The xenograft antigen in complex with GS-1-B4 lectin: crystallization and preliminary X-ray analysis.

The implantation of animal organs is one approach to overcoming the shortage of human donor organs for medical transplantation. Although readily available, non-primate tissues are subject to hyperacute rejection wherein human anti-Galalpha(1-3)Gal antibodies react with haptens present on the transplanted cells' surfaces. The understanding of this interaction on a molecular level will further the development of a strategy for the prevention of hyperacute rejection in xenotransplantation. The Galalpha(1-3)Gal hapten ('xenograft antigen') has been cocrystallized with the Gal-specific B(4) isolectin of Griffonia simplicifolia lectin-1. Crystals were analyzed by cryocrystallography and were found to diffract to moderately high resolution on a rotating-anode X-ray source. They belong to the P2(1)2(1)2 space group, with unit-cell parameters a = 111.0, b = 51.3, c = 76.9 A, and contain two molecules per asymmetric unit.

Effects of cationic charge on three-dimensional structures of intercalative complexes: structure of a bis-intercalated DNA complex solved by MAD phasing.

We characterize intercalative complexes as either "high charge" and "low charge". In low charge complexes, stacking interactions appear to dominate stability and structure. The dominance of stacking is evident in structures of daunomycin, nogalamycin, ethidium, and triostin A/echinomycin. By contrast in a DNA complex with the tetracationic metalloporphyrin CuTMPyP4 [copper (II) meso-tetra(N-methyl-4-pyridyl)porphyrin], electrostatic interactions appear to draw the porphyrin into the duplex interior, extending the DNA along its axis, and unstacking the DNA. Similarly, DNA complexes of tetracationic ditercalinium and tetracationic flexi-di show significant unstacking. Here we report x-ray structures of complexes of the tetracationic bis-intercalator D232 bound to DNA fragments d(CGTACG) and d(BrCGTABrCG). D232 is analogous to ditercalinium but with three methylene groups inserted between the piperidinium groups. The extension of the D232 linker allows it to sandwich four base pairs rather than two. In comparison to CuTMPyP4, flexi-di and ditercalinium, stacking interactions of D232 are significantly improved. We conclude that it is not sufficient to characterize intercalators simply by net charge. One anticipates strong electrostatic forces when cationic charge is focused to a small volume or region near DNA and so must consider the extent to which cationic charge is focused or distributed. In sum, ditercalinium, with a relatively short linker, focuses cationic charge more narrowly than does D232. So even though the net charges are equivalent, electrostatic charges are expected to be of greater structural significance in the ditercalinium complex than in the D232 complex.

Structures of yeast vesicle trafficking proteins.

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.

Structure of DNA-porphyrin complex.

We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.

Clathrate hydrates are poor models of biomolecule hydration.

Clathrate hydrates form the basis of a general model of biomolecule hydration. In clathrate hydrate crystal structures, the size of hydrogen-bonded water rings is highly constrained to five members. The clathrate hydrate model predicts that the size of water rings near biomolecule surfaces is similarly constrained to five members. This report describes a test of this model of biomolecule hydration. We have demonstrated that five-membered water rings are not a general feature of protein or nucleic acid hydration. The clathrate hydrate model appears to be inappropriate for soluble biomolecules.

Asymmetry and dynamics in bis-intercalated DNA.

The bis-intercalator ditercalinium (NSC 366241), composed of two 7 H-pyridocarbazoles linked by a bis(ethylpiperidinium), binds to DNA with a binding constant greater than 10(7) M-1. One distinctive aspect of the 3-D X-ray structure of a DNA-ditercalinium complex is its asymmetry. We propose here that the activity of ditercalinium may be related to structural polymorphism and dynamic conversion between conformers. It was previously reported that activity is closely related to linker composition. Activity increases with increasing conformational restraints of the linker. We suggest these conformational restraints can lead to asymmetry in DNA complexes and that this asymmetry results directly in structural polymorphism. Using the Cambridge Structural Database (CSD) as a source of information about chemical fragments that are analogous to the linker of ditercalinium, we have explored the conformational space available to ditercalinium. The results indicate that the linker is highly constrained and that the DNA complex is intrinsically asymmetric. We propose a reasonable mechanism of ring reversal that is consistent with the conformations of analogous fragments within the CSD.

X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine.

We have determined the x-ray structure of a DNA fragment containing 7,8-dihydro-8-oxoguanine (G(O)). The structure of the duplex form of d(CCAGOCGCTGG) has been determined to 1.6-A resolution. The results demonstrate that GO forms Watson-Crick base pairs with the opposite C and that G(O) is in the anti conformation. Structural perturbations induced by C.G(O)anti base pairs are subtle. The structure allows us to identify probable elements by which the DNA repair protein MutM recognizes its substrates. Hydrogen bond donors/acceptors within the major groove are the most likely element. In that groove, the pattern of hydrogen-bond donors/acceptors of C.G(O)anti is unique. Additional structural analysis indicates that conversion of G to G(O) would not significantly influence the glycosidic torsion preference of the nucleoside. There is no steric interaction of the 8-oxygen of G(O) with the phospho-deoxyribose backbone.

DNA distortion in bis-intercalated complexes.

The bis-intercalators Flexi-Di and ditercalinium are synthetic dimers that bis-intercalate into DNA and cause cell death in prokaryotes from futile and abortive repair of DNA. Each is composed of two 7H-pyridocarbazole units and a linker. Flexi-Di has a flexible spermine-like linker while ditercalinium has a rigid bis(ethylpiperidinium) linker. This report, describing the 2.5-A X-ray structure of Flexi-Di complexed with [d(BrCGCG)]2, appears to be the first report of a three-dimensional structure of a DNA complex with a bis-intercalator with a flexible linker. DNA complex formation with a ditercalinium analog having a flexible linker was not anticipated to yield unstacked and bent DNA as was observed in the previously reported ditercalinium.[d(CGCG)]2 complex. Surprisingly, the DNA in the Flexi-Di complex is bent to a degree exceeding that of the ditercalinium complex. A comparison of the DNA complexes of Flexi-Di and ditercalinium has allowed us to propose a mechanism by which these bis-intercalators distort DNA. We propose that this class of bis-intercalators pulls the internal base pairs into the major groove and pushes the external base pairs into the minor groove. The result is a bend toward the minor groove. It appears that hydrogen bonds between the linker and the internal guanines effectively pull the central base pairs of the complex out into the major groove. At the external regions of the complex, stacking interactions between the chromophores and terminal base pairs effectively push the terminal base pairs into the minor groove. The result of this push/pull combination is to bend the DNA.

Water ring structure at DNA interfaces: hydration and dynamics of DNA-anthracycline complexes.

In crystallographic structures of biological macromolecules, one can observe many hydration rings that originate at one water molecule, pass via hydrogen bonds through several others, and return to the original water molecule. Five-membered water rings have been thought to occur with greater frequency than other ring sizes. We describe a quantitative assessment of relationships between water ring size and frequency of occurrence in the vicinity of nucleic acid interfaces. This report focuses on low-temperature X-ray crystallographic structures of two anthracyclines, adriamycin (ADRI) and daunomycin (DAUN), bound to d(CGATCG) and on several DNA structures published previously by others. We have obtained excellent low-temperature (-160 degrees C, LT) X-ray intensity data for d(CGATCG)-adriamycin and d(CGATCG)-daunomycin with a multiwire area detector. The LTX-ray data sets contain 20% (daunomycin, LT-DAUN) and 35% (adriamycin, LT-ADRI) more reflections than were used to derive the original room-temperature (15 degrees C) structures [Frederick, C.A., Williams, L.D., Ughetto, G., van der Marel, G. A., van Boom, J.H., Rich, A., & Wang, A.H.-J. (1990) Biochemistry 29, 2538-2549]. The results show that five-membered water rings are not preferred over other ring sizes. This assessment is consistent with our observation of broad dispersion W-W-W angles (sigma = 20 degrees). In addition, we report that the thermal mobility, distinct from the static disorder, of the amino sugar of daunomycin and adriamycin is significantly greater than that of the rest of the complex. This mobility implies that if the central AT base pair is switched to a CG base pair, there should be a low energy cost in avoiding the guanine amino group. The energy difference (for the sugar-binding preference) between d(CGTACG) and d(CGCGCG) could be considerably less than 20 kcal/mol, a value proposed previously from computation.