Productive and nonproductive binding to ribonuclease A: X-ray structure oftwo complexes with uridylyl(2 ',5 ')guanosine

Guanine-containing mono- and dinucleotides bind to the active site of ribon uclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, M ills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265-267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 a nd with the sulfate anion located in the P1 site, To investigate the influe nce of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2',5')guanosine ob tained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the Pi site. The X-ray st ructures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Angstrom (R-factor 0.192) and 2.0 Angstrom (R-f actor 0.178), respectively The binding mode of the substrate analogue to th e protein differs markedly in the two complexes. When the sulfate is locate d in P1, we observe retro-binding; whereas when the anion is removed from t he active site, the uridine is productively bound at the B1 site. In the pr oductive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicat es that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal farm. there a re two molecules per asymmetric unit. and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comp arison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the rol e of RNase A active site residues that have recently emerged from site-dire cted mutagenesis studies.