J. Mol. Biol. (1992)

225, 533-542

RNase T1 Mutant Glu46Gln Binds the Inhibitors and 2’AMP at the 3’ Subsite

2’GMP

Joachim Granzin, Ramon Puras-Lutzke, Olfert Landt, Hans-Peter Grunert Udo Heinemann, Wolfram Saenger and Ulrich Hahn? fiir Kristallographie, Fachbereich Chemie Freie liniversitcit Berlin, TakustraJ3e 6 W-1000 Berlin 33, Federal Republic of Gwmany

Institut

(Received 25 October 1991; accepted 30 .Janua,ry 1992) On the basis of molecular dynamics and free-energy perturbation approaches, the GluCBGln (E46Q) mutation in the guanine-specific ribonuclease T, (RNase T,) was predicted to render t,he enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the struct,ures of its two complexes with 2’AMP and 2’GMP. The ribonuclease E46Q mutant is nearly inac+tive towards dinucleoside phosphat)e substrates but shows 17% residual activity towards RNA. Tt binds 2’AMP and 2’GMl’ equally well with dissociation constants of 49 jLM and 37 PM. in contrast to the wild-t?pe enzyme, which strongly discriminates between these two nucleotides, yielding dissociat#mn caonstants of 36pM and 0.6/~. These data suggest tha,t the E46Q mutant binds the nucleotides not’ to the specific recognition site but t,o t’he subsite at His92. This was caonfirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the PhelOO N and 0 atoms, and t,ightly anchored in this position. This interaction may either have locked the guanine recognition site so that B’AMP and B’GMP are unable t.o insert. or t,he contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable t,o function in recognition of eithrr guanine and adenine. h’~yu~r&:

crystal

structure;

molecular

dynamics:

1. Introduction Ribonuclease T, (RNase T,f; EC 3.1.27.3) from Ap~yillus oryznp consists of a single polypeptide chain of 104 amino acid residues, has a relative molecular weight of 11,085, and is one of the beststudied enzymes (Heinemann & Hahn, 1989). Much is known about) its high specificity for guanosine and the hydrolytic action towards RNA, which produces guanosineoligonucleotides with terminal 3’-phosphate groups. The crystal structures of RNase T, complexed with its most potent and specific inhibitors guanylyl-2’,5’-guanosine t Author to whom correspondence should br addressed. 1 Abbreviations used: RNase T, ribonuclease T,: Ade. adenine: 2’.5’GpG. guanylyl-2’.5’-guanosine; B’GMP, 2’-guan?lic acid; 2’AMP. X’-adenylic acid; Ap(‘, adenylyl3’,5’-cytldine: n.m.r.. nuclear magnetic resonance: Gua, guanine: wt, wild-type: E46Q, Glu46Gln; E46Q-2’AMP or E46Qp2’GMP. complexes of RNase TI mutant E46Q with 2’AMP or 2’GMP; GpC, guanylyl-3’,5’-cytidine: (Zuo. guanosine: 3’.5’,pGp. guanosine-3’.5’-bisphosphate.

RNasr

T,: site-direct’ed

mutagenesis

(2’,5’GpG: Koepke et al., 1989) and 2’-guanylic acid (B’GMP: Heinemann & Saenger, 19X2; Arni et al., 1988), with the weakly binding 2’-adenplic acid (2’AMP: Ding et al., 1991), and of the uncomplexed enzyme (Martinez-Oyanedel et al., 1991) have been determined at high resolution. Nuclear magnetic resonance (n.m.r.) spectroscopy showed t,hat the solution structure is identical with that found in the crystals (Hoffmann & Riiterjans, 1988; Schmidt rt al.. 1991). The reasons for the high specificity of RSase T, for guanosine residues are hydrogen-bonding interactions between RNase T, main-chain a,nd sidrchain groups and guanine (Gua). that is. Asn43VH . (iuaX7, Asn44NH GuaOti. Asn45NH . GuaO6, GuaNlH O”‘Glul6. GuaS2Hl W2Glu46, GuaN2Hd OAsn98. supported by sandwich-type stacking of guanine residues between the side-chains of Tyr42 and Tyr45. Attempts to produce mutants of RNase T, with altered specificity have thus far proved futile, except for a report where a mutant showed some

.------.__ higher rat,io of adenine-guanine specificity than the wiltl-type (nt) RNase T, (Ikehara ct (11.. 1985). On the basis of recent t,heoretical molecular dynamics a.nd free-energy pert,urbation calculations, it was suggested that t,he mut>ant Glu46Gln might “have a larger affinity for adenine than guanine” (Hirono & Kollman. 1991). To test this suggestion experimerltally. the K.Xase T, mutant E46Q was prepared by genetic engineering and characterized both biochrrnic~ally t.his study

and c~rystallographically: t’he results are described in this paper.

2. Materials

of

and Methods

Xucleotides and enzyme substrates were from Sigma (Deisenhofen) 01 Pharma-Waldhof (Diisseldorf). Enzymes for cloning work were from Gibco BRL (Berlin). Amersham Ruchler (Braunschweig) or New England Biolabs (Schwalbach). Oligonucleotides were from TIB MOLBIOL (Berlin). All other chemicals were from standard commercial sources. (b)

Site-dirrcted

mutagrnrsis

and

protein,

pwi,fication

The REase T, mutant E46Q was constructed by sitedirected mutagenesis using the polymerase chain reaction (Landt et al.. 1990). The mutagenesis primer was 5’.CAA ATCAAAACCTTGGTAGT-3’ (base substitution underlined) and the standard primers have been AZVO (5’-TAC GGATTCACTGGAACTC-3’) and A2HI (5’Y:ATCTTAGC AGCCTGAAC-3’). Rh’ase T, and the mutant E46Q were both isolated according to the procedure described by Landt’ et al. (1991) from an Escherichia coli overproducing st,rain harbouring pA2Tl (Quaas et al., 1988a.b) or its df.rivatire.

(i) Uinucleotide hydrolysis The hyperchromic effect due to the cleavage by transesterificabion of guanylyl-3’.5’-cytidine (GpC) was monitored at 280 nm to determine the initial velocity (Zabinski

4 (PM)

Enzyme A. RXA

RNase E46Q B. GpC’

RSase E46Q

parameters

~zna, (w/min)

of wt RNase

(m?‘)

RSA hydrolysis was measured following the initial absorbance tlec~reasr at 29X.5 nm in 150 mM-Tris. H(‘I (pH 7.5). 2 rnM-EDTA at 20°C (Oshima et al.. 1976). The absorbanrt~ of the substrate was =1,,,., = 19, and thts enzyme conc>entrations varied between O+i and 31:? phi (Table 1).

Dissoriation constants (Kd) for t,hr binding of d’(:MP or %‘AMP to w-t KXasr T, or the mutant, E46Q were det,ermined by fluorescence-quenching titration curve’s (Kell? pt a,Z.. 1976). The measurements were performed in 3 ml stirring quartz-cuvettes with a path length of 10 mm using a Shimadzu RF-5000 speotrofluorophotometer. Inhibitor (final concarntrations between 0.2 x 1W6 and 10 4 M) was added to lW6 M-protein solutions. Fluorescenc*e was monitored after an equilibration period of 3 min. The excitation wavelength was 295 nm and the fluorrscrncar was tieasurrd at the emission maximum (328 nm). The reaction buffer was 100 mar-sodium acetate. pH 5.6 (the optimum pH for inhibition). thermostated at %O”(“. I)ata were fitted by non-linear regression (LeathrrharroH. 1987). (P) ~‘rystallizntion~

(f th.r

snzpm

inhibitor

T, and the mutant

Wh’m

(min--‘.W

I)

‘I’, (wt)

Specific activity (AAmx/

E46Q

Relative activity

min,mg)

(“b of wt value)

582 I@0

100 17

hydrolysis$

188 116

93 6.5

18,532 19.8

cwr~pl~srs

The recombinant’ RlYase T, mutant E46Q was cocr,vstallizrd with d’GMP and I’AMP by rniwodialvsis

hydrolysis?

T, (wt)

.~~-.-

Kr Wale. 1976: (irunert rt CL/.. 1991) at 12 differenr I,clnc~rntrat,iorls IwtWPt~n 04i x 10 i1 allI substrate 3.2 x lW4 M as tripli~atc~s. The enzyme c~onl~rnt rat ions spectrophotomrtrically using t>htt were dfatermined absorption coefficient 17.300 M- I per csm for Kh’ascJ T, ai 278 nm (Cieorgalis et nl.. 1991): thr final concaentrations for E46Q and nt RXasr T, were 3.3 x IO-’ and 5 x 10-” 31. respectively. Xll kinet)icL experiments were performetl with a Shimadzu I‘\‘- I60 A spec,trophotomrter in Tris but&r (20 mM-Tris, H(‘I. pH 7.5. 2 rnM-ET)TA) thermostated at 20°C in quartz cuvrttes with a path length of 10 mm. or L’ mm for higher sllbstratci c~otlc~t~ntr’;~tic,rrs.Kinetic, lbar;i~ meters. giv(kn in Table 1. \vCrtl clvaluat,ed Ilsitlg tht> program ESZFITTEK (Leatherbarrow. 19X7)

Table 1 Kinetic

..-

9.86 x 10’ 1.7 x 105

1CM) @2

7 Measured according to the method of Oshima et al. (1976) by monitoring the absorbance increase at 298.5 nm in 150 mM-Tris’ HCl (pH 7.5), 2 mM-EDTA at 20°C. Yeast RNA (1.9 A,,,., Unit) was used in the presence of enzyme concentrations between @6 and 315 PM. $ Michaelis-Menten kinetics were measured by monitoring the hyperchromicity at 280 nm caused by the decrease of stacking interactions on dinucleoside phosphate hydrolysis (Zabinski & Walz, 1976). Standard assay conditions were: 20 mwTris.HCl (pH 7.5), 2 miwEDTA; 20°C; between @6 x 10m6 and 3.2 x 1O-4 x-substrate. Enzyme concentrations varied between 5 to 330 niw. Kinetic constants were determined using the program ENZFITTER (Leatherbarrow. 1987).

Inhibitor

Binding

qf RNaae

according to the procedure of Heinemann et al. (1980). Solutions containing 1 mM-E46Q and (20PI) 13.5 mM-2’GMP. or 1.3 rnM-E46Q and 14.5 mM-2’AMP were dialyzed against 5 ml reservoirs containing 507, (v/v) ‘t-methyl-2,4-pentanediol, 1 mM-Z’GMP or 2’AMP in 20 mivl-sodium acetate, 2 mM-calcium acetate buffer (pH 4.2). The dialysis membrane used (SpectraiPor 3) had a relative molecular-weight cutoff of 3500. The crystals of the 2 mutant-inhibitor complexes are prismatic. Both belong to orthorhombic space group P2,2,2, with the lattice parameters a = 49,16(l) A. b = 46.76( 1) A. c = 41.09( 1) A for the complex of the mutant enzyme with 2’(:MP (E46Q2’GMP) and a = 49,10(l) ,A. h = 46.69(2) A, c = 41,11(l) A for the complex of the mutant tnzymr with 2’AMI’ ( EltiQ-YAMP) (I A = 0.1 nm): standard deviations of the last digit are given in parentheses. Crystals grew to a size of 046 mm x 0.16 mm x 0.16 mm (E46&-2’GMP) and 0.44 mm x 0.4 mm x 0.56 mm (E46Q- Y.AMP). (f) X-ray

data

collection

and

(g)

Initial

model

and

group, and density for bases and phosphate groups of the inhibitors in the subsite close to His92. The final stage of refinement’ included for E46&-2’GMP all non-hydrogen atoms of the protein molecule except Asn98(N6). and 81 water molecules. For E46&-2’AMP. no electron densities were present for Lys25(c”. NC), IA~s41(Cd, C”, NV). Asn83(CY, O’, Nd) and t’he .4sn98 sidechain: for the side-chain of Gln85. electron density around 8X? was interpreted as disordered and given half OCCUpancy; 85 water molecules were located. The S’GMP ribose is poorly defined as indicated by high temperature factors of C-3’. C-4’. C-5’. The final R factors calculated for all reflections were R = 0.148 for E46Q-2’GMP and R = 6142 for E46&-2’AMP in the above specified resolution range and a-cutoff. The mean errors in atomic positions were estimated by the method of Luzzati (1952) t,o be (1.15 (0.14) A%.

3. Results and Discussion (a) Enzyme

processirry

The 2 X-ray diffraction data sets were recorded with a Delft Instruments FAST television area detector using graphite monochromatized (‘UK, radiation of a Delft Instruments FR 571 generator wit’h rotating anode at a setting of 45 kV and 65 mA with a focal spot, size of 0.2 mm x 2.0 mm. The crvstals were mounted in an arbitrary orientation on the diffractometer cradle (K geometry, CADP). The orientation and the lattice parameters were determined from data c~ollectrd with small scans at 2 different o angles using the program MADXES (Messerschmidt, B Pflugrath, 1987). Data of each compound were collected in a single detector setting with a defined misset in h’ between goniometer and crystallographic co-ordinate system to record data in possible blind regions. The total w range in both cases was 95” in steps of 0.1” per frame. Non-uniformity caorrection was performed pixel by pixel and dark current, was integrated in the total background correction. Data processing was done using t)he program MADKES by evaluation of the diffraction spots in boxes of 15 x 15 (E46Q- S’GMP) and 21 x 21 (E46&-2’8MP) pixels. The resulting integrated LP-corrected intensities were fitt’ed by profile analysis according to the method of Kabsch (1988). The final dat,a sets showed an overall El,,,,, of 3.07; for (E46&-2’GMP) (3.69; (E46Q-2’AMP)) in F2 for 11,009 (( E46&-2’GMP) (11,554 (E46&-2’AMP)) different reflect’ions and cont,ained 5721 (6822) unique reflections between IO and 2.0 (187) A corresponding to an overall completeness of 85”,, (83.3’?,,,) for data with I”, 2 la(FO), rejhement

structure refinements for both complexes (ET&--2’CMP and E46&-2’AMP) were performed with st,ereochemically restrained least-squares methods using the refinement package TNT (Tronrud et al.. 1987). The structure of uncomplexed RXase T, (MartinezOyanrdel et al.. 1991) served as starting model: the isomorphous difference between the diffraction data of RNase T, and E46Q-2’GMP and E46&-2’AlMP was 229b after anisotropic scaling. The refinements were combined with computer graphics assisted manual-model revisions (FRODO: Jones. 1978). After several rounds of refinement, the difference> electron density clearly showed the (la’+ position coordinated to the Asp1 5 cxarboxylatr

53.5

T, Glu4601n

kinetics

Kinetic data of wt RNase T, and of the E46Q mutant are listed in Table 1. Wit-h GpC as substrate, the E46Q mutant is nearly inactive, as shown by the k,,JK,,, rat,ios. These values are comparable wit,h the data reported by Steyaert et al. (1991). If, however. the biological substrate RNA is used, the mutant still exhibits 17% activity relative to the wb enzyme. Using ApC as substrate, no activity ( specificity. (c)

Prystal

structure

analysis

The final atomic parameters and structure amplitudes are deposited with the Brookhaven Protein Data Bank (accession numbers: IRGL: IRGLSF).

Table 2 Binding

KNase KNasr E46Q E46Q

parameters

T, T,

of 2’GMP

I’GMP YAMP 4’GMP Z’AMP

and 2’AMP

0.6 362 37. I 491

to wt RNase

T, nnd the E46Q

-8.35 -5% - 594 -5.78

t AG was calculated f’rom the dissociation (7’ = 293.15 K: I cal = 4184 .I).

constant

mutant

2~31) 0, I6 (&)

using

the equation:

AC = - RT In( I//C,):

Table 3 Statistics

of r$nement procedures

in the E46&-2’GMP (amp) complexes

Bond length deviations (A) Bond annle deviations (“1 Torsion lngle deviations’(“)? Trigonal atom non-planarity Planar groups (A) Chiral centers

(A)

and E46Q-2’AMP

Average deviation f’rom standard value

r.111.s. gw

(gmp)

amp

O-010 0409 2.033 1.784 25.422 24850 0003 0403 0309 0008 None has the wrong hand

km 0043 9.35 59.0 (kOO.5 o-0”

A

amp wo27 7.38 62.88 0004 041 !l

t So restraints

A summary of refinement and weighting details is given in Table 3. For the analysis of hydrogen-bonding interactions, the positions of hydrogen atoms covalently bonded to the sp2-hybridized nitrogen atoms were calculated using the program MOLEDT (BIOSYM, 1988) for the amino acids, for guanine and adenine. Cut-off values for possible hydrogen-bond interactions were: donor-acceptor (D-A) distance 5 3.5 A, hydrogen-acceptor distance 5 2.9 A. and angles D-H . A 2 90”. Since E46&-2’GMP and E46&-2’AMP are isomorphous to native RNase T, (root-mean-square (r.m.s.) deviations of the backbone atoms are 62 or 0.3 A for the wt RNase T,-2’GMP and wt RNase T,-B’AMP complexes, respectively), no significant conformational changes for main-chain atoms are to be expected. In consequence, the overall architecture is similar to uncomplexed RNase T,. In contrast to most of the other RNase T, structures with “empty” guanine recognition sites, the side-chain of Va178 is not disordered (MartinezOyandedel et al., 1991; Ding et aE., 1991). (d) Binding

of 2’GMP

and 2’AMP

In both complexes with RNase T, E46Q, the nucleotides are not bound to the specific guanine recognition site. They are located in the subsite previously identified in the complexes between wt RNase T, and (Guo), (Lenz et aE., 1991), 2’,5’GpG (Koepke et aE., 1989), 2’AMP (Ding et al., lQQl), and

in the mutant Tyr45Trp complexed with 2’AMP (Koellner et al., 1991). The possible hydrogen bonding contacts between E46Q and 2’AMP, 2’GMP are given in Table 4. This subsite is occupied by the nucleotide N in a substrate GpN and serves to orient the scissilephosphodiester bond (Lenz et aZ., 1991). It is formed by the imidazole group of His92, on which the base of N is stacked. In the present two complexes of E46Q with 2’AMP and S’GMP, this stacking is supported by hydrogen bonds formed for adenine between Gly740 and AdeN6H, and Arg77Nq2 and AdeN7, and for guanine between Gly740 and GuoN2H. The adenine group of B’AMP is in the same position and forms the same hydrogen-bonding interactions as in the complex with wt RNase T, (Ding et al., 1991). The hydrogen bonds formed between E46Q and 2’GMP are different, not only becausethe functional groups of guanine differ, but also because the nucleotides are in different conformations (Table 5). They adopt their preferred orientations, anti for B’AMP and syn (with the base rotated by about 180”) for 2’GMP (Saenger, 1984), as shown in Figure l(a) and (b). In both nucleotides, the ribose moiety adopts the C-2’-endo form with maximum puckering amplitude z,,, (Table 5) as known from small molecule crystal structures (Altona & Sundaralingam, 1972). This conformation is stabilized in E46&-2’AMP by intracomplex hydrogen bonding O-5’ . Asn360d/Nd (Table 4), and in both complexes by hydrogen bonding between ribose O-5’ and Gl~1020”~ of a symmetry-related

I.9 2.0 2.2 2.4 2.4

I 54 146 140 126 92

95

t 1,2+r.

l/-“--f/,

--z.

intermolecular molecule, with an additional O-5’ NAsp49 contact in E46&-2’GMP. The phosphak groups are anchored by hydrogen bonding to the catalytically important amino acid side-chains of Tyr38: His46, Glu58, ,4rg77 and His92 as in the complexes of B’AMP with wt RNase T, (Ding et al., 1991) and with the mutant RNase T, Tyr45Trp (Koellner et al.. 1991). At the pH of crystallization. pH 4.2. the phosphat,e is mono prot,onated. The proton resides probably on 03P, which is in hydrogen-bonding contacts with both Glu58 carboxylatr oxygen atoms; since the latt,er are less acidic than a phosphate group. we cannot exclude that. in fact. the proton is shifted more towards or bound to the carboxylate group, the phosphate now caarrying two negative charges.

(e)

Architecture

of catalytic

binding The

and

guanine

sites

geometry of the catalytic site is similar to described previously for the complex between wt RSase T, and 2’AMP (Ding rt al.. 1991). This holds also for the hydrogen-bonding scheme between the Y-phosphate groups of 2’GMP and B’AMP, and for the water molerules L%:at181 (E46Q-2’GMP) and Wat 192 (E46QYAMP. Table 4). which have their correspondence in the wt RNase T, complex with X’AMP (1)ing et ccl.. 1991). The puaninr-recognition site. however. shows some differences with respect, to the “empty” wt R,Nase TI. The segment of the polypept’ide chain formed by amino acid residues 42 to 46 adopts that

Table 5 Torsion E&Qp%‘C/M

anylrs of the ribow group in fhP P (qmp) and E&Q-%‘A MI’ (amp) complexrs His

**poo osBf 3 Asp49 .C

...a*

His40

nearly the same conformation in E46Q as in wt RN&se T,. The side-chain of Tyr4.5 is held in position by two hydrogen bonds, Tyr45O” . N’Asn99. and Tyr450V. . . Wat . . . OAsn98 (Table 6). The conformations of the Glu/Gln46 side-chains are

different in the wt and mutant. enzymes and stabilized by hydrogen bonding to the PhelOO accept,sa hydropeptide. In wt’ RNase T,, Glu460”’ gen bond from PhelOONH, and 61~460”~ point’s “away” from the guanine-recognit,ion site and hydrogen bonds to two water molecules (Fig. 2(a)). Tn E46Q. Gln46 is anchored in its position by tight hydrogen bonding between the amide and the PhelOO peptide groups, Gln46N”H . OPhrlOO and Gln46O” HNPhelOO (Table 6). The guanine-binding site is tilled by four water molecules in wt RNase T, (Fig. 2(a)). Two of these water molecules. bridging Asn43 peptide NH and 0 with Tyr450q a,nd Asn980, Watl40 and Wat’l94. have their equivalents in E46&-2’GMP (Wat 145. Wat179: see Fig. 2(b)), and in E46&-P’AMI’ (Wat121, Watl55: see Fig. 2(c)). One of the two remaining water molecules in wt RNase T,. M’at 129 in Figure 2(a), is moved “up” towards the mainchain segment Asn44-Tyr45 in E46Q (Fig. 2(b) and (c)), and the position of the other Watl65 in wt RNase T, is pushed further into the binding site in E46Q-2’GMP (Fig. 2(b)): in the 2’AMPt;;;plex n in these movements are even wider E46&-Z’GMP, so that a third water molecule. W’at140, can be accommodated (Fig. 2(c)). (f) Conclusions The result’sof the present study are surprising and remarkable, and differ largely from t.he theoretical predictions. There is ample reason why this is so.

Figure 1. Section of the crystal structures of complexes bet,ween E46Q and (a) 2’GMP and (b) Z’AMP. Possible hydrogen bonds formed between the S/-phosphate groups and the catalytic sites, and those formed between the nucleoside moieties (which are in different conformations, syn. guanosine and alzti adenosine) and the Y’-subsite in RNase T, are shown. The latter involves stacking of the guanine and adenine moieties, respectively. on the imidasole group of His92. Water molecules are shown as filled spheres, hydrogen bonds are indicated by broken lines. and residues of symmetry-related molecules by dotted lines. Drawn using SCHAKAL (Keller. 1988).

The crystallographic and biochemical data are in full agreement. The dissociation constants (Table 2) indicate that binding of 2’C:MP t,o wt R,Nase T, is strong

because

guaninr

is locat,ed

in the

specific>

recognition site: as shown bv the corresponding LX-ray studies (Heinemann & Saenger, 1982; Arni et al., 1988). The

binding

of 2’AMP

to wt RNase

T, is

much weaker because, as shown by another X-ray analysis. this nucleotide does not occupy t,he

Inhibitor

Binding

of RNase

T, Blu4M~ln

X39

Asns9

Figure 2. Comparison of guanine-recognition sites in (a) wt RNase T,, and in E46Q complexed with (b) P’GMP and (c) 2’AMP. The side-chain orientations of Glu46 and Gln46 (drawn with filled lines) are different in wt and mutant RNase T,, but the side-chain of Tyr45 retains its position, stabilized by hydrogen bonds to the side-chain of Asn99 and to 2 water molecules bonded to Asn43N, 0 (not valid for 2’AMP as there is no hydrogen bond between Watl21 and Wat155, which are separated by 42 A). The positions of the other water molecules in the guanine-binding site vary. and their number increases from 2 in wt RNase T’~ and E46&-Z’GMP to 3 in E46&-2’AMP.

guanine recognition site. but is located in the subsitr of the enzyme (Ding et nl.. 1991). As similar dissociation constants were also observed wit’h YAMP and 2’GMP binding to the mutant E46&, a comparable subsite binding was suggested, and is indeed proven by the two crystal structures described in this study. This behaviour is also paralleled by the enzyme kinetic studies (Table 1). There is an apparent discrepancy in the relative activity of wt and mutant enzymes towards the dinucleoside-phosphate Gp(’ and the polynucleotide RNA. It can be explained by tighter binding of the latter to RNase binding t’o Tl. which we associate with phosphat’e positive charges of His27 and L,ys41 at the periphery of IZNase T,. The guaninr-binding site adopts essentially the same geometry as in “empty” RNase T,, with no guanine complexed. The only, and important, difference is the tight hydrogen-bonding of Gln46

amide to the PhelOO peptide groups. It, appears that this interaction is so strong that it does not permit the sit’e to open and to accommodat’e guanine or adenine, i.e. t#he induced-fit like conformational changes of the recognition site (Kostrewa et al., 1989). in particular the flip of a peptide group that forces t’he cp, $ torsion angles of Asn44 in the energetically less favourable left-handed helix conformation, cannot occur. Or. w&h a different viewpoint, one could argue that the specific hydrogen bonding Glu46 . guanine is so crucial for guanine binding that substit,ution for Gln46 abolishes the ability of RXase ‘I’, to properly recaognize and bind its substrate. For wt RNase T,, the simulations of Hirono 8r Kollman (1990, 1991) yielded a value for AAC&, with B’GMP and 2’AMP of +3.14 kcaljmol. which reproduces closely the experimental value of + 3.07 kcal/mol of Campbell & Ts’o (1971), and both are in reasonable agreement with t’he value deter-

Table 6 Possible

hydrogen

E46&-Z’GMP

bonding (gmp)

I) Residue Tyr42

Asn43 Am43

&XI43 Am44

Am44 Tyr45 Tyr45

Qln46

Oln46

C&46

residue

Relevant

water

molecules:

in, the guaninP-recogn,ltion (amp)

E46Q-2’AMP

.A@,

sitr

I) H amp

WI’

I I/ fh
he complex with two guanosines. and model building of the c~omplex with the substrate (+I)(;. ,I. liiol. C”hewL. 266, 7661 -7667. Luzzat i. \‘. (1952). Traitement statistiqur drs erreurs dans la tlrt)ermination drs stru(‘tures cristallines. Acta C’rystrrllogr. 5. 802-810. ~lart,in~~z-Ovartrtlel. J.. Choe. H.-\V.. Hcinrmann. I’. B Sarnger. \V. (1991). Ribonuclease Tl nith free rrcognition and caat,alytic site: crvst,aI structure analysis at I.5 .A resolution. .J. Mo1. Rio/. 222. X35-352.

_-----.------.Messerschmidt, A. & Pflugrath. J. W. (1987). (Irystal orientation and X-ray pattern prediction routines for macroarea-detector diffractometer systems in molecular crystallography. ,I. Appl. (‘rystalloyr. 20. 306-315. Oshima. T.. Uenishi. N. & Imahori. K. (1976). Simple assay methods for ribonuclease Tl . T2. and nuclease

1’1. Anal. Biochem. 71, 632-634. Quaas,

R.. McKeown. Y., Stanssens. I’.. Frank. R., Bliicker. H. & Hahn, U. (1988a). Expression of the chemically synthesized gene for ribonuclease Tl in Escherichia coli using a secretion cloning vector. Eur.

J. Biochem. 173, 617-622. Quaas,

R., Grunert, H.-P.. Kimura, M. & Hahn, LJ. (19883). Expression of ribonuclease Tl in Escherichia coli and rapid purification of the enzyme. Nucleosides LVucleotides, 7. 619-623. Saenger. W;. (1984). Principles of AVucleic Acid Structures. Springer-Verlag, Sew Pork. Berlin. Heidelberg. Tokyo.

Edited

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Schmidt. J. 11.. Thiiring, H., Werner. A.. Riiterjalrs. H.. Quaas. R. & Hahn. C. (1991). l’u,o-dimt?nsionaI ‘H. “S-XMR investigation of uniformly ’ S?j-labeleci ribonuclease Tl : complete assignment of “N WSOll anc:rs. k:?(r. .I. Biochcm. 197. 643S653. Steyaert. ,I.. Opsomer. C’.. \Vyns. I,. h Sta,nsxens. P (1991). Quantitative analysis of the contribution ot (:I~46 and Asn98 to the guanosine specificity of ribonuclrasr Tl. Biochemistry. 30, 4944499. Tronrud. I>. lC(:., Ten Eyck. I,. F. & Matthews, I