Additivity of Protein-Guanine Interactions in Ribonuclease T1

Loverix, Stefan; Doumen, J.; Steyaert, Jan

doi:10.1074/jbc.272.15.9635

Cited by 19 publications

(30 citation statements)

References 49 publications

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“…This decrease in enzymatic activity has already been described for other variants containing substitutions in the guanine binding segment (24,26,44). RNase T1-8/3 exhibits a 10-fold lower ApC cleavage activity than determined for GpC.…”

Section: Discussionmentioning

confidence: 70%

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

1999

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Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.

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Section: Discussionmentioning

confidence: 70%

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

1999

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“…The structural problem of the hammerhead apparently does not arise simply from the small size of this ribozyme; even a protein such as RNase T1, which is similar in molecular mass to the hammerhead and carries out the same reaction, is much less sensitive to mutation (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) and much more catalytically proficient. At 25°C, k cat is ∼2 × 10 4 and ∼1 min -1…”

Section: Implications and Conclusionmentioning

confidence: 99%

“…Mutations at 19 different positions located at or near the active site have been characterized for this enzyme (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38), yet mutations at only two positions decreased k cat with GpC by more than 100-fold (38); these positions correspond to the active site His40 and His92 that have been proposed to act in general acid and base catalysis (27,38).…”

Section: Introductionmentioning

confidence: 99%

A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations

et al. 1998

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Introducing abasic nucleotides at each of 13 positions in the conserved core of the hammerhead ribozyme causes a large decrease in the extent of catalysis [Peracchi, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522]. This extreme sensitivity to structural defects is in contrast to the behavior of protein enzymes and larger ribozymes. Several additional differences in the behavior of the hammerhead relative to that of protein enzymes and larger ribozymes are described herein. The deleterious effects of the abasic mutations are not relieved by lowering the temperature, by increasing the concentration of monovalent or divalent metal ions, or by adding polyamines, in contrast to effects observed with protein enzymes and large RNA enzymes. In addition, the abasic mutations do not significantly weaken substrate binding. These results and previous observations are all accounted for by a "core folding" model in which the stable ground state structure of the hammerhead ribozyme complexed with the substrate is a partially folded state that must undergo an additional folding event to achieve its catalytic conformation. We propose that the peculiar behavior of the hammerhead arises because the limited structural interconnections in a small RNA enzyme do not allow the ground state to stably adopt the catalytic conformation; within the globally folded catalytic conformation, limited structural interconnections may further impair catalysis by hampering the precise alignment of active site functional groups. This behavior represents a basic manifestation of the well-recognized interconnection between folding and catalysis.The hammerhead (Figure 1) is the smallest known naturally occurring ribozyme (see refs 3-5 for review), and the only one for which a complete three-dimensional structure is known to atomic resolution (6, 7). Previously, we began to address the structural basis of hammerhead catalysis through a "subtraction mutagenesis" approach, in which each residue in the conserved core was individually replaced by an abasic nucleotide analogue (Chart 1) (8,9). Removing individual bases from DNA duplexes induces local, context-dependent rearrangements (10-16), and removing large amino acid side chains from protein cores (e.g., via alanine scanning mutagenesis) can result in various rearrangements or can leave a cavity within the core (17)(18)(19). Analogously, abasic mutagenesis in the hammerhead core could reveal how this ribozyme responds functionally to the introduction of structural defects and to changes in local packing.Nearly all of the 13 positions in the hammerhead ribozyme core that were substituted with an abasic residue gave large decreases in the extent of catalysis (8) ( Figure 1B). In contrast, mutations at only a small subset of "catalytic" residues in proteins typically yield a large decrease in activity (see below). The characterization of the abasic hammerhead variants reported herein was carried out in an effort to understand why so many abasic substitutions in the hammerhead produce s...

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“…3,5,6,12,14 All of these findings suggest that the PRS may have achieved its optimal structure for guanine recognition. The free energy change through mutation of Tyr45 (Tyr45Ala) of the guanine-RNase T1 complex suggests that the stacking interaction between the phenolic side chain of Tyr45 and the aromatic ring of guanine is less important compared with the multiple H-bonding between the PRS and the guanine moiety.…”

Section: Introductionmentioning

confidence: 89%

Molecular Basis of the Recognition Process: Hydrogen-Bonding Patterns in the Guanine Primary Recognition Site of Ribonuclease T1

Wang

Leszczyński

2006

J. Phys. Chem. B

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Investigation of the intrinsic H-bonding pattern of the guanine complex with a sizable segment (from Asn43 to Glu46) of the primary recognition site (PRS) in RNase T1 at the B3LYP/6-311G(d,p) level of theory enables the electronic density characteristics of the H-bonding patterns of the guanine-PRS complexes to be identified. The perfect H-bonding pattern in the guanine recognition site is achieved through the guanine complex interactions with the large segment of the PRS. Two significant short H-bonds, O epsilon 1...HN1 and O epsilon 2...HN2, have been identified. The similar short H-bond distances found in the anionic GC- base pair and in this study suggest that the short hydrogen-bond distances may be characteristic of the multiple H-bonded anionic nucleobases. The H-bonding energy distribution, the geometric analysis of the H-bonding pattern, and the electron structure characteristics of the H-bonds in the guanine PRS of RNase T1 all suggest that the O epsilon 1...HN1 and O epsilon 2...HN2 side-chain H-bonds dominate the binding at the guanine recognition site of RNase T1. Also, the geometry evidence, the electron structure characteristics, and the properties of the bond critical points of the H-bonds reveal that the side-chain H-bonding and the main-chain H-bonding are mutually intensifying. Thus the positive cooperativity between Asn43 to Tyr45 and Glu46 is proposed.

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Additivity of Protein-Guanine Interactions in Ribonuclease T1

Cited by 19 publications

References 49 publications

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations

Molecular Basis of the Recognition Process: Hydrogen-Bonding Patterns in the Guanine Primary Recognition Site of Ribonuclease T1

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Additivity of Protein-Guanine Interactions in Ribonuclease T1

Cited by 19 publications

References 49 publications

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment,

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment,

A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations

Molecular Basis of the Recognition Process: Hydrogen-Bonding Patterns in the Guanine Primary Recognition Site of Ribonuclease T1

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Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,