Dissection of the structural and functional role of a conserved hydration site in RNase T1

The reoccurrence of water molecules in crystal structures of RNase T1 was i nvestigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and function al characteristics of one of these conserved hydration sites (WAT1) were an alyzed by protein engineering, X-ray crystallography, and O-17 and H-2 nucl ear magnetic relaxation dispersion (NMRD). The position of WAT1 and its sur rounding hydrogen bond network are unaffected by deletions of two neighbori ng side chains. In the mutant Thr93Gln, the Gln93N epsilon 2 nitrogen repla ces WAT1 and participates in a similar hydrogen bond network involving Cys6 , Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds m ay explain why evolution has preserved a water molecule, rather than a side -chain atom, at the center of this intricate hydrogen bond network. Compari son of the O-17 NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order para meter of 0.45. The effects of mutations around WAT1 on the kinetic paramete rs of RNase T1 are small but significant and probably relate to the dynamic s of the active site.