In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1; P212121, but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A ̊, b = 62.31 A ̊, c = 43.70 A ̊ for PEG-derived crystals and a = 32.76 A ̊, b = 55.13 A ̊, c = 43.29 A ̊ for phosphate-derived crystals, compared to a = 48.73 A ̊, b = 46.39 A ̊, c = 41.10 A ̊ for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo ≥ σ(Fo) of 3712 reflections in the resolution range 10 to 2.2 Å (R = 15.8%) for the PEG-derived crystal and based on Fo ≥ σ(Fo) of 6258 reflections in the resolution range 10 to 1.8 Å (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92NεH ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two β-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both β-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 Oη; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that Φ Ψ-angles of Asn44 are in αL-conformation (that is observed in wild-type enzyme when guanine is bound). In spite of the structural differences observed for the guanine binding of RNase T1 His92Ala, the binding affinity for the inhibitor 2′-GMP is similar as for wild-type enzyme. © 1992.
