Ribonuclease T1 with free recognition and catalytic site: Crystal structure analysis at 1.5 Å resolution

doi:10.1016/0022-2836(91)90215-R

Journal of Molecular Biology

Volume 222, Issue 2, 20 November 1991, Pages 335-352

https://doi.org/10.1016/0022-2836(91)90215-R Get rights and content

Abstract

The free form of ribonuclease T₁ (RNase T₁) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 Å nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T₁, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T₁ crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T₁ of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the α-helix, where their minor components close 3₁₀-type turns, and in β-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T₁ with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T₁-catalyzed phosphate transesterification.

References (48)

R. Arni et al.
Three-dimensional structure of the ribo-nuclease T1^∗2′-GMP complex at 1.9 Å resolution
J. Biol. Chem
(1988)
E.N. Baker et al.
Hydrogen bonding in globular proteins
Progr. Biophys. Mol. Biol
(1984)
C. Ceccarelli et al.
A survey of OH … O hydrogen bond geometries determined by neutron diffraction
J. Mol. Struct
(1981)
U. Heinemann et al.
Crystallographic study of one turn of G/C-rich B-DNA
J. Mol. Biol
(1989)
W.A. Hendrickson
Stereochemically restrained refinement of macromolecular structures
Methods Enzymol
(1985)
J. Koepke et al.
Three-dimensional structure of ribo-nuclease T1 complexed with guanylyl-2′,5′-guanosine at 1.8 Å resolution
J. Mol. Biol
(1989)
A. Lenz et al.
Evidence for a substrate binding subsite in ribonuclease T1: crystal structure of the complex with two guanosines, and model building of the complex with the substrate GpG
J. Biol. Chem
(1991)
A.D. MacKerell et al.
A time-resolved fluorescence, energetic and molecular dynamics study of ribo-nuclease T1
Biophys. Chem
(1987)
R. Malin et al.
Structurally conserved water molecules in ribonuclease T1
J. Biol. Chem
(1991)
R. Preiβner et al.
Occurrence of bifurcated three-center hydrogen bonds in proteins
FEBS Letters
(1991)

W. Saenger

Structure and catalytic function of nucleases

Current Opinion Struct. Biol

(1991)

S. Sugio et al.

Three-dimensional structure of the ribo-nuclease T1 · 3′-guanylic acid complex at 2.6 Å resolution

FEBS Letters

(1985)

P.H. Axelson et al.

Molecular dynamics of tryptophan in ribonuclease-T1 II Correlation with fluorescence

Biophys. J

(1989)

P.V. Balaji et al.

Computer modeling studies of ribonuclease T1-guanosine complexes

Biopolymers

(1990)

BIOSYM Technologies Inc

J. Ding et al.

Crystal structure of ribonuclease T₁ complexed with adenosine 2′-monophosphate at 1.8 Å resolution

Eur. J. Biochem

(1991)

F. Egami et al.

Specific interactions of base-specific nucleases with nucleo-sides and nucleotides

Mol. Biol. Biochem. Biophys

(1980)

B.C. Finzel

Incorporation of fast Fourier transforms to speed restrained least-squares refinement of protein structures

J. Appl. Crystallogr

(1987)

H.-P. Grunert et al.

Studies on ribonuclease T1 mutants affecting enzyme catalysis

Eur. J. Biochem

(1991)

U. Heinemann et al.

Structural and functional studies of ribonuclease T1

U. Heinemann et al.

Specific protein-nucleic acid recognition in ribonuclease T1-2′-guanylic acid complex: an X-ray structure

Nature (London)

(1982)

U. Heinemann et al.

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis

J. Biomol. Struct. Dynam

(1983)

H. Hirono et al.

Calculation of the relative binding free energy of 2′GMP and 2′AMP to ribonuclease T₁ using molecular dynamics/free energy perturbation approaches

J. Mol. Biol

(1990)

E. Hoffmann et al.

Two-dimensional ¹H-n.m.r investigation of ribonuclease T1

Eur. J. Biochem

(1988)

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Table 1 lists the collected νW3 and χ2,1 values of 26 Trp residues in 18 proteins. The proteins examined are azurin [15,16], horse heart cytochrome c [17,18], colicin immunity protein Im7 [19,20], photoactive yellow protein [21,22], ribonuclease T1 [23,24], nonspecific DNA-binding protein Sac7d [25,26], human serum albumin [27,28], triosephosphate isomerase (TIM) and its complex with 2-phosphoglycolate [9,29], Yersinia protein tyrosine phosphatase (PTPase) [30,31], human galectin-1 (hGal-1) and its complex with lactose [32,33], bacteriorhodopsin (bR) [34,35], blue light photoreceptor AppA [36,37], Escherichia coli redox sensor [38], outer membrane protein A (OmpA) [39,40], sperm whale deoxy-myoglobin (Mb) [41,42], and human deoxy-hemoglobin (Hb) [6,43]. The proteins from azurin to hGal-1 in the above list contain single Trp residues and the assignment of the observed W3 Raman band is straightforward.
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The fourth water molecule, which was found in the PRS, was in its position comparable to the Oϵ1 of Glu46 of the wt–2′-GMP complex. This water is also present in structures in which the Glx46 is pulled out of the PRS for example in the wt with empty PRS [12,13] or in variant 9/5 [21]. In RNase T1-wt with an empty PRS, the peptide bond of amino acids 43 and 44 is turned by about 140° [12].
Ribonuclease T1 is an enzyme that cleaves single-stranded RNA with high specificity after guanylyl residues. Although this enzyme is a very good characterized protein with respect to structure and enzymatic function, we were only recently successful in generating RNase T1-RV, a variant where the specificity was changed from guanine to purine. As this change of substrate specificity was made at the cost of activity, the aim was now to further improve the overall activity of the enzyme. Therefore, we have substituted the tryptophan in position 59 by tyrosine. This substitution led to an increase of enzymatic activity in comparison to variant RV to 425%. As the extent of this enhancement is unique so far we have crystallized and analyzed the structure of this variant in order to get more insights into the reasons for this. Here, we present the crystal structure of this so-called RNase T1-R2 at 2.1 Å resolution. The structure was determined by molecular replacement using the coordinates of the RV variant (PDB entry: 1Q9E). The data were refined to an R-factor of 18.7% and R_free of 24%, respectively. The asymmetric unit contains three molecules and the crystal packing is very similar to that of variant RV.

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^☆: This work forms part of the Ph.D. thesis of José Martinez-Oyanedel.

^☆☆: These studies were supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 9 and Leibniz-Programm) and by the Fonds der Chemischen Industrie.

^†: Present address: Laboratorio de Biofisica Molecular, Universidad de Concepcion, Casilla 4077, Correo 3, Concepcion, Chile.

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Journal of Molecular Biology

ArticleRibonuclease T1 with free recognition and catalytic site: Crystal structure analysis at 1.5 Å resolution☆,☆☆

Abstract

J. Biol. Chem

Progr. Biophys. Mol. Biol

J. Mol. Struct

J. Mol. Biol

Methods Enzymol

J. Mol. Biol

J. Biol. Chem

Biophys. Chem

J. Biol. Chem

FEBS Letters

Current Opinion Struct. Biol

FEBS Letters

Molecular dynamics of tryptophan in ribonuclease-T1 II Correlation with fluorescence

Biophys. J

Computer modeling studies of ribonuclease T1-guanosine complexes

Biopolymers

Crystal structure of ribonuclease T1 complexed with adenosine 2′-monophosphate at 1.8 Å resolution

Eur. J. Biochem

Specific interactions of base-specific nucleases with nucleo-sides and nucleotides

Mol. Biol. Biochem. Biophys

Incorporation of fast Fourier transforms to speed restrained least-squares refinement of protein structures

J. Appl. Crystallogr

Studies on ribonuclease T1 mutants affecting enzyme catalysis

Eur. J. Biochem

Structural and functional studies of ribonuclease T1

Specific protein-nucleic acid recognition in ribonuclease T1-2′-guanylic acid complex: an X-ray structure

Nature (London)

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis

J. Biomol. Struct. Dynam

Calculation of the relative binding free energy of 2′GMP and 2′AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches

J. Mol. Biol

Two-dimensional 1H-n.m.r investigation of ribonuclease T1

Eur. J. Biochem

Article
Ribonuclease T₁ with free recognition and catalytic site: Crystal structure analysis at 1.5 Å resolution☆,☆☆

Crystal structure of ribonuclease T₁ complexed with adenosine 2′-monophosphate at 1.8 Å resolution

Calculation of the relative binding free energy of 2′GMP and 2′AMP to ribonuclease T₁ using molecular dynamics/free energy perturbation approaches

Two-dimensional ¹H-n.m.r investigation of ribonuclease T1