Computer modeling studies on the subsite interactions of ribonuclease T1.

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Computer Modeling Studies on the Subsite Interactions of Ribonuclease T1 a

P. V. Balaji & V. S.R. Rao a

b

Molecular Biophysics Unit

b

Jawaharlal Nehru Center for Advanced Scientific Research , Indian Institute of Science , Bangalore , 560 012 , India Published online: 21 May 2012.

To cite this article: P. V. Balaji & V. S.R. Rao (1992) Computer Modeling Studies on the Subsite Interactions of Ribonuclease T1 , Journal of Biomolecular Structure and Dynamics, 9:5, 971-989, DOI: 10.1080/07391102.1992.10507971 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507971

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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 5 (1992), ®Adenine Press (1992).

Computer Modeling Studies on the Subsite Interactions of Ribonuclease T 1


P.V. Balaji and V.S.R. Rao# Molecular Biophysics Unit #Jawaharlal Nehru Center for Advanced Scientific Research Indian Institute of Science Bangalore 560 012, India Abstract The modes ofbinding of pGp,ApG, CpG and UpG to the enzyme ribonuclease T 1were determined by computer modeling. Essentially two binding modes are possible for all the four ligands- one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5' -phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5' -phosphate moieties occupy the same sites on the enzyme as 5' -GMP when bound to RNase T 1• The ribose moiety of pGp adopts a C3'endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5' -GMP in the RNase T 1- 5' -GMP complex. Based on these results, a mechanism for the release ofthe product subsequent to cleavage ofthe substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5' -side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the lN subsite for adenine.

Introduction The occurrence and influence of enzyme subsites on the overall binding and catalytic process has been elucidated in studies on a variety of hydrolytic enzymes (1,2). Extensive studies on bovine pancreatic ribonuclease, RN ase A, and staphylococcal nuclease have indicated the presence of electrostatic subsites for binding the substrate phosphate groups in these enzymes (3-9). Although extensive studies have been carried out on the three dimensional structure of the complexes of ribonuclease (RN ase) T 1 (EC 3.1.27.3) with various inhibitors (10) and its mechanism of action (ll), the enzyme sub sites have not been characterised though the presence of subsites was indicated from the RNase T 1 catalysed hydrolytic studies of rabbit reticulocyte RNA as early as in 1970 (12). From UV difference spectral studies on the binding of guanosine 3',5'-bis phosphate (pGp), adenyl3',5'-guanine (ApG), cytidyl3',5'-guanine (CpG) and uridyl 3',5'-guanine (UpG) to RNase T 1, a subsite specific for the adenine moiety of ApG was proposed (13). These studies also ruled outthe existence of the 5' -phosphate specific sub site proposed earlier from spectrophotometric and gel filtration studies (14). Although other kinetic studies have also indicated the

971

972

Balaji and Rao

existence of subsites in RNase T 1 (15-17), the nature of the amino acid residues constituting these subsites could not be established. Recently from 1H-nmr studies it was noted that pGp, which has both 3'- and 5'phosphate groups, adopts a C3'-endo-anti conformation when bound to RNase T 1 similar to 5'-GMP and not 3'-GMP (18) eventhough 3'-GMP is a strongerinhibitor ofRNase T 1 than 5'-GMP (19)which is somewhat surprising. Hence to characterise the subsites ofRNase T 1 and to investigate the unexpected mode of binding of pGp to RNase T 1, computer modeling studies on the complexes ofRNase T 1 with pGp, ApG, CpG and UpG have been carried out.


Methods The atomic coordinates for RNase T 1 were taken from the 1.9 A resolution x-ray crystallographic study of the RNase T 1 - 2'-GMP complex (20). Since the coordinates for only the non-hydrogen atoms were available from this study, the coordinates for the polar hydrogens were generated using standard bond lengths and bond angles (21). All the CH, CH 2, and CH 3 groups in the protein were treated as 'united atoms'. The coordinates forpGp,ApG, CpG and UpGwere generated using the standard geometry (22,23). The torsion angles used to generate the coordinates of the ribose moiety were taken from Ref. 24. In the x-ray crystallographic study of the RNase T 1 - 2'-GMP complex (20), most of the water molecules were found around the surface of the protein and sparsely in the active site and the inhibitor binding site was found to be part of an apparently underhydrated surface portion. Hence instead of including the solvent molecules explicitly, the effect of solvent in damping the electrostatic interactions was modelled by using a distance dependant dielectric constant which weighs the short range interactions more than the long range interactions. Computational Details

The calculations were carried out in three steps: (1) Contact criteria- The sterically allowed orientations for the guanine base and guanosine were determined. For this, all the amino acid residues that fall within a sphere of radius 10 Afrom the center of the base in the active site were considered. The three Eulerian rigid body rotation angles - . a and \j/- were used to define the orientation of the base in the binding site. (2) Energy minimisation in torsion angle space - Starting from the orientations obtained as sterically allowed (Step 1), energy minimisation was carried out in the torsion angle space. The variable ligand torsion angles shown in Figure 1 and the side chain torsion angles of 37 amino acid residues including those involved in guanine recognition and in catalysis were allowed to move during energy minimisation. The details of the procedure adopted for contact criteria and energy minimisation in torsion angle space are given in reference 25. (3) Energy minimisation in Cartesian coordinate space- Only a few low energy con-

973

Interactions of Ribonuclease T1

3' H

01-t-02


q; OJ

I

H I

I

Guanosi,. J, 5- bis ( Phosphateo)(pGp)

i

05

1

02

, I

057-P~OJ

I 01 02

\

H

1

03

1

oz'

J

I

I

H

NucS.otidyl Guanine ( NpG) ( N =Ade or Cyt or Ura) Figure 1: Schematic diagrams of guanosine 3',5'-bisphosphate (pGp) and nucleotidyl3',5'-guanosine (NpG) studied in the present work along with the nomenclature used.

formers obtained by energy minimisation in torsion angle space (Step 2) were selected for energy minimisation in Cartesian coordinate space due to severe restriction on the available computer time.

Potential Energy Functions The total energy (E101) was calculated by considering the intramolecular protein and

974

Balaji and Rao

Table I Conformation of pGp and the Proposed Hydrogen Bonding Scheme in the RNase T 1 - pGp Complexes Conformational Angles (degrees)


Mode of Binding*

Hydrogen Bonding Scheme 2

C4 -N9 -Cl'-04' C3' -C2' -02' -02'H

74.9 - 43.3

- 63. - 75.4

C4' -C3'-03'-P3

-156.5

158.2

C3' -C4' -C5' -05'

- 66.

-175.3

C4' -C5' -05' -P5

- 90.7

-173.

C3' -03' -P3 -03 03' -P3 -03 -03H

66. 165.2

50.9 -177.7

C5' -05' -P5 -03

-163.6

-116.9

05' -P5 -03 -03H C4' -04'-C1'-C2'

59.6 - 26.9

- 52.2 11.5

04' -C1'-C2'-C3'

35.7

- 35.1

Cl' -C2' -C3' -C4'

- 29.5

43.5

C2' -C3' -C4' -04'

15.3

- 39.3

C3' -C4' -04' -C I'

7.1

17.6

2 Guanine NIH ... E46 N2Hl ... N98 N2H2 ... E46 ... N44 06 ... Y45 ... N43 N7 ... N43

OEl 0 OE2 N-H N-H N-H HD21

Ribose 02' ... E58 HE2 03' 04'

E46 N98 E46 N44 Y45 N43

OEI 0 OE2 N-H N-H N-H

H40 HE2 N98 HD22

3'-Phosphate ... N36 HD21 01 ... Y38 HH 02

... R77 HE ... R77 HH21 ... H92 HE2

5'-Phosphate

Pseudorotation Phase Angle P H8-H1' Dist (A) ENERGY (kcaVmol) Ligand Protein Interatcion Total

149.1

4.4

2.6

3.7

- 25.8 -170.8 -144.9 -341.5

- 33. -183.1 -156.1 -372.2

01

Y38 HH E58 HE2 R77 HE

02

H92 HE2

03

R77 HH21

03H ... N98 001

*Mode 1: Substrate mode of binding Mode 2: Inhibitor mode of binding

ligand energies and the intermolecular interaction energy between the enzyme and the inhibitor. In step 2, contributions from van der Waals (Evdw), electrostatic (Eele), hydrogen bonding (~b) and torsional (E1or) terms were considered. In addition to these, bond stretching (Eb1) and angle bending (Eba) energy terms were also included in Step 3. The form of the potential function used is as follows:

The form of the function for the various terms and the parameters used along with



975

Figure 2: Stereo diagrams of the active site residues of the enzyme and the product pGp in the minimum energy conformations of the RNase T 1 - pGp complex (Protein atoms are connected by thin lines and those of pGp by thick lines); (a) Substrate mode of binding (top); (b) Inhibitor mode of binding (bottom).

the details of the procedures for energy minimisation in Cartesian coordinate spaces have been described elsewhere (26).

Results Two modes of binding of pGp to RNase T 1 can be envisaged: One similar to that of 3'-GMP with the 3'-phosphate group of pGp occupying the primary phosphate binding, pl and the second similar to that of5'-GMP wherein the 5'-phosphate of pGp occupies the p 1 site. These two modes of binding will be henceforth referred to as the substrate and inhibitor modes of binding respectively. Potential energy of the RNase T 1 - pGp complexes in both the modes was minimised considering both the C2'- and C3' -endo pucker forms for the ribose. The conformational angles of pGp


976

Balaji and Rao

Figure 3: Pseudorotation cycle of furanose ring in nucleosides. Values of the phase angle Pare given in multiples of 36°. Envelope (E) and twist (T) forms alternate every 18°. On the periphery of the cycle, riboses with signs of endocyclic torsion angles are indicated: (+) Positive; (-)Negative and (0) Angle close to 0°. (Adopted from Altona, C. and Sundaralingam, M.,.l. Am. Chern. Soc. 94, 8205-8212 (1972)).

and the proposed hydrogen bonding scheme in the minimum energy complexes are given in Table I. Stereo diagrams of the active site residues ofRNase T 1 and pGp in these complexes are shown in Figures 2a and 2b. The pseudorotation phase angle P which characterises the ribose pucker (Figure 3) is also given in Table I for both the minimum conformers of the RNase T 1 - pGp complex. It is interesting to note that when pGp binds with its 5' -phosphate group in the pl site (inhibitor mode of binding), the ribose favours a C3' -endo pucker form. Even when the energy minimisation in the Cartesian coordinate space is carried out starting from a C2'-endo pucker form, the ribose moiety repuckers to C3'-endo via the 04' -endo pathway of pseudorotation (27). This enzyme induced ribose repuckering was monitored at every 25 iterations of minimisation and the variation of the phase angle P and the rms gradient as a function of the number of iterations is plotted in Figure 4. Snapshots showing the different stages of ribose repuckering are shown in Figure 5. The potential energy of the complexes of RNase T 1 with the dinucleotides ApG, CpG and UpG was also minimised first in the torsion angle space and subsequently

977


-

180

1.2

150

1.0

120

0.8 Ien

r

A.

'I .i ~


A.

90

0.8

80

0.4 2.

30

0.2

li

!

~

f

~

0

'V

J l a.

~

:I

I

A.

0.0

0 I

0

I

I

I

II

I

I

I

I

I

I

I

l500

I

I

I

I

I

I

I

I

I

I

1000

I

I

I

I, I

I

I

I

If I, I

1l500

I

I

I

I

I

I

2000

I

I

I

I

I

I

I

I

2l500

No. of IWcrllone Figure 4: Variation of the phase angle and the RMS gradient plotted as a function of the number of iterations during the course of minimisation of the RNase T 1 - pGp complex (Inhibitor mode ofbinding). The curve for the phase angle is shown in thin line and that for the RMS gradient in thick line.

in the Cartesian coordinate space. The conformation of the bound dinucleotides in the minimum energy complexes and the proposed hydrogen bonding scheme for the substrate mode of binding are given in Tables II and III and in Tables IV and V for the inhibitor mode of binding. Stereo diagrams of the active site residues of RNase T 1 and the bound dinucleotide in both the modes of binding are shown in Figures 6a-6f for all the three RNase T 1 - dinucleotide complexes.

Discussion The hypothetical scheme proposed by Walz and Terenna (13) for subsite nomenclature in RN ase T 1 has been used for discussion of subsites in the present study and is as shown below:

L ..._2N_2p_lN_lp_G_pl_Nl_p2_N2_...=-oJ Here G represents the guanine specific primary binding site and p 1 represents the binding site for the 3' -phosphate group of the guanine binding in the G site. Nl represents the binding sub site for the base on the 3' -side of guanine bound at the G site.

978

Balaji and Rao

(a) p - 168.s0 C2'-ENDO


(b) p - 127.1

0

(c) p- 102.0

04'-ENDO

(d) p -

47.7'

(e) p -

12.s0

CJ'-ENDO


979

Figure 5: Stereo snapshots of the active site residues ofRNaseT 1 and the product pGp taken after (a) 0, (b) 100, (c) 600, (d) 725 and (e) 1000 iterations of minimisation. Note the change in the ribose pucker from

C2'-endo to C3'-endo via 04'•endo.

Similarly lp, lN ..... stand for the subsites for binding the phosphates and bases on the 5' -side of the guanine bound at the G site.


Earlier x-ray crystallographic (20,28) and theoretical studies (25,26) have indicated that the amino acid residues Tyr42-Asn43- Asn44-Tyr45-Glu46 and Asn98 constitute the G site and the residues Tyr38, His40, Glu58, Arg77 and His92 constitute the p 1 site, the site of hydrolysis. RNase T1-pGp Complex

The guanine moiety of pGp occupies the primary base binding site of the enzyme in both the substrate and inhibitor modes of binding and forms similar hydrogen bonds with the enzyme (Table I). The ribose forms one hydrogen bond with Glu58 in the substrate mode of binding and one hydrogen bond each with the residues His40 and Asn98 in the inhibitor mode of binding. The amino acid residues Asn36, Tyr38, Glu58, Arg77, His92 and Asn98 interact with the phosphate groups in both the modes of binding. Since 3'-GMP is a stronger inhibitor compared to 5'-GMP (29), it was expected that pGp, having with both 3'- and 5'-phosphate groups, will bind to RNase T 1 with its 3' -phosphate group in the p 1 site. But in the energetically favoured conformer of the RNase T 1 - pGp complex (Table I; inhibitor mode of binding), the 5'-phosphate ofpGp occupies the same pl site and forms the same hydrogen bonds with the enzyme as the phosphate group in the RN ase T 1 - 5'-GMP complex (25,26) whereas the 3' -phosphate of pGp in this mode occupies a site different from that of the phosphate in the RNase T 1 - 3'-GMP complex (25,26) and does not form any hydrogen bonds with the protein. These results also suggest that the addition of a 3' -phosphate group to 5' -GMP would not alter its orientation whereas the addition of a 5' -phosphate group to 3' -GMP will lead to flipping of the ligand in the binding site. Table I also shows that the C3'-endo pucker(Phase angle P = 4.4°) is preferred for the ribose moiety of pGp in the energetically favoured mode as in the RNase T 1 - 5'GMP complex (26). In fact, when minimisation was carried out starting from C2'endo pucker and with the 5'-phosphate group in the pl site (inhibitor mode of binding), the ribose moiety repuckers to C3' -endo via the 04' -endo pathway of pseudorotation (Figures 3-5). It can be seen from Figure 4 that the repuckering is complete at the end of about 1000 iterations of minimisation itself indicating that the RNase T 1 - pGp (C2' -endo) complex is energetically less favoured compared to the RNase T 1 - pGp (C3'-endo) complex. The glycosyl torsion angle ofpGp in the RNase T 1 - pGp complex (inhibitor mode of binding) is -63 o and falls in the -sc range as in the case of the RNase T 1 - 5' -GMP complex (25,26,30). Thus the present results suggest that pGp binds to RNase T 1 similar to 5'-GMP. Interestingly, 1H-nmr studies also have indicated that pGp binds to RNaseT 1 in a mode similar to 5'-GMP and not3'-GMP(l8). These studies

980

Balaji and Rao Table II Conformation of ApG, CpG and UpG in their complexes with RNase T 1 Substrate mode of binding Dinucleotide

ApG

CpG

UpG

-

G04' G02'H G03'H G05' p N03' NC3' NC4' N05' N05'H N02'H NC4*

57.8 -170.3 176. 163.2 -176.7 76.7 63.7 -171.2 146.1 79. - 61.3 145.5

54.5 -176.8 175.9 166.8 -178.3 77.4 71.9 -169.3 148.8 73.2 - 60.7 -150.1

54.5 -176.7 175.9 166.9 -178.1 77.5 71.4 -169.4 149.5 73.8 - 60.5 -152.2

GC4' G04' - GCl' G04' - GCl' - GC2' GCl' - GC2' - GC3' GC2' - GC3' - GC4' GC3' - GC4' - G04' Pseudorotation Phase Angle P

-

GC2' GC3' GC4' G04' GCl'

- 37.7 15.9 9.6 - 32.2 44.4 77.3

- 40.1 20.4 4.9 - 28.8 43.6 83.5

- 40. 20.3 5.1 - 28.9 43.6 83.3

NC4' - N04' - NCl' N04' NC2' - NCl' NCl' - NC2' - NC3' NC2' NC3' - NC4' NC3' NC4' N04' Pseudorotation Phase Angle P

-

NC2' NC3' NC4' N04' NCl'

- 42.6 23.8 1.5 - 26.1 42.4 88.1

- 44.4 27.1 1.8 - 23.9 41.9 92.3

- 44.5 27.3 1.9 - 23.9 42. 92.4

- 23.9 -175.5 - 85.3 -284.7

- 36.5 -188.7 - 82.9 -308.2

- 40.1 -188.7 - 83. -311.8

Conformational Angles (degrees) (N =A or Cor U)


GC4 GC3' GC2' GC3' GC4' GC5' G05' p

NC3' NC4' NC3' N04'

-

-

-

-

-

-

GN9 GC2' GC3' GC4' GC5' G05' p

N03' NC4' NC5' NC2' NCl'

-

-

-

-

-

GCl' G02' G03' GC5' G05' p N03' NC3' NC5' N05' N02' NN9

-

-

Ribose Torsion Angles (degrees)

-

-

-

-

ENERGY (kcal/mol) Ligand Protein Interaction Total * N04' - NCl' - NNl - NC2 in case of CpG and UpG.

also revealed that ribose favours a C3' -endo conformation and the phosphate is placed near the amino acid residues same as those found from the present study. Thus the results obtained from present study are in agreement with the nmr studies. Recently, the crystal structure of the RNase T 1 - pGp complex was determined at a low resolution of 3.2A (31) and it was found that pGp binds to RNase T 1 approximately in the same mode as predicted by theory. However, there are slight differences in the exact orientation of the ligand. Binding ofApG, CpG and UpG to RNase T1

Tables II and IV show that all the three dinucleotides ApG, CpG and UpG favour to

981

Interactions of Ribonuclease T1 Table III Proposed Hydrogen Bonding Scheme in the RNase T 1 - ApG, RNase T 1 - CpG and RNase T 1 - UpG Complexes Substrate mode of binding Dinucleotide


GUANINE NlH N2Hl N2H2 06

N7 RIBOSE (Gua) 02'H PHOSPHATE 02 RIBOSE (A I C I U) 05'H

ApG

UpG

CpG

E46 N98 E46 N43 N44 Y45 N43 N43

OEI 0 OE2 N-H N-H N-H N-H HD21

E46 N98 E46

OEl 0 OE2

E46 N98 E46

OEI 0 OE2

N44 Y45 N43 N43

N-H N-H N-H HD21

N44 Y45 N43 N43

N-H N-H N-H HD21

E58

OE2

E58

OE2

E58

OE2

N98

HD21

N98

HD21

N98

HD21

N99

ODI

N99

ODI

N99

ODI

bind to RNase T 1 in the inhibitor mode of binding. The possible hydrogen bonds between these nucleotides and the enzyme are nearly the same (Table V). This suggests that all these dinucleotides assume nearly the same orientation in the binding site ofRNase T 1 (Figures 6b, 6d and 6f). Energy minimisation calculations carried out in the torsion angle space on the complexes ofRNase T 1 with ApGp, CpGp, UpGp (Table VI) show that these ligands favour to bind toRN ase T 1 in the inhibitor mode suggesting that the presence of the phosphate group on the 3'-side of guanine will not alter the mode of binding of ApG, CpG and UpG. The preference of these dinucleotides for the inhibitor mode of binding has important implications in the release of the products subsequent to cleavage of the substrate by RNase T 1 and this is discussed later. The present calculations also indicate that in ApG and UpG, the base on the 5'-side of guanine is placed close to the amino acid residues Ser72, Pro73 and Gly74 in the energetically favoured inhibitor mode of binding (Figures 6b and 6f). Computer modelling studies on the binding of the substrate dinucleotides GpA, GpC, GpG and GpU to RNase T 1 showed that in these complexes, the second base (i.e., the base on the 3'-side of guanosine bound at the G site) occupies the same site. Thus the residues Ser72, Pro73 and Gly74 may constitute the Nl site. Subsites of RNase T1

A trinucleotide, for example of the type ApGpN, when bound to RNase T 1 should occupy the lN (A), lp(5'-phosphate)G(gua),pl (3'-phosphate)andNl (N)sites for cleavage to take place. This corresponds to the substrate mode of binding of pGp or NpGp to RNase T 1• It can be seen from Tables I and III that in the substrate mode of binding, Asn98 forms a hydrogen bond with the 5'-phosphate group of pGp and the phosphate group of ApG, CpG and UpG. It is likely that this hydrogen bond between the Asn98 side chain and the 5'-phosphate group contributes significantly to

982

Balaji and Rao Table IV Conformation of ApG, CpG and UpG in their complexes with RNase T 1 Inhibitor mode of binding Dinucleotide

ApG

CpG

UpG


Conformational Angles (degrees) (N =A or Cor U) GC4 GC3' GC2' GC3' GC4' GC5' G05' p NC3' NC4' NC3' N04'

-

-

GN9 GC2' GC3' GC4' GC5' G05' p N03' NC4' NC5' NC2' NC1'

-

-

-

-

GCl' G02' G03' GC5' G05' p N03' NC3' NC5' N05' N02' NN9

-

-

G04' G02'H G03'H G05' p N03' NC3' NC4' N05' N05'H N02'H NC4*

- 70.2 - 79.2 58.5 170. 176.3 - 83.9 173.9 -173.8 80.3 - 94.6 29.5 -159.8

- 35.1 -163.3 14.3 - 57.8 162.6 153.7 - 78. - 70.4 ,85.1 - 95.8 161.5 -170.5

- 74.4 47.2 69.1 163.7 175.3 - 81.5 167.2 -175.4 77.9 - 82. 3.8 -152.5

-

GC2' GC3' GC4' G04' GC1'

- 46.4 35. - 11.6 - 15.3 38.8 104.7

- 43.6 27.5 3.1 - 22.3 41.7 94.1

- 45.8 31.8 - 7.1 - 19.7 41.2

- 35.7 8.5 19.1 - 39.6 47. 65.8

- 44.5 34.2 - 13.1 - 12.4 34.5 107.8

- 18.4 - 11.3 33.8 - 44.4 39.8 41.2

- 12.2 -186.3 -119. -317.5

- 31.8 -174.7 -119.2 -325.7

- 24.6 -185.2 -124.7 -334.4

Ribose Torsion Angles (degrees)

-

GC4' GC1' - G04' G04' GC2' - GC1' GC1' GC2' - GC3' GC2' GC3' - GC4' GC3' G04' - GC4' Pseudorotation Phase Angle P

-

-

-

NC4' NC1' - N04' NC2' N04' - NC1' NC1' NC3' - NC2' NC2' NC4' - NC3' NC3' N04' - NC4' Pseudorotation Phase Angle P

-

-

NC2' NC3' NC4' N04' NCl'

ENERGY (kcal/mol) Ligand Protein Interaction Total

*

99.

N04' - NC1' - NN1 - NC2 in case ofCpG and UpG.

the binding energy in the substrate mode of binding. Hence Asn98 can be considered as the sub site for the 5'-phosphate group, the 1p site. Spectrophotometric and gel filtration studieson the interaction ofthioguanosine 3',5'-bis phosphate and thioguanosine 2',3'-cyclic phosphate-5'-phosphate with RNase T 1 also indicated the presence of such a 5'-phosphate specific subsite in RNase T 1 (14). The lack of evidence for this sub site in the UV difference spectral studies ofWalz and Terenna (13) can be understood in the light of the present calculations which show that dinucleotides of the type NpG which these authors have used prefer to occupy the G, p1 and N1 sites (corresponding to the inhibitor mode of binding) when bound to

983

Interactions of Ribonuclease T1 TableV Proposed Hydrogen Bonding Scheme in the RNase T 1 - ApG, RNase T 1 - CpG and RNase T 1 - UpG Complexes Inhibitor mode of binding Dinucleotide


GUANINE NIH N2Hl N2H2 06 N7 RIBOSE (Gua) 05' PHOSPHATE 01

02 RIBOSE (A I C I U) 02' 02'H 03' 05'H Ade I Cyt IUra 04 N7

ApG

E46 N98 E46 N44 Y45 N43

N98

OEI 0 OE2 N-H N-H N-H

E46 N98 E46 N44 Y45 N43 N43

OEI 0 OE2 N-H N-H N-H HD21

HD22

R77 H92

HH21 HE2

H92 N98

HE2 ODI

N36

ODI

S72

CpG

Y38 R77 R77 H92

HH HE HH21 HE2

N36

HD22

N36 N98

HD22 ODI

UpG

E46 N98 E46 N44 Y45

OEI 0 OE2 N-H N-H

N43

HD21

N98

HD22

R77 H92

HH21 HE2

H92 N98

HE2 ODI

N36

ODI

S72 G74

HG N-H

HG

RNase T 1 and not the IN, lp and G sites (corresponding to the substrate mode of binding) as assumed by Walz and Terenna for interpreting their results. The existence of such a 5' -phosphate specific subsite constituted by Lys66 has been indicated in RNase A also (5,32).1t can also be seen from Table III that a hydrogen bond between the 5'-nucleoside ribose and Asn99 side chain is present in all the three RNase T 1 NpG complexes in the substrate or productive mode of binding. The present calculations also show that the adenine of ApG has good stacking interaction with the phenyl ring ofTyr45 (Fig. 6a) in the substrate mode ofbinding.lt is interesting to note that when ApG binds to RNase T 1 in the substrate mode of binding, adenine, Tyr45, guanine and Tyr42-Phel00 are placed in such a way that they lead to very good stacking interaction between the aromatic rings (Figure 6a). In fact the guanine and the adenine bases sandwich the phenyl ring ofTyr45 and restrict its movement. These factors contribute significantly to the binding energy. The cytosine and the uracil moieties of CpG and UpG respectively will be positioned in an 'edge to face' orientation with respect to the phenyl ring ofTyr45 when these nucleotides are bound to RNase T 1 in the substrate mode of binding (Figures 6c and 6e). These pyrimidine bases cannot have a 'face to face' stacking interaction with the


984

Balaji and Rao N99

S72

Figyre 6: Stereo diagrams of the active site residues of the enzyme and the inhibitor NpG in the minimum energy conformations of the RNaseT 1 -ApG (a,b), RNaseT 1 -CpG(c,d) and RNaseT 1 - UpG Legend continued on next page

985



S72

JJS72

7JS72

(e,f) complexes. (Protein atoms are connected by thin lines and those ofNpG in thick lines). a, c and e: Substrate mode of binding; viewed down the x-axis. b, d and f: Inhibitor mode ofbinding; viewed down the z-axis.

986

Balaji and Rao Table VI* Conformational angles and rigid body rotation parameters of ApGp, CpGp and UpGp in their complexes with RNase T 1 Dincleotide

ApGp CpGp UpGp ApGp CpGp UpGp Substrate mode

Translational Parameters X y

Inhibitor mode

14.879 14.842 14.889 15.308 15.32 15.291 33.458 33.517 33.403 33.18 33.356 33.222 21.052 2l.ll5 2l.l3l 21.391 21.46 21.433

z Rotational Parameters (degrees)


ct>

e

"'

Conformational Angles (degrees) (N = A or C or U) G04' GCI' GC4 GN9 G02'H GC3' GC2' G02' G03' GC3' GC4' - G03'H GC3' G03' P3 03 G03' 03H P3 03 G05' GC3' GC4' GC5' G05' GC4' GC5' P5 GC5' G05' P5 - N03' NC3' G05' N03' P5 NC3' NC4' N03' P5 NOS' NC5' NC3' NC4' NOS'H NC4' NC5' NOS' N02' N02'H NC3' NC2' NC4 t N04' NCI' NN9

-

-

ENERGY (kcal/mol) Ligand Protein Interaction Total

221.9 147. 56.8

209.8 146.5 45.9

229.9 147.1 64.1

161.7 150.5

133.4 - 49.3 -155.3 81.3 -172.4 -56. -ll8. -163. - 83.9 176.9 -177.6 -179.2 - 74.3 -132.5

122.5 -51.3 -157.3 86.2 -178.8 - 49.7 -175.7 - 62.6 - 79.7 -108.6 -178.3 -178.7 - 84.9 -ll8.6

134.8 - 49.4 -157.7 94.2 68.4 - 52.9 - 82. -176.4 - 83. -161.8 -176.9 -178.2 - 69.8 38.4

- 45.6 - 48.1 - 79.8 - 79.6 165.8 165.8 167.1 166.4 -81.3 - 86.1 -172.5 -172.2 139. ll5.2 -157.1 -108.2 150.8 -167. -104.1 -154.9 57.5 46.9 -163.6 -136. - 42.9 - 64.4 -170.4 33.8

- 46. - 79.7 166.1 167.9 - 80.1 -172. 122.6 -120.5 -169.5 -153.5 43.3 -131.5 - 61.4 -173.3

- 18.4 -317. - 97. -432.4

- 18.2 -317.5 -102.6 -438.3

- 18. -315.7 -104.8 -438.5

-

- 10.7 -325.5 -ll4.4 -450.6

-I.

9.4 -325. -113. -447.4

165.3 152.5 .2

- 16.2 -327.5 -107.3 -451.

162.8 152.4 0.

* Conformational angles correspond to the minimum energy conformers arrived at by energy minimisation in torsion angle space. X, Y and Z are the three translational parameters and cf>, eand 111 are the three rotational parameters. t N04' - NCl' - NNl - NC2 in case of CpGp and UpGp.

phenyl ring ofTyr45 due to the restriction imposed by the 2-oxo group and the ribose pucker on the glycosidic torsion angle. This suggests that the stacking interaction with Tyr45 is possible only with the adenine base implying that Tyr45 constitutes the adenine specific IN site. Such an adenine specific interaction was indicated from kinetic studies also (15). RNase T 1 contains as many as twelve acidic amino acid residues (6 aspartic and 6 glutamic acids) and only five basic amino acid residues (3 histidines, I lysine and I arginine) (33) and consequently is an highly acidic protein carrying a net negative charge at pH 7 where it shows maximum activity (34,35). Of the five basic amino acid residues, three (His40, Arg77 and His92) are clustered in a narrow zone forming the



987

Figure 7: Schematic representation of the subsite interactions in RNase T 1•

pl site. Although Lys41 is close to the primary guanine binding site, it does not interact with either the base or the ribose and chemical modification studies (19) have also suggested that modification of this residue has no effect on the enzyme activity. His27, which is at the C-terminal of the lone a-helix is away from the active site and cannot have any interaction with the substrate. Hence the presence of electrostatic subsites in RNase T 1 for the binding of the negatively charged substrate RNA as in the case of RNase A and Staphylococcal nuclease is highly unlikely. However RN ase T 1 contains four phenyl alanine and nine tyrosine residues. In the light ofthe results obtained from the present calculations, it appears that RN ase T 1 utilises the stacking and hydrophobic interactions between phenyl alanine and tyrosine residues and the nucleotide bases for the tight binding of the substrate RNA

Mechanism of Product Release A trinucleotide substrate of the type NpGpN when bound to RNase T 1 will occupy the lN, lp, G, pl and Nl sites and the phosphodiester bond between the phosphate occupying the pl site and the nucleoside occupying the Nl site will be hydrolysed. After the cleavage, the orientation of the resulting product NpGp will be in the energetically less favoured mode since in the preferred mode of binding the 5'phosphate ofNpGp occupies the p 1 site as discussed earlier. Hence the orientation ofthe product in this mode may facilitate itself to release from the enzyme active site.

988

Balaji and Rao

Conclusions The main conclusions drawn from the present computer modeling studies can be briefly summarised as follows:


1. Molecules of the type pGp,ApG, CpG, UpG,ApGp, CpGp and UpGp can bind to RNase T 1 in essentially two modes: an energetically favourable mode called the inhibitor mode of binding and a weak binding mode or the substrate mode of binding. The inhibitor mode of binding of these nucleotides is similar to the mode of binding of 5'-GMP to the enzyme. The ribose moiety ofpGp favours a C3'-endo pucker form in the energetically favoured mode of binding. 2. The amino acid residues Tyr45 and Asn98 constitute the IN and lp subsites and the IN subsite is specific for adenine and not cytosine or uracil. The residues Ser72, Pro73 and Gly74 constitute the Nl site (Figure 7).

3. Hydrophobic and stacking interactions rather than electrostatic interactions are likely to play a key role in the binding of the substrate RNA to the enzyme RNase T 1.

Acknowledgements The authors thank Prof. Dr. Wolfram Saenger, Institute for Crystallography, Free University Berlin, Germany for providing the atomic coordinates and some of the preprints of ribonuclease T 1• The authors also thank Prof. Girjesh Govil, Tata Institute of Fundamental Research (TIFR), Bombay for very generously allowing them to use the IRIS-4D Silicon graphics workstation of 500 MHz FTNMR facility funded by the Department of Science and Technology, India and located at TIFR, Bombay. References and Footnotes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

J.D. Allen and J.A Thoma, Biochem. J. 159, 105 (1976). J. Krout, Ann. Rev. Biochem. 46, 331 (1977). J.R. Li and F.G. Walz, Jr., Arch. Biochem. Biophys. 161,227 (1974). Y. Mitsui, Y. Urata, K. Torii and M. Irie, Biochim. Biophys. Acta 535, 299 (1978). F. Sawada and M. Irie, J. Biochem. 66, 415 (1969). A McPherson, G. Brayer and R. Morrison, Biophys. J. 49,209 (1986). A McPherson, G. Brayer, M. Cascio and R Williams, Science 232, 765 (1986). A McPherson, G.D. Brayer and R.D. Morrison,J. Mol. Bioi. 189,305 (1986). P. Cuatrecasas, M. Wilchek and AB. Anfinsen, Science 162, 1491 (1968). C.N. Pace, U. Heinemann, U. Hahn and W. Saenger,Angew. Chern. Int. Ed. Engl. 30,343 (1991). P.V. Balaji, W. Saenger and V.S.R. Rao, J. Biomol. Struct. Dyn.9, 215 (1991). J.C. Pinder and W.B. Gratzer, Biochem. 9, 4519 (1970). F.G. Walz, Jr., and B. Terenna, Biochem. 15,2837 (1976). F. Sawada, T. Samejima and M. Saneyoshi, Biochim. Biophys. Acta 299, 596 (1973). H.L. Osterman and F.G. Wa1z, Jr., Biochem. 18, 1984 (1979). M. Zabinsky and F.G. Wa1z, Jr., Arch. Biochem. Biophys. 175, 558 (1976). H. Watanabe, E. Ando, K. Ohgi and M. Irie,J. Biochem. 98, 1239 (1985). F. Inagaki, I. Shimada and T. Miyazawa, Biochem. 24, 1013 (1985). K. Takahashi and S. Moore, Enzymes XV, 433 (1982). R. Ami, U. Heinemann, R. Tokuoka and W. Saenger,J. Bioi. Chern. 263, 15358 (1988). E.N. Baker and R.E. Hubbard, Prog. Biophys. Mol. Bioi. 44,97 (1984).


Interactions of Ribonuclease T1 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

R. Taylor and 0. Kennard.J Mol. Str 78, 1 (1982). W. Saenger, In Principles of nucleic acid structure, Springer Verlag, Berlin (1984). C. Arnott and D.W.L. Hukins,Biochem. J 130,453-465 (1972). P.V. Balaji, W. Saenger and V.S.R. Rao, Biopolymers 30, 257 (1990). P.V. Balaji, W. Saenger and V.S.R Rao, Cu". Sci. 60,363 (1991). C. Altona and M. Sundaralingam,J Am. Chern. Sci. 94,8205 (1972). S. Sugio, T. Amisaki, H. Ohishi and K Tomita,J Biochem. 103,354 (1988). K Takahashi,J Biochem. 72, 1469 (1972). I. Shimada and F. lnagaki,Biochem. 29,757 (1990). Prof. W. Saenger (private communication). R.de Llorens, C. Arus, X Pares and C.M. Cuchillo, Prot. Engg. 2, 417 (1989). K Takahashi, J Biochem. 98, 815 (1985). K Sato and F. Egami,J. Biochem. 44,753 (1957). M. Irie,J Biochem. 56, 495 (1964).

Date Received: September 24,1991

Communicated by the Editor M. Sundaralingam

989

Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes.

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Computer modelling studies on the mechanism of action of ribonuclease T1.

CD studies on ribonuclease A - oligonucleotides interactions.

Time-resolved fluorescence studies of ribonuclease T1 in reversed micelles.

Proton and phosphorus nuclear magnetic resonance studies of ribonuclease T1.

Subsite mapping of enzymes. Depolymerase computer modelling.

Effect of ribonuclease T1 on ribosomal subunits of rat liver.

The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

Improving purification of recombinant ribonuclease T1.

The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

Ribose recognition by ribonuclease T1: difference spectral binding studies with guanosine and deoxyguanosine.

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1.

Three-dimensional structure for the beta 2 adrenergic receptor protein based on computer modeling studies.

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Studies on the specificity of ribonuclease from Rhizopus sp.

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

Kinetic studies on the cyclization of T7 and T1 DNA's.

Methylated nucleotide sequences in HeLa-cell ribosomal ribonucleic acid. Correlation between the results from 'fingerprinting' hydrolysates obtained by digestion with T1 ribonuclease and with T1 plus pancreatic ribonuclease.

Computer modeling of polysaccharide-polysaccharide interactions: an approach to the kappa-carrageenan-mannan case.