Molecular replacement using DNA helical symmetry.

The efficiency of molecular-replacement methods in the structure analysis of B-DNA is markedly increased if a knowledge of the structural properties and helical symmetry of B-DNA is incorporated into molecular-replacement procedures. The separation of the most significant or most robust parameters, such as the location of helices in the unit cell, from the less well defined parameters, such as rotation around the helix axis, further improves the reliability of molecular replacement and avoids frameshift errors in the positioning of the model. This approach has been applied successfully to solve novel structures of four B-DNA decamers in various space groups.

NarL dimerization? Suggestive evidence from a new crystal form.

The structure of the Escherichia coli response regulator NarL has been solved in a new, monoclinic space group, and compared with the earlier orthorhombic crystal structure. Because the monoclinic crystal has two independent NarL molecules per asymmetric unit, we now have three completely independent snapshots of the NarL molecule: two from the monoclinic form and one from the orthorhombic. Comparison of these three structures shows the following: (a) The pairing of N and C domains of the NarL molecule proposed from the earlier analysis is in fact correct, although the polypeptide chain connecting domains was, and remains, disordered and not completely visible. The new structure exhibits identical relative orientation of N and C domains, and supplies some of the missing residues, leaving a gap of only seven amino acids. (b) Examination of corresponding features in the three independent NarL molecules shows that deformations in structure produced by crystal packing are negligible. (c) The "telephone receiver" model of NarL activation is confirmed. The N domain of NarL blocks the binding of DNA to the C domain that would be expected from the helix-turn-helix structure of the C domain. Hence, binding can only occur after significant displacement of N and C domains. (d) NarL monomers have a strong tendency toward dimerization involving contacts between helixes alpha 1 in the two monomers, and this may have mechanistic significance in DNA binding. Analogous involvement of helix alpha 1 in intermolecular contacts is also found in UhpA and in the CheY/CheZ complex.

Structure of the Escherichia coli response regulator NarL.

The crystal structure analysis of the NarL protein provides a first look at interactions between receiver and effector domains of a full-length bacterial response regulator. The N-terminal receiver domain, with 131 amino acids, is folded into a 5-strand beta sheet flanked by 5 alpha helices, as seen in CheY and in the N-terminal domain of NTRC. The C-terminal DNA-binding domain, with 62 amino acids, is a compact bundle of 4 alpha helices, of which the middle 2 form a helix-turn-helix motif closely related to that of Drosophila paired protein and other H-T-H DNA-binding proteins. The 2 domains are connected by an alpha helix of 10 amino acids and a 13-residue flexible tether that is not visible and presumably disordered in the X-ray structure. In this unphosphorylated form of NarL, the C-terminal domain is turned against the receiver domain in a manner that would preclude DNA binding. Activation of NarL via phosphorylation of Asp59 must involve transfer of information to the interdomain interface and either rotation or displacement of the DNA-binding C-terminal domain. Docking of a B-DNA duplex against the isolated C-terminal domain in the manner observed in paired protein and other H-T-H proteins suggests a stereochemical basis for DNA sequence preference: T-R-C-C-Y (high affinity) or T-R-C-T-N (low affinity), which is close to the experimentally observed consensus sequence: T-A-C-Y-N. The NarL structure is a model for other members of the FixJ or LuxR family of bacterial transcriptional activators, and possibly to the more distant OmpR and NtrC families as well.

How proteins recognize the TATA box.

The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses from Saccharomyces cerevisiae and Arabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein molecule with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T.A base-pairs are required rather than C.G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box in order to bind properly; and (4) why the second half of the TATA box can be more variable than the first.

Crystal structure of a covalent DNA-drug adduct: anthramycin bound to C-C-A-A-C-G-T-T-G-G and a molecular explanation of specificity.

A 2.3-A X-ray crystal structure analysis has been carried out on the antitumor drug anthramycin, covalently bound to a ten base pair DNA double helix of sequence C-C-A-A-C-G-T-T-G-G. One drug molecule sits within the minor groove at each end of the helix, covalently bound through its C11 position to the N2 amine of the penultimate guanine of the chain. The stereochemical conformation is C11S, C11aS. The natural twist of the anthramycin molecule in the C11aS conformation matches the twist of the minor groove as it winds along the helix; a C11aR drug would only fit into a left-handed helix. The C11S attachment is roughly equatorial to the overall plane of the molecule, whereas a C11R attachment would be axial and would obstruct the fitting of the drug into the groove. The six-membered ring of anthramycin points toward the 3' end of the chain to which it is covalently attached or toward the end of the helix. The acrylamide tail attached to the five-membered ring extends back along the minor groove toward the center of the helix, binding in a manner reminiscent of netropsin or distamycin. The drug-DNA complex is stabilized by hydrogen bonds from C9-OH, N10, and the end of the acrylamide tail to base pair edges on the floor of the minor groove. The origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds but from the low twist angles adopted by purine-purine steps in a B-DNA helix. Binding of anthramycin induces a low twist at T-G in the T-G-G sequence of this DNA-drug complex, by comparison with the structure of the free DNA. The origin of anthramycin's preference for adenines flanking the alkylated guanine arises from a netropsin-like fitting of the acrylamide tail into the minor groove.

Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set.

We hypothesize that inclusion of denatured proteins in the set of reference native proteins may better represent the unordered form in the current circular dichroism (CD) analyses of proteins involving unfolding ones. Adding three denatured-protein spectra and one oligopeptide spectrum to 16 reference protein spectra markedly improved the correlation coefficients (r) between CD calculations and X-ray determinations for the unordered form and, to a lesser extent, for beta-turn, but the r-values for alpha-helix and beta-sheet decreased slightly. With 20 reference proteins the estimates of the unordered form of denatured proteins were significantly improved. Thus, we suggest that as a compromise the new set of reference proteins be used for estimating the changes in conformation for unfolding proteins. However, the current use of 16 reference native proteins appears to be adequate for CD analysis of native proteins and the expansion to 20 reference proteins including denatured ones may not enhance the analysis of native proteins.

The crystal structure of the trigonal decamer C-G-A-T-C-G-6meA-T-C-G: a B-DNA helix with 10.6 base-pairs per turn.

The B-DNA decanucleotide C-G-A-T-C-G-6meA-T-C-G has been crystallized under the same conditions used earlier for C-G-A-T-C-G-A-T-C-G, but is found to adopt a new trigonal P3(2)21 packing mode instead of the expected orthorhombic P2(1)2(1)2(1) form. Unit cell dimensions a = b = 33.38 A, c = 98.30 A, gamma = 120 degrees, imply ten base-pairs or one complete decamer double helix per asymmetric unit. The 2282 two-sigma data to 2.0 A refine to R = 17.2% with 45 water molecules, 1.5 hexavalent hydrated magnesium complexes, and 0.5 chloride ion per asymmetric unit. Neighboring helices interlock backbone chains and major grooves, crossing at an angle of 120 degrees in a manner that yields an excellent model for a Holliday junction. Local helix parameters differ markedly in the trigonal and orthorhombic structures, with the trigonal helix exhibiting behavior closer to that expected of B-DNA in solution. The trigonal form has an average of 10.6 base-pairs per turn, in contrast to 9.7 base-pairs per turn in the orthorhombic cell. A comparison of all known B-DNA decamer and dodecamer crystal structure analyses indicates that, the greater the cell volume per base-pair (and hence the more open the crystal structure), the closer the mean helix twist approaches an expected 10.6 base-pairs per turn.

Some problems of CD analyses of protein conformation.

The circular dichroic (CD) spectra of 16 reference proteins were analyzed by the Provencher-Glöckner method (Biochemistry 20, 31, 1981) with the lower-wavelength limit raised from 190 to 235 nm at 5-nm intervals. Fifty-one data points at 1-nm intervals were taken between 190 and 240 nm. Variations of the correlation coefficients (r) and root-mean-square (RMS) deviations between X-ray diffraction results and CD analyses showed no definite trend with shorter wavelength ranges. The CD spectra (190-240 nm) were also analyzed by assigning the secondary structure of X-ray results according to the Levitt-Greer method (J. Mol. Biol. 114, 181, 1977) and the Kabsch-Sander method (Biopolymers 22, 2577, 1983). The r and RMS values based on the Levitt-Greer assignment were good and comparable to those based on the secondary structure given by crystallographers, but the Kabsch-Sander assignment seemed to give unsatisfactory results. The choice of reference proteins remains one of the uncertainties in the CD analysis. The five most significant orthogonal spectra (190-240 nm) calculated from the 16 reference proteins and those based on another 16 proteins used by Hennessey and Johnson (Biochemistry 20, 1085, 1981) were similar to each other, but different in intensities. These methods still cannot recognize a failed analysis of unknown proteins without X-ray results to check their reliability.

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. III. Estimation of the protein secondary structure.

Infrared spectra of 13 globular proteins have been obtained in the 1800-1480-cm-1 region for H2O solutions. A method for estimating protein secondary structure from the ir spectrum has been developed. The method can also be used for estimating polypeptide and fibrous protein conformation. For the globular and fibrous proteins and polypeptides analyzed, the correlation coefficients between the ir and x-ray estimates of ordered helix, disordered helix, ordered beta-structure, disordered beta-structure, turns, and remainder were 0.98, 0.80, 0.99, 0.87, 0.90, and 0.92 respectively.