Modes of Binding of 2′-AMP to RNase T1 A Computer Modeling Study

Abstract

The modes of binding of adenosine 2′-monophosphate (2′-AMP) to the enzyme ribonuclease (RNase) T₁ were determined by computer modelling studies. The phosphate moiety of 2′-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites - (1) The primary base binding site where the guanine of 2′-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3′-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2′-AMP to RNase T₁ were found to be of nearly the same energy implying that in solution 2′-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T₁ - 2′-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T₁ catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2′-AMP and 2′-GMP with RNase T₁ reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme - 2′-GMP complex.     

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

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.     

An exposed tyrosine residue of RNase T1 and its involvement in the interaction with guanylic acid

A photo-CIDNP spectrum of RNase T₁ showed that 4 out of the total 9 tyrosines are accessible to the photosensitive dye, while none of the histidine and tryptophan residues are accessible. By comparison with the results of nitration of tyrosine side chains followed by peptide analysis, it can be concluded that Tyr 45 is mostly exposed on the surface of RNase T₁. On addition of 2'-GMP, the signal of Tyr 45 shifts upfield and is remarkably broadened, which suggests that the phenyl ring of Tyr 45 stacks on the guanine ring of 2'-GMP. Similar phenomena were observed on addition of 3'-GMP and 3'-dGMP.     

Solution Structure of Rnase T1 and Its Complexes with Nucleotides

Abstract

The solution structure of RNase T₁ and its complexes with 2′-GMP and. 3′-GMP have been investigated by combined use of 2D-NMR spectroscopy and restrained molecular dynamics calculations (MD). An analysis of the nuclear Overhauser effects (NOEs) observed indicates the presence of one a helix as well as of two antiparallel β sheets. Interaction of the nucleotides with the active site leads to changes of the backbone conformation of the amino acids involved. However, the interaction between the protein and 3′-GMP is not as strong as the interaction with 2′-GMP, possibly because of weaker binding.     

Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1

Abstract

The mechanism of action of ribonuclease (RNase) T₁ is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T₁ and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T₁ with the reaction intermediate guanosine 2′, 3′-cyclic phosphate (G>p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T₁ - GpC (substrate) complex was found to be O4′-endo and not C3′-endo as in the RNase T₁ - 3′-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T₁ has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T₁ - GpC and RNase T₁ - G>p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.     

Disordered Single Crystal Evidence for a Quadruple Helix Formed by Guanosine 5′-Monophosphate

Abstract

The disodium salt of guanosine 5′-monophosphate (5′-GMP) has been crystallized earlier in an orthorhombic array. We have obtained a new crystal form of 5′-GMP at pH 8 which reveals a clear helical nature, with guanine bases stacked perpendicular to the helix axis. Although the X-ray pictures show partial disorder, they can be indexed on a hexagonal net with a = b = 28.6 Å,c = 9.8 Å, V= 6942ų(1Å = 0.1 nm). The probable space group is P6₄, and past experience with ca. 600 ų per base in oligonucleotide crystals suggests that the cell contains 12 GMP molecules. The crystal packing parameters and the intensity distribution agree with a model of three hydrogen-bonded guanine tetrads in the unit cell, stacked so as to build a quadruple helix similar to that proposed earlier from fiber studies (Zimmerman, S.B., J. Mol. Biol. 106, 663–672 (1976)).     

The effect of monovalent cations on the association behavior of guanosine 5'-monophosphate, cytidine 5'-monophosphate, and their equimolar mixture in aqueous solution

¹H- and ³¹P-nmr spectroscopy have been used to investigate the self-association of M₂(5′-CMP) [M = Li⁺, Na⁺, K⁺, Rb⁺, or (CH₃)₄ N⁺; 5′-CMP = cytidine 5′-monophosphate], the self-association of Li₂(5′-GMP) (5′-GMP = guanosine 5′-monophosphate), and the heteroassociation of 5′-GMP and 5′-CMP (1 : 1 mole ratio) in aqueous solution as a function of the nature of the monovalent cation. Proton spectral differences for the different 5′-CMP salts exhibit a cation-size dependence and have been ascribed to a change in the stacking geometry. An average stacking association constant of 0.63 ± 0.24M^?1 at 1°C, consistent with the weak stacking interactions of the cytosine bases, was determined for the 5′-CMP salts. Heteroassociation of 5′-GMP and 5′-CMP follows the reverse of the cation order for the formation of ordered aggregates of 5′-GMP. Heteroassociation occurs in the presence of Li⁺, Na⁺, and Rb⁺ ions, but only self-association occurs for the K⁺ nucleotides. Li₂(5′-GMP), which does not form ordered species, self-associates to form disordered base stacks with a stacking constant of 1.63 ± 0.11M^?1 at 1°C.     

E. COLI RNASE HI AND THE PHOSPHONATE-DNA/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3′–O–CH₂–P–O–5′ or 3′–O–P–CH₂–O–5′) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3′-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg⁺⁺ · 4H₂O chelate complex (bound in the active site) were analyzed in detail. Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.     

Enzymatic analysis of guanine nucleotides in tissues and cells

GTP and GDP concentrations can be determined by a simple and specific spectrophotometric assay that uses commercially available enzymes. The conversion of GTP to GDP catalyzed by nucleosidediphosphate kinase in the presence of ADP enables the subsequent use of guanylate kinase which is coupled with hexokinase and glucose-6-phosphate dehydrogenase as indicator enzymes. Guanylate kinase which is highly specific for GDP and 5′-GMP (Miech, R. P., and Parks, R. E., Jr. (1965) J. Biol. Chem.240, 351–357) is also used for the determination of 5′-GMP and of the sum of all acid-soluble guanine 5′-nucleotides. The latter are hydrolyzed by snake venom phosphodiesterase and assayed as 5′-GMP. The assays are highly reproducible with standard deviations of less than 2% when performed in the optimal range between 2 and 100 nmol of guanine nucleotide per cuvette. The sensitivity can be increased by use of dual wavelength measurements of fluorimetry or by following the generation of ATP with the luciferase-catalyzed luminescence. Contents of guanine nucleotides and of total nucleoside 5′-triphosphates were measured in liver, kidney, brain, and skeletal muscle of the rat. The effect of guanosine and of inhibitors of inosinate dehydrogenase (virazole and mycophenolate) on the level of GTP and GDP was examined in ascites hepatoma cells in suspension.     

Structure and Conformation of the Branch Core Triribonucleotide Containing 2′-5′ and 3′-5′ Phosphodiester Linkages (A2′p5′G 3′p5′C) in Solution,Essential for Yeast mRNA Splicing,Deduced from 1H-NMR

Abstract

The non-exchangeable ¹H-NMR signals of the branch core trinucleotide of the lariat branch site (A^2′p5′G _3′p5′C), 1) and its derivatives 2 and 3 are completely assigned using one- and two- dimensional NMR techniques including NOE, COSY, NOESY, ¹H-¹HINADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T₁ measurements, the following properties of the trimers are deduced.(i)The unique stacking behavior of the trimers is S^1′N _3′N, and the sugar rings exist predominantly in the N-conformation (3′-endo-2′-exo).(ii)The sugar-base orientations appear to be anti.(iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21°C and pH 7.2.(iv) The calculated values for the torsion angles ε^t andγ⁺ for the trimers are 201–203° and 71–86%, respectively, while the percent β¹ values are higher for the guanine (87–92%) than the cytosine residues (73–77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

Regulation of hormone stimulation of adipose tissue lipolysis by guanosine triphosphate

Guanosine triphosphate (GTP) enhanced the rate of mobilization of free fatty acids from isolated rat epididymal fat cells and potentiated the lipolytic response to norepinephrine, adrenocorticotropic hormone, glucagon, and theophylline. ITP, CTP, UTP, and TTP also increased basal and norepinephrine-stimulated lipolysis but to a lesser extent than GTP. ATP differed from the other nucleotides by inhibiting norepinephrine-stimulated lipolysis. The degree of phosphorylation of the guanine was important for activity since GTP was more active than GDP which, in turn, was more active than GMP in potentiating hormone-sensitized free fatty acid mobilization. Cyclic 3′, 5′-GMP, guanine, and guanosine were inactive in this regard. Activation of lipolysis by GTP occurred immediately upon addition of the nucleotide. The lipolytic response to GTP alone or in combination with norepinephrine or theophylline was exquisitely sensitive to inhibition by prostaglandin E₂. Nicotinic acid also inhibited the GTP response but to a lesser extent than prostaglandin E₂ and the β-blocker, propranolol, had no effect. Lipolytic concentrations of GTP in combination with norepinephrine increased intracellular levels of cAMP. By some as yet unknown mechanism GTP and GDP sensitized the adenylate cyclase of adipocytes to the actions of both agonists and antagonists.     

Ordered forms of 5′-8-aminoguanylic acid

5′-8NH₂GMP forms an ordered structure in moderately acid (pD 4.7) solution. We propose for this ordered form a novel hemiprotonated G·G structure with a twofold rotation axis and three hydrogen bonds between each pair of guanine residues. Gel formation does not occur with this nucleotide in either neutral or acid solution. In neutral solution 5′-8NH₂GMP also forms a regular, ordered structure, quite different from the acid form and similar to that formed by 5′-GMP under the same neutral conditions. We suggest that this ordered structure consists of a regularly stacked array of planar tetramers, similar to that proposed for 3′-GMP at pH 5.²     

Interaction of Purine Nucleotides with Cobalt-Hexammine,Cobalt-Pentammine and Cobalt- Tetrammine Cations. Evidence for the Rigidity of Adenosine and Flexibility of Guanosine and Deoxyguanosine Sugar Conformations

Abstract

The interaction of adenosine-5′-monophosphate (5′-AMP), guanosine-5′-monophosphate (5′-GMP) and 2′-deoxyguanosine-5′-monophosphate (5′-dGMP) with the [Co(NH₃)₆]³⁺, [CO(NH₃)₅C1]²⁺ and [CO(NH₃)₄C1₂]⁺ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and ¹H-NMR spectroscopy.

The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH₃) through the N-7 and the PO₃ ^2- groups of the AMP and via O-6, N-7 and the PO₃ ^2- of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2′-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine - AMP complexes, whereas a mixture of the C2′-endo/anti and C3′-endo/anti sugar puckers were observed for the Co(NH₃)₆-GMP, Co(NH₃)₅-GMP and a C3′-endo/anti conformer for the Co(NH₃)₄-GMP complexes. The deoxyribose showed an O4′-endo/anti conformation for the free dGMP anion and a C3′-endo/anti for the Co(NH₃)₆-dGMP, Co(NH₃)₅-dGMP and Co(NH₃)₄-dGMP complexes.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Nonspecific base recognition mediated by water bridges and hydrophobic stacking in ribonuclease I from Escherichia coli

The crystal structure of Escherichia coli ribonuclease I (EcRNase I) reveals an RNase T2-type fold consisting of a conserved core of six beta-strands and three alpha-helices. The overall architecture of the catalytic residues is very similar to the plant and fungal RNase T2 family members, but the perimeter surrounding the active site is characterized by structural elements specific for E. coli. In the structure of EcRNase I in complex with a substrate-mimicking decadeoxynucleotide d(CGCGATCGCG), we observe a cytosine bound in the B2 base binding site and mixed binding of thymine and guanine in the B1 base binding site. The active site residues His55, His133, and Glu129 interact with the phosphodiester linkage only through a set of water molecules. Residues forming the B2 base recognition site are well conserved among bacterial homologs and may generate limited base specificity. On the other hand, the B1 binding cleft acquires true base aspecificity by combining hydrophobic van der Waals contacts at its sides with a water-mediated hydrogen-bonding network at the bottom. This B1 base recognition site is highly variable among bacterial sequences and the observed interactions are unique to EcRNaseI and a few close relatives.     

Stimulation of mammalian adenylate cyclase activity with guanosine 5′-tetraphosphate and other guanine nucleotides

Guanosine 5′-tetraphosphate (GTP₄) stimulated mammalian adenylate cyclase activity at concentrations down to 1 μM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP₄ stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3′, 5′-monophosphate and 5′-guanylylimidodiphosphate (GMP · PNP) also stimulated mammalian adenylate cyclase activity. GMP · PNP irreversibly activated, whereas GTP₄ and GTP reversibly activated adenylate cyclase. Adenosine 5′-tetraphosphate (ATP₄) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP₄. The formation of cyclic AMP by GTP₄-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H⁺), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP₄ was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The β-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP₄, thus, suggesting that GTP₄ and β-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP₄ or GMP. PNP, thus indicating that GTP₄ resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to β-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2′-monophosphate, guanosine 3′-monophosphate or guanosine 2′,5′-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.     

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.