Guanine-containing mono- and dinucleotides bind
to the active site of ribonuclease A in a nonproductive
mode (retro-binding) (Aguilar CF, Thomas PJ, Mills 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 and with the sulfate
anion located in the P1 site. To investigate the influence
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 obtained 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 P1 site. The X-ray
structures of the complexes with and without sulfate in
P1 were refined using diffraction data up to 1.8 Å
(R-factor 0.192) and 2.0 Å (R-factor
0.178), respectively. The binding mode of the substrate
analogue to the protein differs markedly in the two complexes.
When the sulfate is located in P1, we observe retro-binding;
whereas when the anion is removed from the active site,
the uridine is productively bound at the B1 site. In the
productive complex, the electron density is very well defined
for the uridine moiety, whereas the downstream guanine
is disordered. This finding indicates that the interactions
of guanine in the B2 site are rather weak and that this
site is essentially adenine preferring. In this crystal
form, there are 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 comparison 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
role of RNase A active site residues that have recently
emerged from site-directed mutagenesis studies.
