CRYSTAL-STRUCTURE OF RIBONUCLEASE A-CENTER-DOT-D(APTPAPAPG) COMPLEX -DIRECT EVIDENCE FOR EXTENDED SUBSTRATE RECOGNITION

The crystal structure of the complex between ribonuclease A and d(ApTp ApApG) has been solved by x-ray crystallography using the molecular re placement method. The model includes, besides the enzyme, the d(ApTpAp A) 5'-segment (A(1)T(2)A(3)A(4)) and 68 solvent molecules. The R-facto r for the strongest 87% of the measured data that partially extends to 2.3-Angstrom resolution is 0.207. The A(1) position is well defined; the 5'-O of the deoxyribose establishes a hydrogen bond with a solvent molecule that is, in turn, bonded to the epsilon-amino group of Lys(6 6). The base (B-0 site) is well ordered; it interacts with a symmetry- related enzyme molecule. In the crystal, the phosphate group at the p( 0) site has no direct charge compensation. However, Lys(66) is not too far, and, in solution, it could bind to it. The T-2 (R(1)B(1)p(1)) Si te is occupied as in other complex structures, and it is defined by ve ry good electron density. The A(3) Site shows that the adenine moiety interacts with Asn(71) and Gln(69) and that the phosphate at p(2) form s a salt bridge with Lys(7). The most consistent model for the base of A(4) (B-3), both in terms of electron density and stereochemistry, sh ows that it forms a hydrogen bond with Gln(69) and a g(-)g(-) array wi th the base at B-2. The stacking of B-2 and B-3 may be a general featu re of the binding of polyribonucleotides to ribonuclease A. The side c hains of GLn(69), Asn(71), and Glu(111) may thus constitute a malleabl e binding site capable of establishing various hydrogen bonds dependin g on the nature of the stacked bases. There is no evidence for the 3' G(5) site in the electron density map.