Syntheses of DNA duplexes containing a C-C interstrand cross-link

Short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link were prepared on controlled pore glass supports using a DNA synthesizer. The C-C cross-link was introduced via a convertible nucleoside on the support or by using a protected C-C cross-link phosphoramidite. An orthogonal protection scheme allowed selective chain growth in either a 3'-->5' or 5'-->3' direction. The cross-linked duplexes were purified by HPLC and characterized by MALDI-TOF mass spectrometry and/or by enzymatic digestion.     

Linker histones do not interact with DNA containing a single interstrand cross-link created by cisplatin

During our earlier investigations we have observed a prominent preference of the linker histone H1 for binding to a cis-platinated DNA (a synthetic fragment with global type of platination in respect to targets for cisplatin) comparing with unmodified and trans-Pt-modified DNA. In the present work we report our recent experimental results on the binding of the linker histones H1 and H5 to a cisplatin-modified synthetic DNA fragment containing a single nucleotide target d(GC/CG) for inter-platination. Surprisingly, no preferential binding of linker histones to cis-inter-platinated DNA was observed by means of the electromobility-shift assay. The same negative results were obtained with a part of the linker histone molecule suggested to be responsible for DNA-binding--its globular domain. Contrary, the data with another nuclear protein with similar DNA-binding properties as linker histones--HMGB1--showed a strong afinity for interaction with DNA containing interstrand cross-links.     

Solution structure of a nitrous acid induced DNA interstrand cross-link

Nitrous acid is a mutagenic agent. It can induce interstrand cross-links in duplex DNA, preferentially at d(CpG) steps: two guanines on opposite strands are linked via a single shared exocyclic imino group. Recent synthetic advances have led to the production of large quantities of such structurally homogenous cross-linked duplex DNA. Here we present the high resolution solution structure of the cross-linked dodecamer [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined), determined by 2D NMR spectroscopy, distance geometry, restrained molecular dynamics and iterative NOE refinement. The cross-linked guanines form a nearly planar covalently linked 'G:G base pair' with only minor propeller twisting, while the cytidine bases of their normal base pairing partners have been flipped out of the helix and adopt well defined extrahelical positions in the minor groove. On the 5'-side of the cross-link, the minor groove is widened to accommodate these extrahelical bases, and the major groove becomes quite narrow at the cross-link. The cross-linked 'G:G base pair' is well stacked on the spatially adjacent C:G base pairs, particularly on the 3'-side guanines. In addition to providing the first structure of a nitrous acid cross-link in DNA, these studies could be of major importance to the understanding of the mechanisms of nitrous acid cross-linking and mutagenicity, as well as the mechanisms responsible for its repair in intracellular environments. It is also the shortest DNA cross-link structure to be described.     

NMR solution structure of a DNA dodecamer containing a transplatin interstrand GN7-CN3 cross-link.

The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links.     

Crystal structure at 1.63 A resolution of the native form of porcine beta-trypsin: revealing an acetate ion binding site and functional water network

The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine beta-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 A resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-HellipsisO hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cbeta-Ogamma-His-57 Nepsilon angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.     

Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein

Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.     

Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.     

Thermal and thermodynamic properties of duplex DNA containing site-specific interstrand cross-link of antitumor cisplatin or its clinically ineffective trans isomer

The effect of the single, site-specific interstrand cross-link formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair DNA duplex is reported. The cross-linked or unplatinated 20-base pair duplexes were investigated with the aid of differential scanning calorimetry, temperature-dependent UV absorption, and circular dichroism. The cross-link of both platinum isomers increases the thermal stability of the modified duplexes by changing the molecularity of denaturation. The structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. This entropic cross-link-induced stabilization of the duplex is partially but not completely compensated by the enthalpic destabilization of the duplex. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from the formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability, with this destabilization being enthalpic in origin. By contrast, the interstrand cross-link of transplatin is enthalpically almost neutral with the cross-link-induced destabilization entirely entropic in origin. These differences are consistent with distinct conformational distortions induced by the interstrand cross-links of the two isomers. Importantly, for the duplex cross-linked by cisplatin relative to that cross-linked by transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. It has been proposed that the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different for each and might also be associated with the distinctly different antitumor effects of these platinum compounds.     

Crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly at 1.4 A resolution: implications for collagen hydration

The use of polypeptide models has proved to be a valuable tool to obtain accurate information on the collagen triple helix. Here we report the high resolution crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly. The structure has been refined to an R(factor) of 0.137 and an R(free) of 0.163 using synchrotron diffraction data extending up to 1.4 A resolution. The polypeptide triple-helical structure binds a large number of water molecules, in contrast with a previous structure determination at lower resolution. The highly hydrated nature of this polypeptide confirms a number of previous studies conducted both in solution and in the crystal state. In addition, neighboring polypeptide triple helices are directly bound in the crystal through Hyp-Hyp hydrogen-bonding interactions. This finding supports the idea that Hyp residues may be important for the assembly of the triple helices in the collagen fibrils and may stabilize the fibrils by mediating direct contacts between neighboring molecules.     

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics,hydration, sequence,and ionic effects

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG]] or cis-[Pt(NH(3))(2) [d(ApG]] cross-links, and their corresponding unmodified duplexes. The platinated duplexes are less stable and unfold with lower T(M)s (and Delta G degrees s) in enthalpy-driven reactions, which indicates a loss of favorable base-pair stacking interactions. The folding thermodynamics and hydration effects for the first set of decamers containing the d(GpG) cross-link was investigated by a combination of titration calorimetry, density, and ultrasound techniques. The hydration parameters showed an uptake of structural water by the platinated duplex and a release of electrostricted water by the control duplex. Relative to the unmodified duplex, the folding of the platinated duplex at 20 degrees C yielded a positive Delta Delta G degrees term [and positive Delta Delta H-Delta(T Delta S) compensation] and a negative differential volume change. The opposite signs of the Delta Delta G degrees and Delta Delta V terms confirmed its uptake of structural water. Further, solvent-accessible surface areas calculations for a similar pair of dodecamer duplexes indicated that the modified duplex has a 503 oeA(2) higher polar and nonpolar surface area that is exposed to the solvent. Therefore, the incorporation of a platinum adduct in duplex DNA disrupts favorable base-pair stacking interactions, yielding a greater exposure of aromatic bases to the solvent, which in turn immobilizes structural water. The overall results correlate nicely with the results reported in the available structural data of nuclear magnetic resonance solution studies.     

Crystal structure of lactoperoxidase at 2.4 A resolution

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R_cryst and R_free factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H_2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution.

The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.     

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Processing of a psoralen DNA interstrand cross-link by XPF-ERCC1 complex in vitro

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5' to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3'-side of an ICL. The ICL-specific 3'-incision, along with the 5'-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.     

Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution

BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.     

Crystal structure of human alpha-lactalbumin at 1.7 A resolution

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.     

Crystal structure of muconolactone isomerase at 3.3 A resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.