CRYSTAL-STRUCTURES OF RIBONUCLEASE-HI ACTIVE-SITE MUTANTS FROM ESCHERICHIA-COLI

In order to investigate the relationships between the three-dimensiona l structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of th ree functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional struc tures were determined by x-ray crystallography. Although the entire ba ckbone structures of these mutants were not affected by the replacemen ts, very localized conformational changes were observed around the Mg2 +-binding site. The substitution of an amide group for a negatively ch arged carboxyl group in common induces the formation of new hydrogen b ond networks, presumably due to the cancellation of repulsive forces b etween carboxyl side chains with negative charges. These conformationa l changes can account for the loss of the enzymic activity in the muta nts, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of r everse transcriptases from retroviruses, including human immunodeficie ncy virus, the concepts of the catalytic mechanism deduced from this s tructural analysis can also be applied to RNase H activity in reverse transcriptases.