SUBSITE BINDING IN AN RNASE - STRUCTURE OF A BARNASE TETRANUCLEOTIDE COMPLEX AT 1.76-ANGSTROM RESOLUTION

A set of subsites in barnase has been proposed from kinetic studies. A specific substrate analog, the tetradeoxynucleotide, CGAC, has been d esigned from this information. We report the crystal structure of its complex with barnase at 1.76-Angstrom resolution. The structure was so lved by molecular replacement from a model of free barnase and refined to a crystallographic R factor of 19.0%. The stoichiometry of the asy mmetric unit dimeric complex is [barnase:d(CGAC)](2), with 2-fold nonc rystallographic symmetry. Each barnase molecule binds one oligonucleot ide, whereby the recognition site is occupied by guanine, and all thre e phosphate groups of the nucleotide make electrostatic interactions w ith basic residues in a strongly electropositive region at the bottom of the active site. The active-site His102 packs against the adenine b ase of the nucleotide in an almost identical manner to the guanine bas e in the barnase-d(GpC) complex and defines a possible subsite in the Michaelis complex. The overall protein structure is unchanged on formi ng the complex with d(CGAC), but there are small differences in the ac tive site and in crystal packing regions. The protein coordinates will be useful for theoretical calculations since some disorder induced by packing constraints in the crystals of the free enzyme are absent in the crystals of the complex. The interface of the dimer is formed by a His102-adenine-adenine-His102 face-to-face ring stack directly on the 2-fold axis. The edge of the adenine-adenine stack packs closely onto the face of a 3'-cytosine-3'-cytosine interaction, which has a ''base -pair''-like conformation but too great a separation of the bases to f orm hydrogen bonds. This unusual arrangement is the major stabilizing interaction within the dimeric complex, since there are no direct prot ein-protein interactions. Using the structure of the complex as a star ting point for model building, the nature of the enzyme-substrate and enzyme-transition state complexes is investigated.