Three-dimensional structure of a human pancreatic ribonuclease variant, a step forward in the design of cytotoxic ribonucleases

We have determined the crystal structure of a human pancreatic ribonuclease or RNase 1 variant at 1.65 Angstrom resolution. Five residues in the N-ter minal region were substituted by the corresponding amino acids of the bovin e seminal RNase. in addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in i mproving the expression of this enzyme as a recombinant protein and in achi eving its crystallisation. The determination of the crystal structure revea led the characteristic RNase fold including a V-shaped beta -sheet and thre e alpha -helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that o f its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human ribon uclease with the ribonuclease inhibitor, together with inhibition assays, i ndicate that, in contrast to RNase A, the modification of the loop beta4 be ta5 is not enough to avoid inhibition. This study represents the first crys tallographic approach to the human enzyme, and should constitute an invalua ble tool for the design of ribonudease variants with acquired cytotoxic pro perties. (C) 2000 Academic Press.