. The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.

Sequence alignments of a number of epsilon subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two epsilon subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the epsilon and gamma subunits in the complete F1-ATPase complex. In the crystal structure we observe what is apparently a stable interface between the two domains of the epsilon subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the epsilon subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

For a series of antitumor-active 5-substituted 9-aminoacridine-4-carboxamide topoisomerase II poisons, we have used X-ray crystallography and stopped-flow spectrophotometry to explore relationships between DNA binding kinetics, biological activity, and the structures of their DNA complexes. The structure of 5-F-9-amino-[N-(2-dimethylamino)ethyl]-acridine-4-carboxamide bound to d(CGTACG)(2) has been solved to a resolution of 1.55 A in space group P6(4). A drug molecule intercalates between each of the CpG dinucleotide steps, its protonated dimethylamino group partially occupying positions close to the N7 and O6 atoms of guanine G2 in the major groove. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Intercalation unwinds steps 1 and 2 by 12 degrees and 8 degrees, respectively compared with B-DNA, whereas the central TpA step is overwound by 10 degrees. Nonphenyl 5-substituents, on average, decrease mean DNA dissociation rates by a factor of three, regardless of their steric, hydrophobic, H-bonding, or electronic properties. Cytotoxicity is enhanced on average 4-fold and binding affinities rise by 3-fold, thus there is an apparent association between kinetics, affinity, and cytotoxicity. Taken together, the structural and kinetic studies imply that the main origin of this association is enhanced stacking interactions between the 5-substituent and cytosine in the CpG binding site. Ligand-dependent perturbations in base pair twist angles and their consequent effects on base pair-base pair stacking interactions may also contribute to the stability of the intercalated complex. 5-Phenyl substituents modify dissociation rates without affecting affinities, and variations in their biological activity are not correlated with DNA binding properties, which suggests that they interact directly with the topoisomerase protein.