Three-Dimensional Structure of Ribonuclease T₁ Complexed with an Isosteric Phosphonate Substrate Analogue of GpU:  Alternate Substrate Binding Modes and Catalysis^,

Abstract

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Probing Functional Perfection in Substructures of Ribonuclease T1: Double Combinatorial Random Mutagenesis Involving Asn43, Asn44, and Glu46 in the Guanine Binding Loop

“…It is remarkable that only five variants (out of 799 possibilities) in the present study had enzyme activity comparable to that for wild type particularly when possible combinatorial effects were included. A primary role of the PRS is to anchor the substrate and provide favorable contacts of the guanosyl 2′-OH group at the active site (37). It is possible that this limited role of the PRS in catalysis might be optimal for RNase T 1 and cannot be dramatically improved beyond the minor enhancements observed for some variants in the present study.…”

Crystal structure of toxin HP0892 from Helicobacter pylori with two Zn(II) at 1.8 Å resolution

“…His83 in YoeB, 1 His92 in RNase T1, 53 and Arg81 in RelE, 54 which has been shown to be essential to the RNase activity. As discussed earlier, His86 shows the maximum reorientation and H86A HP0892 exhibited threefold less binding affinity with zinc ions (Supporting Information Fig.…”

“…Similar results were observed where mutation of His50 and His63 in YafQ also resulted in the complete abolishment of mRNase activity in vivo. 11 The Tyr38 and His40 in RNase T1 serve as a general acid in RNase activity, 53 and they are in proximity to His47 and His60 in HP0892. However, His47 and His60 in HP0892 do not colocalize structurally with any of the catalytic residues of YoeB.…”

The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

“…Also, the imidizoyl moiety of H49 contacts the oxygen atom of the nucleophilic 2′ hydroxyl group in the structural model (Figure 1B)(2) and in the structure of RNase T1 bound to a substrate analogue. 2,25 This contact may help nucleophilic activation by the putative general base (E95). The H49Q mutation, expected to maintain hydrogen-bonding capabilities, restores only 16-fold of the 2190-fold k 2 loss for k rel (SRL) observed for the H49A mutant.…”