CRYSTALLOGRAPHIC ANALYSES OF AN ACTIVE HIV-1 RIBONUCLEASE H DOMAIN SHOW STRUCTURAL FEATURES THAT DISTINGUISH IT FROM THE INACTIVE FORM

An active recombinant preparation of the carboxy-terminal ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase has produced crysta ls of several different forms, including a trigonal prism form (P3(1); a = b = 52.03, c = 113.9 angstrom with two molecules per asymmetric u nit) and a hexagonal tablet form (P6(2)22 or P6(4)22; a = b = 93.5, c = 74.1 angstrom with one molecule per asymmetric unit). The former app ears to be isomorphous with crystals of a similar, but inactive, versi on of the enzyme that was used for a prior crystal structure determina tion [Davies, Hostomska, Hostomsky, Jordan & Matthews (1991). Science, 252, 88-95]. We have also obtained a structure solution for this crys tal form and have refined it with 2.8 angstrom resolution data (R = 0. 216). We report here details of our crystallization studies and some i nitial structural results that verify that the preparation of active H IV-1 RNase H yields a protein that is not just enzymatically, but also structurally, distinguishable from the inactive form. Evidence sugges ts that region 538-542, which may be involved in the catalytic site an d which is disordered in both molecules in the prior structure determi nation, is ordered in the crystal structure of the active enzyme, alth ough the ordering may include more than one conformation for this loop . It should also be noted that, in the crystal structure of the trigon al form, RNase H monomers associate to form noncrystallographic twofol d-symmetric dimers by fusing five-stranded mixed beta sheets into a si ngle ten-stranded dimerwide sheet, an assembly that was not remarked u pon by previous investigators.