CRYSTAL-STRUCTURE OF RNASE-A COMPLEXED WITH D(PA)4

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 .ANG. resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 .ANG.. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 .ANG. resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 .ANG. resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5'' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5'' terminus by a salt link to lysine 98, passes near arginine 39 and then through a solvent region to terminate with its 3'' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5'' end on the opposite side of the protein from the active site in an electropositive anion trap that included lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5'' phosphate of one tetramer and the 3'' bases of two other tetramers and this may help to stabilize the crystalline complex. If the sites of interaction between the protein and the d(pA)4 fragments are mapped on the surface of the protein, they describe a nearly continuous path into and through the active site, across the surface of the enzyme and finally into the basic amino acid cluster on the opposite side of the protein. Such a virtual DNA strand could explain the observation that, when RNase binds to a long single strand of nucleic acid, it can cover or protect 11 to 12 bases along the polynucleotide chain. The path would also account for the observation that at least seven individual electrostatic interactions are involved in the binding of RNase to single-stranded nucleic acids.