Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.

New parameters for the refinement of nucleic acid-containing structures.

Structures at atomic resolution (up to 1.0 A) which contain bases, sugars or the phosphodiester linkage, were selected from the Nucleic Acid Database or the Cambridge Structural Database to build a nucleic acid dictionary from X-ray refined structures. The dictionary consists of the average values for bond distances, bond angles and dihedral angles. The variance of the sample is used to provide information about the expected r.m.s. deviations of the refined parameters. A dictionary was constructed for refinement trials in X-PLOR. The dictionary includes RNA and DNA in C2'-endo and C3'-endo sugar pucker conformations, as well as values for the backbone dihedrals. Tests were performed on the dictionary using three structures: a B-DNA, a Z-DNA and a protein-DNA complex. During the course of refinement, all three structures showed significant improvements as measured by r.m.s. deviations and R factors when compared to the previous DNA dictionary.

Structure of a new crystal form of a DNA dodecamer containing T.(O6Me)G base pairs.

The structure of the synthetic dodecamer (d[CGTGAATTC(O6Me)GCG])2 has been determined to a resolution of 2.25 A and refined to a final R factor of 16.7%. The volume of the unit cell is significantly smaller by 16% than the original Drew and Dickerson parent dodecamer [Drew, H. R., Wing, R. M., Takano, T., Broka, C., Tanaka, S., Itakura, K., & Dickerson, R. E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7318-7322]. The double helix is in a different position in the unit cell, rotated by -85.9 degrees, and translated by 9.9 A around the helical axis with respect to the parent structure. The intermolecular arrangement of helices, characterized by double hydrogen bonded guanine-guanine minor groove interactions, remains conserved. The molecular geometry exhibits several significant changes that are related to the changed position of the helix and the presence of two mismatched base pairs with O6-methylguanine. Both mispairs are found in a symmetrical T(anti).(O6Me)G(anti) conformation, and the methyl groups are in proximal orientation. The hydration pattern of the structure is different and can be related to changes in the minor groove geometry. An incorrect model that was isomorphous to the parent dodecamer could be refined to a low R factor. Characteristics of the refinement and of the geometry that are indicative of incorrect structures have been analyzed.

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.

Crystal structure of a mispaired dodecamer, d(CGAGAATTC(O6Me)GCG)2, containing a carcinogenic O6-methylguanine.

The crystal structure of the synthetic deoxydodecamer d(CGAGAATTC(O6Me)GCG)2 has been determined and refined to an R-factor of 16.9% with data up to 2.9-A resolution. This sequence contains two mismatched base pairs between O6-methylguanine and adenine with the arrangement A(syn).(O6-Me)G(anti) which differs from the geometry observed in solution by NMR. The intermolecular arrangement is equivalent to the other isomorphous deoxydodecamers. However, the weakening of some significant crystal packing contacts was observed and related to the effect of stacking between the mispaired adenine and the adjacent guanine in the sequence. The structure is highly hydrated, with a total of 49 solvent molecules located. The methyl group and the mismatched base-pair geometry locally disrupt the B-DNA-type solvent network with two solvent molecules found close to the N1 and N6 of the mispaired adenine.