the tetramethyl-N-methyl phenanthrolinium-iodoCpG complex, the asymmetric unit contains two TMP molecules, two iodoCpG molecules, 17 water molecules and 2 methanol molecules, a total of 141 atoms. In both structures, the two iodoCpG molecules are hydrogenbonded together by guanine-cytosine Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoCpG structure are separated by about 6.7 Å; this separation results from intercalative binding by one ellipticine (or TMP) molecule and stacking by the other ellipticine (or TMP) molecule above or below the base-pairs. Base-pairs within the paired nucleotide units are related by a twist of 10 to 12 °. The magnitude of this angular twist is related to conformational changes in the sugar-phosphate chains that accompany drug intercalation. These changes partly reflect the mixed sugar puckering pattern observed: C3′ endo (3′–5′) C2′ endo (i.e. both iodocytidine residues have C3′ endo conformations, whereas both guanosine residues have C2′ endo conformations), and additional small but systematic changes in torsional angles that involve the phosphodiester linkages and the C4′C5′ bond.The stereochemistry observed in these model drug-nucleic acid intercalative complexes is almost identical to that observed in the ethidium-iodoUpA and -iodoCpG complexes determined previously (Tsai et al., 1975a,b,1977; Jain et al., 1977). This stereochemistry is also very similar to that observed in the 9-aminoacridine-iodoCpG and acridine orange-iodoCpG complexes described in the preceding papers (Sakore et al., 1979 Reddy et al., 1979). We have already proposed this stereochemistry to provide a unified understanding of a large number of intercalative drug-DNA (and RNA) interactions (Sobell et al., 1977a,b), and discuss this aspect of our work further in this paper.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.