Three-dimensional structure of the ternary complex between ribonuclease T1, guanosine 3',5'-bisphosphate and inorganic phosphate at 0.19 nm resolution (original) (raw)
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Dissection of the structural and functional role of a conserved hydration site in RNase T1
Protein Science, 2008
The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites~WAT1! were analyzed by protein engineering, X-ray crystallography, and 17 O and 2 H nuclear magnetic relaxation dispersion~NMRD!. The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93NE2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the 17 O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 8C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.
Structural analysis of an RNase T1 variant with an altered guanine binding segment
Journal of Molecular Biology, 1999
The ribonuclease T 1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the speci®c inhibitor 2 H-GMP. The crystal structure has been re®ned to a crystallographic R factor of 0.198 at 2.3 A Ê resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A Ê , 2 H-GMP is ®xed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2 H-GMP is located about 4.2 A Ê apart from its position in wild-type ribonuclease T 1-2 H-GMP complexes, allowing a Ca 2 , coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine af®nity and turnover rate. The presence of Ca 2 was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.
Structural analysis of an RNase T1 variant with an altered guanine binding segment1
J Mol Biol, 1999
The ribonuclease T 1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the speci®c inhibitor 2 H-GMP. The crystal structure has been re®ned to a crystallographic R factor of 0.198 at 2.3 A Ê resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A Ê , 2 H-GMP is ®xed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2 H-GMP is located about 4.2 A Ê apart from its position in wild-type ribonuclease T 1-2 H-GMP complexes, allowing a Ca 2 , coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine af®nity and turnover rate. The presence of Ca 2 was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.
Protein Science, 1994
The interactions of RNase A with cytidine 3'-monophosphate (3"CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.
Solution structure of RNase P RNA
RNA, 2011
The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.
European Journal of Biochemistry, 1993
The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 NEH' activates 02'-H, (d) Glu58 carboxylate acts as base and His92 NEH' as acid in a general acid-base catalysis.
RNase-Stable RNA: Conformational Parameters of the Nucleic Acid Backbone for Binding to RNase T1
Biological Chemistry, 2000
An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in singlestranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCAtetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.