Preliminary crystallization studies of calmodulin-dependent protein phosphatase (calcineurin) from bovine brain.

Calcineurin is a serine/threonine protein phosphatase which catalyzes the hydrolysis of both phosphoseryl/phosphothreonyl and phosphotyrosyl proteins as well as low molecular weight compounds such as p-nitrophenyl phosphate. It is a hetero-dimeric protein consisting of a 60 kDa A chain and 19 kDa B chain. Calcineurin A is organized into functionally distinct domains such as a catalytic domain, a calcineurin B binding domain, a calmodulin-binding domain, and an inhibitory domain. Calcineurin B has four EF-hand calcium binding domains with a secondary structure that is homologous to calmodulin but its metal binding properties are more similar to troponin-C. The N-terminal myristoyl group of calcineurin B might play a role in the interaction between subunits A and B during phosphorylation/dephosphorylation processes. Crystals of size 0.125 x 0.07 x 0.03 mm and 0.7 x 0.03 x 0.02 mm have been obtained for calcineurin and the A subunit respectively. Crystals of calcineurin show strong diffraction to 5.3 A and weak diffraction to 3.0 A on rotating anode operated at 50 kV and 100 mA. Further work is in progress to improve the X-ray diffraction quality of these crystals and to obtain well diffracting crystals of calcineurin B.

Crystal structure of the PvuII restriction endonuclease.

Crystal structures have now been determined for the R.PvuII restriction endonuclease as a protein-DNA complex [Cheng et al., EMBO J. 13 (1994) 3927-3935; this report] and in apo-form [Athanasiadis et al., Nature Struc. Biol. 1 (1994) 469-475; our unpublished result]. The structures indicate how the interaction with DNA might proceed [Riddihough, Nature 370 (1994) 78].

X-ray structures of the B-DNA dodecamer d(CGCGTTAACGCG) with an inverted central tetranucleotide and its netropsin complex.

The crystal structures of the B-DNA dodecamer d(CGCGTTAACGCG) duplex (T2A2), with the inverted tetranucleotide core from the duplex d(CGCGAATTCGCG) [A2T2, Dickerson & Drew (1981). J. Mol. Biol. 149, 761-768], and its netropsin complex (T2A2-N) have been determined at 2.3 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), unit-cell dimensions of a = 25.7, b = 40.5 and c = 67.0 A, for T2A2 and a = 25.49, b = 40.87, c = 67.02 A for T2A2-N and are isomorphous with A2T2. The native T2A2 structure, with 70 water molecules had a final R value of 0.15 for 1522 reflections (F > 2sigma), while for the netropsin complex, with 87 water molecules, the R value was 0.16 for 2420 reflections. In T2A2, a discontinuous string of zig-zagging water molecules hydrate the narrow A.T minor groove. In T2A2-N, netropsin binds in one orientation in the minor groove, covering the TTAA central region, by displacing the string of waters, forming the majority of hydrogen bonds with DNA atoms in one strand, and causing very little perturbation of the native structure. The helical twist angle in T2A2 is largest at the duplex center, corresponding to the cleavage site by the restriction enzymes HpaI and HincII. The sequence inversion AATT-->TTAA of the tetranucleotide at the center of the molecule results in a different path for the local helix axis in T2A2 and A2T2 but the overall bending is similar in both cases.

Structure of PvuII endonuclease with cognate DNA.

We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation.

Expression, purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site.

We have overexpressed the type II restriction endonuclease PvuII (R-PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2(1)2(1)2(1) with cell constants a = 95.8 A, b = 86.3 A, c = 48.5 A, and diffract X-rays to at least 2.7 A. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit.

Structural comparison of the B-DNA dodecamers d(CGCGTTAACGCG) and d(CGCGAATTCGCG) with T2A2 and A2T2 tracts.

The x-ray structure of the deoxy oligonucleotide dodecamer d(CGCGTTAACGCG) recently determined in our laboratory shows that the helical parameters of the central TTAA segment are significantly different compared to the central AATT in d(CGCGAATTCGCG). The roll in the central TA step of the T2A2 dodecamer opens towards the minor groove while the AT step of the A2T2 dodecamer opens towards the major groove. Also, the roll angles at the steps 4 and 8 (GT and AC in T2A2) and (GA and TC in A2T2) are in opposite directions. The high cup and helical twist angles at the central base-pair of T2A2 decreases the base stacking interactions compared to A2T2. Tilt angles within the tetranucleotide segments TTAA and AATT have opposite signs. In spite of the local differences caused by the sequence inversion (TTAA----AATT), the two dodecamers exhibit similar overall bending. The top third is more bent than the bottom third relative to the central segment. This asymmetric bending in the two dodecamers is mainly due to crystal packing interactions.