100
Biochimica et Biophysica Acta, 580 ( 1 9 7 9 ) 1 0 0 - - 1 0 7 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 3 8 2 5 9
1H NMR STUDY ON THE INTERACTION OF CUPRIC ION WITH RIBONUCLEASE A
L U I G I S P O R T E L L I a a n d V I N C E N Z A VITI b,*
a Laboratorio delle Radiazioni and b Laboratorio di Biologia Cellulare ed Immunologia, Istituto Superiore di Sanitd, Viale Regina Elena 299, Roma (Italy) (Received J a n u a r y 2 5 t h , 1 9 7 9 )
Key words: Cu 2+; RNAase A; Histidine; IH-NMR; (Interaction)
Summary The reassignment of the 1H NMR C-2 histidine signals of the bovine pancreactic ribonuclease A has required a revision of the 1H NMR data on the role of the different histidines in their interaction with the Cu 2+. The results of our measurements carried out at p2H 5.5 and 7.0 reduce the importance o f His-12 as main site of interaction. At p2H 5.5 a very strong binding site involves His119, while a weaker one contains certainly His-105. On the contrary, at p2H 7.0 the histidines 105 and 119 seem to possess binding constants of the same order of magnitude and in addition they provide stronger ligands for the Cu 2+ than His-12. The comparison with X-ray data in the crystal shows numerous analogies. Finally, preliminary results on the competitive inhibition effects between the Cu 2+ and 2',3'-cytidine monophosphoric acid are discussed.
Introduction Although the interaction between Cu 2+ and RNAase A has been extensively investigated, some discrepancies are still present in the literature a b o u t the binding sites of the metal ion to the enzyme. From the main results reported on solution studies (for a review see for example Ref. 1) it appears that the Cu 2+ mainly interacts with the histidine residues of the protein. The first C-2 histidine resonance assignment of the 1H NMR spectra of the RNAase A was also used to identify the order of interaction of these residues with the Cu 2+
* T o w h o m c o r r e s p o n d e n c e should be addressed. Abbreviations: RNAase A, bovine pancreatic ribonuclease A; 2',3'-CMP, 2',3'-cytidine monophosphoric acid.
101 [2--4]. A few years ago a new assignment of the histidine peaks was independently made by different groups [6--10]. It was found that the assignment of the C-2 His-119 and His-12 resonances, in phosphate-free solutions and with pure ~vater as a solvent, is reversed with respect to the previous one [2]. In this communication we report our findings of 1H NMR studies on the effects of Cu 2+ on the different histidines of RNAase A at the p2H values 5.5 and 7.0 starting from the new assignment. Preliminary results a b o u t the interaction between the binary system RNAase A/Cu 2+ and 2',3'-CMP are also reported with the aim of a better understanding of the competitivity between the Cu 2+ and the inhibitor in the active site. Materials and Methods RNAase A and 2',3'-CMP were purchased from Sigma and made phosphate free b y chromatography on a Sephadex G-25 fine column (Pharmacia) using 0.1 M ammonium carbonate (pH 8.2) as eluent. Then, the lyophylized RNAase A and nucleotide inhibitor were applied to a column of Chelex 100 ion-exchange resin 200--400 mesh (BioRad) at room temperature. Cu(NO3)2 • 3H20 obtained from Merck, was dissolved in 2H:O (99.7%) and lyophylized twice. Solutions of 2.52 • 10 -3 M o f lyophylized RNAase A were prepared in 0.3 M NaC1 in 2H:O (99.9%). Before the 1H NMR run the samples were heated in a water bath for 5 min at 65 ° C in order to exchange the NH resonances. All binary systems (RNAase A/Cu :÷) were obtained b y adding aliquot amounts of a Cu:+-concentrated solution to the RNAase A solution. The Cu :+ concentration was ranging from 3 . 1 0 -s to 1 - 1 0 -4 M. The ternary systems were also prepared by adding aliquot amounts of a 2',3'-CMP-concentrated solution to the binary systems. The p:H value of the solution was measured just before each 1H NMR run with a Radiometer PHM 63 digital pH meter. The p2H was adjusted to 5.5 and 7.0 with 1 M :HCI or NaO2H (Stohler). The p:H values indicated are pH meter readings in 2H:O without correction for isotope effect. Fourier transform ~H NMR spectra were recorded at 34°C with a Varian XL-100-15 FT spectrometer, interfaced with a Varian 620/L-100 computer equipped with a 1.24 • 106 word disk accessory. In order to minimize the problems arising from the limited dynamic range o f the computer, the spectra were obtained under double precision conditions. Chemical shifts (in ppm) were evaluated with respect to DSS in 2H:O. A D u p o n t 310 Curve Resolver was used to analyse overlapping histidine resonances in the 1H NMR spectra. Results
The interaction of RNAase A with Cu :÷ has been studied in aqueous solutions o f different Cu 2÷ concentrations up to [Cu2÷]/RNAase A] molar ratio 1 : 25. The very downfield region o f the 1H NMR spectra of RNAase A under the effect of increasing Cu 2÷ concentrations is shown in Fig. 1A and B at p2H 5.5 and 7.0, respectively. In Fig. 1A (a) we can observe the four well-resolved C-2 histidine peaks
102
C-2 His
A
C-2 His ' r"T-r"--'q
f
C-4HJ Iosl
A J
b
I 9
i [ 8.5 8 ppm from DSS
I Z5
t
8.5
I
l
8 7.5 ppm from DSS
Fig. 1. F o u r i e r t r a n s f o r m 1H N M R s p e c t r a f r o m 7.5 t o 9.0 p p m o f 2 . 5 2 • 10 -3 M R N A a s e A in 2 H 2 0 , 0 . 3 M NaCI, at 3 4 ° C , in t h e p r e s e n c e of d i f f e r e n t Cu 2+ c o n c e n t r a t i o n s . (A) M e a s u r e m e n t s a t p 2 H 5.5. Mola~ r a t i o s [ C u ~ ' ] / [ R N A a s e A ] : a, 0; b , 1 : 84; c, 1 : 63; d, 1 : 4 2 , a n d e, 1 : 25. (B) M e a s u r e m e n t s a t p 2 H 7.0 w i t h m o l a r r a t i o s as in ( A ) .
assigned in agreement with Refs. 6--10. The C-4 His-105 resonance is also evident. Linewidths Avln and chemical shifts 5 are given in Table I. In the presence of the lowest Cu 2÷ concentration examined ([Cu2÷]/ [RNAase A] 1 : 84) the C-2 H i s - l l 9 resonance is no more observable whereas the C-2 and C-4 His-105 and C-2 His-12 peaks are slightly broadened (Fig. 1A (b)). From a curve analysis the spectrum results in the overlapping o f only three absorption curves possessing Lorentzian shape and centered at the C-2 His-105, His-12 and His-48 resonance positions (See Fig. 2A). Similar 1H NMR spectra decomposition is also possible for the solutions with a higher metal ion to enzyme molar ratio. The resulting linewidths are reported in Table I. As we can observe, a parallel broadening of the C-2 His-105 and His-12 resonances occurs by stepwise increasing of the molar ratio [Cu2÷]/[RNAase A]: it results larger for His-105 than for His-12 (Fig. 1A (c--e) and Table I). The C-4 His-105 absorption band behaves as the C-2 proton of the same residue. On the contrary, no effect has been observed on His-48.
I
A]
* F r o m R e f . 3.
(ppm) K ( H z • M -1 ) r (A)
0 1:84 1:63 1:42 1:25
[ Cu2+] [RNAase
8.74 + 0.02 3.3 - 105 5.6
(119)
8 . 6 2 -+ 0 . 0 2 n.e. n.e.
His-ll9
His-105
HVll 2
8-+1 n.d. n.d. n.d. n.d.
(105)
6-+1 1 7 -+ 1 20- + 2 2 4 -+ 2 4 0 -+ 4
AVl/2
p2H 5.5
8 . 5 4 -+ 0 . 0 2 1.3 • 105 6.5
His-12
8+1 1 2 -+ 2 13-+ 2 1 8 -+ 2 2 0 -+ 4
AVl/2(12)
(48)
8 . 2 7 -+ 0 . 0 2 2.8 • 103 *
His-48
18-+2 1 8 -+ 2 1 8 -+ 2 1 8 -+ 2 1 8 -+ 2
HPl/2
8 . 0 6 -+ 0 . 0 2 7.5 - 105 4.8
His-105
6-+1 2 9 -+ 2 3 7 -+ 4 n.d. n.d.
HVll 2(105)
p2H 7.0
7 . 9 4 -+ 0 . 0 2 4.5 • 105 5.3
His-119
6-+1 2 0 -+ 2 2 5 -+ 4 3 0 -+ 4 n.d.
AVl/2(119)
H a l f - h e i g h t l i n e w i d t h s , c h e m i c a l s h i f t s , r a t e s o f b r o a d e n i n g a n d d i s t a n c e s f r o m t h e C u 2+ o f t h e C - 2 h i s t i d i n e r e s o n a n c e s o f a 2 . 5 2 • 1 0 - 3 M R N A a s e t h e p r e s e n c e o f d i f f e r e n t C u 2+ c o n c e n t r a t i o n s a t 3 4 ° C a n d a t p 2 H 5 . 5 a n d 7 . 0 A P l / 2 is i n H z . n . d . , n o t d e t e c t a b l e ; n . e . ; n o t e v a l u a b l e .
TABLE
in
7 . 8 1 -+ 0 . 0 2 6 • 104 7.5
His-12
6-+1 8 -+ 2 8-+ 2 1 0 -+ 2 1 0 -+ 2
AVl/2(12)
A in 2H20
104
A
i
9
I
l
~
8.5 8 ppm from DSS
l
I
8.5 8 7.5 ppm from DSS
Fig. 2. E x p e r i m e n t a l a n d f i t t e d c u r v e s o f t h e h i s t i d i n e r e s o n a n c e s o f R N A a s e A . T h e f i t t i n g w a s o b t a i n e d w i t h a D u p o n t 3 1 0 c u r v e r e s o l v e r , a s s u m i n g e q u a l a r e a s f o r all p e a k s . (A) p 2 H 5 . 5 a n d [ C u 2 + ] / [ R N A a s e A ] m o l a r r a t i o 1 : 2 5 . (B) p 2 H 7 . 0 a n d m o l a r r a t i o s [ C u 2 + ] / [ R N A a s e A ] : I, 1 : 2 5 ; II, 1 : 4 2 .
At p2H 7.0 the presence of increasing amounts of Cu 2÷ in the RNAase A solution causes different linebroadening of the C-2 histidine peaks as well (Fig. 1B (a--e)). At lower Cu 2÷ concentration a decomposition of the absorption
.
, 9.5
I 9
•
I 8.5
_4
I 8
°
t 7.5
ppm from DSS Fig. 3. F o u r i e r t r a n s f o r m I H N M R s p e c t r a f r o m 7.5 t o 9.5 p p m o f 2 . 5 2 • 10 -3 M R N A a s e A in 2 H 2 0 , 0.3 M NaC1, at 3 4 ° C , in t h e p r e s e n c e o f 1 0 -4 M Cu 2+ a n d o f d i f f e r e n t 2 ' , 3 ' - C M P c o n c e n t r a t i o n s , a t p 2 H 5.5: a, only R N A a s e A; b , [ C u 2 + ] / [ R N A a s e A] m o l a r r a t i o 1 : 25; c, [ C u 2 + ] / [ R N A a s e A ] / [ 2 ' , 3 ' - C M P ] m o l a r r a t i o 1 : 25 : 2 5 , a n d d, 1 : 25 : 50.
105 spectra into three overlapping curves is possible. They are centered at the positions as C-2 His-105 H i s - l l 9 and His-12 peaks and possess the linewidths given in Table I. On the other hand, at higher Cu 2÷ concentrations some of the signals are not more detectable, as is indicated in Table I. In Fig. 2B the fitted curves are shown for the two different situations. The effects of increasing amounts of 2',3'-CMP in the solution with the highest [Cu2÷]/[RNAase A] molar ratio (1 : 25) at p2H 5.5 is shown in Fig. 3. The resonance of His-12 shifts downfield and slightly widens out until it disappears under the envelope of His-105 when the 2',3'-CMP concentration is raised up to 2.52 • 10 -4 M (Fig. 3c). In the presence of 5 • 10 -3 M of nucleotide the same peak is shifted further downfield up to 9.14 ppm. The His-105 remains at the same position as in the absence of both Cu 2÷ and nucleotide and a small sharpening o f the peak is now observable. H i s - l l 9 remains undetectable. No results are available at present on the same ternary system at p2H 7.0. The Cu 2÷ maintains very broad peaks even at high nucleotide concentration.
Discussion The IH NMR spectra shown in Fig. 1A and B and the rates o f broadening of the C-2 histidine protons with Cu 2÷ concentration, K=dAvln/d[Cu2+], reported in Table I, indicate a different behavior of the histidine residues at the two p~H studied. At p2H 5.5 the rates of broadening of the C-2 His-105 and His-12 peaks are comparable to each other, e.g. K(105) = 3.3 • l 0 s Hz • M -1. and K(12) = 1.3 • l 0 s Hz • M -1 . Moreover it is not possible to determine that o f the C-2 H i s - l l 9 prot o n resonance. In fact its corresponding absorption band is not detectable even in the presence of the lowest Cu 2÷ concentration: certainly it should be at least two order o f magnitude larger. As a consequence it results in a very strong interaction site between the Cu 2÷ and the protein which is located on the H i s - l l 9 while two weaker ones are located on the His-105 and His-12. Between the last two the His-105 manifests a somewhat stronger interaction than His-12. Up to 1 • 10-4 M Cu 2÷ concentration no effect has been observed on the C-2 His-48 peak. This last result is in agreement with the small rate o f broadening observed by Inhat [3] working at the same p2H with higher Cu 2÷ concentrations. However, our former findings are somewhat in contrast to the conclusions reached by Joyce and Cohn [5] working on the Cu2÷-RNAase A complex at pH 5.0 and with other results obtained by Inhat [3] and by Roberts and Jardetzky [4] working at p2H 6.0 and 5.5, respectively. In the light of the present findings the observations o f Joyce and Cohn [5] can be interpreted in a different way. In fact the enhancement of the strong site with consequent loss of the weak sites due to an absence or modification o f His-12 could reflect the localization o f the strong site on H i s - l l 9 . On the other hand, the loss of one of the two weaker sites as a consequence of the c a r b o x y m e t h y l a t i o n of H i s - l l 9 could simply imply an increase in affinity towards the Cu 2÷ o f one of the two weaker sites when the original is lost. As far as Inhat's and Roberts and Jardetzky's results are concerned, the dif-
106
ferent assignment of the C-2 proton resonances of H i s - l l 9 and His-12, are a consequence of different solution conditions, and the difference in the working p2H does n o t allow any direct comparison between our and their data. The behavior o f the histidines of the active site of RNAase A in solution is now somewhat in agreement with that observed in the crystal studies, as we will discuss further below. At p2H 7.0 there is no evidence for such a strong interaction site with Cu 2÷ which is comparable to the His-119 at p2H 5.5. Probably this is due to a conformational change of the protein. However, as we can see from the rate of broadening values reported in Table I, in this case His-105 provides a stronger ligand than His-12 and H i s - l l 9 . The binding constant of His-12, also in this case, is the smallest one. Unfortunately, at this p2H, data in solution are n o t available in the literature for a comparison. Binding of the Cu 2÷ to RNAase S was studied in the crystal both at pH 5.5 and 7.0 [11]. In analogy with our results, X-ray data indicate a possibility of interaction of the Cu 2÷ mainly with histidines 119 and 105. In particular, near pH 5.5 both measurements imply the localization of a very strong interaction site on His-119 and of a weaker one on His-105. At both pH His-12 is the less interested in the binding with the metal ion. For the crystal this last result was attributed mainly to the presence of sulfate. However, at pH 7.0 this site of interaction disappears in the crystal while in our study K(12) is greatly reduced. Probably the conformational rearrangement that occurs around pH 7.0 might be the main reason for this behavior. The distance r between the Cu 2÷ and the C-2 protons of the different histidines has also been calculated and given in Table I *. Until now experimental evaluation of these distances have not been reported, therefore we cannot make any comparison. We can observe only that the C-2 His-105 proton reduces its distance from Cu 2÷ less than 1 ]k when the p2H is raised from 5.5 to 7.0. On the other hand, for the same range of p2H H i s - l l 9 and His-12 show more drastic variations. Moreover, it is worth noting that for the C-4 His-105 proton the same distance from the Cu 2÷ has been calculated as for the C-2. This supports the hypothesis that the histidine imidazole N-3 is directly involved in the interaction with the metal ion. Finally, we would like to discuss our preliminary results on the behavior of the histidines when the 2',3'-CMP is added to the Cu2+-RNAase A complex at p2H 5.5. The studies present in the literature suggest inhibition of enzymatic activity due to a competition between inhibitor and Cu 2÷ [5,13,14]. The strongest binding site, though not identified, seems to be involved [14]. In our case His-12 suffers the same shift and broadening than in the absence of Cu 2÷, His105 becomes sharper than in the presence of Cu 2÷ while H i s - l l 9 remains very broad and therefore undetectable even at high inhibitor concentration. This last point clearly needs further investigation. At present we can only suggest that 2',3'-CMP acts on His-12 and His-105 in the same way in the presence and in the absence o f Cu :÷ while it has no effect on His-119. * T h e c a l c u l a t i o n o f r h a s b e e n m a d e f o l l o w i n g R e f . 3, u s i n g for t h e c o r r e l a t i o n t i m e t h e v a l u e 1 • 1 0 - 9 s given b y Ref. 12.
107
Acknowledgement We thank Mr. S. Ioppolo for his help in purifying the materials.
References 1 Breslow, E. (1974) in Metal Ions in Biological Systems (Siegel, H., ed.), Vol. 3: High Molecular Complex, p. 133, M. Dekker, Inc., New York 2 Meadows, D.H., Jardetzsky, O., Epand, R.M., RuterJans, H.H, and Scheraga, H.A. (1968) Proc. Natl, Acad. Sci. U.S. 60, 766--772 3 Inhat, M. (1972) Biochemistry 1 1 , 3 4 8 - - 3 4 9 4 Roberts, G.C.K. and J a r d e t z s k y , O. (1970) Adv~ Protein Chem. 24, 447--545 5 Joyce, B.K. and Cohn, M. (1969) J. Biol. Chem. 244, 811--821 6 0 h e , J., Matsuo, H., Saidyama, F. and Narita, K. (1974) J. Bioehem. (Tokyo) 75, 1 1 9 7 - - 1 2 0 0 7 Bradbury, J.H. and Teh, J.S. (1975) Chem. Commun. 936--937 8 Patel, D.L., Cannel, L.L. and Bavey, F.A. (1975) Biopolymers 1 4 , 9 8 7 - - 9 9 7 9 Markley, J.L. (1975) Acc. Chem. Res. 8, 70--80 10 Shindo, H., Hayes, M.B. and Cohen, J.S. (1976) J. Biol. Chem. 251, 2644--2647 11 Allewell, N.M. and Wyckoff, H.W. (1971) J. Biol. Chem. 246, 4 6 5 7 - - 4 6 6 3 12 Benz, F.W., Roberts, G.C.K., Feeney, J. and Ison, R.R. (1972) Biochim. Biophys. Acta 278, 233--238 13 Alger, T.D. (1970) Biochemistry 9, 3 2 4 8 - - 3 2 5 4 14 Breslwo, E. and Girotti, A.W. (1970) J. Biol. Chem. 245, 1 5 2 7 - - 1 5 3 6
Biochimica et Biophysica Acta, 580 ( 1 9 7 9 ) 1 0 0 - - 1 0 7 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 3 8 2 5 9
1H NMR STUDY ON THE INTERACTION OF CUPRIC ION WITH RIBONUCLEASE A
L U I G I S P O R T E L L I a a n d V I N C E N Z A VITI b,*
a Laboratorio delle Radiazioni and b Laboratorio di Biologia Cellulare ed Immunologia, Istituto Superiore di Sanitd, Viale Regina Elena 299, Roma (Italy) (Received J a n u a r y 2 5 t h , 1 9 7 9 )
Key words: Cu 2+; RNAase A; Histidine; IH-NMR; (Interaction)
Summary The reassignment of the 1H NMR C-2 histidine signals of the bovine pancreactic ribonuclease A has required a revision of the 1H NMR data on the role of the different histidines in their interaction with the Cu 2+. The results of our measurements carried out at p2H 5.5 and 7.0 reduce the importance o f His-12 as main site of interaction. At p2H 5.5 a very strong binding site involves His119, while a weaker one contains certainly His-105. On the contrary, at p2H 7.0 the histidines 105 and 119 seem to possess binding constants of the same order of magnitude and in addition they provide stronger ligands for the Cu 2+ than His-12. The comparison with X-ray data in the crystal shows numerous analogies. Finally, preliminary results on the competitive inhibition effects between the Cu 2+ and 2',3'-cytidine monophosphoric acid are discussed.
Introduction Although the interaction between Cu 2+ and RNAase A has been extensively investigated, some discrepancies are still present in the literature a b o u t the binding sites of the metal ion to the enzyme. From the main results reported on solution studies (for a review see for example Ref. 1) it appears that the Cu 2+ mainly interacts with the histidine residues of the protein. The first C-2 histidine resonance assignment of the 1H NMR spectra of the RNAase A was also used to identify the order of interaction of these residues with the Cu 2+
* T o w h o m c o r r e s p o n d e n c e should be addressed. Abbreviations: RNAase A, bovine pancreatic ribonuclease A; 2',3'-CMP, 2',3'-cytidine monophosphoric acid.
101 [2--4]. A few years ago a new assignment of the histidine peaks was independently made by different groups [6--10]. It was found that the assignment of the C-2 His-119 and His-12 resonances, in phosphate-free solutions and with pure ~vater as a solvent, is reversed with respect to the previous one [2]. In this communication we report our findings of 1H NMR studies on the effects of Cu 2+ on the different histidines of RNAase A at the p2H values 5.5 and 7.0 starting from the new assignment. Preliminary results a b o u t the interaction between the binary system RNAase A/Cu 2+ and 2',3'-CMP are also reported with the aim of a better understanding of the competitivity between the Cu 2+ and the inhibitor in the active site. Materials and Methods RNAase A and 2',3'-CMP were purchased from Sigma and made phosphate free b y chromatography on a Sephadex G-25 fine column (Pharmacia) using 0.1 M ammonium carbonate (pH 8.2) as eluent. Then, the lyophylized RNAase A and nucleotide inhibitor were applied to a column of Chelex 100 ion-exchange resin 200--400 mesh (BioRad) at room temperature. Cu(NO3)2 • 3H20 obtained from Merck, was dissolved in 2H:O (99.7%) and lyophylized twice. Solutions of 2.52 • 10 -3 M o f lyophylized RNAase A were prepared in 0.3 M NaC1 in 2H:O (99.9%). Before the 1H NMR run the samples were heated in a water bath for 5 min at 65 ° C in order to exchange the NH resonances. All binary systems (RNAase A/Cu :÷) were obtained b y adding aliquot amounts of a Cu:+-concentrated solution to the RNAase A solution. The Cu :+ concentration was ranging from 3 . 1 0 -s to 1 - 1 0 -4 M. The ternary systems were also prepared by adding aliquot amounts of a 2',3'-CMP-concentrated solution to the binary systems. The p:H value of the solution was measured just before each 1H NMR run with a Radiometer PHM 63 digital pH meter. The p2H was adjusted to 5.5 and 7.0 with 1 M :HCI or NaO2H (Stohler). The p:H values indicated are pH meter readings in 2H:O without correction for isotope effect. Fourier transform ~H NMR spectra were recorded at 34°C with a Varian XL-100-15 FT spectrometer, interfaced with a Varian 620/L-100 computer equipped with a 1.24 • 106 word disk accessory. In order to minimize the problems arising from the limited dynamic range o f the computer, the spectra were obtained under double precision conditions. Chemical shifts (in ppm) were evaluated with respect to DSS in 2H:O. A D u p o n t 310 Curve Resolver was used to analyse overlapping histidine resonances in the 1H NMR spectra. Results
The interaction of RNAase A with Cu :÷ has been studied in aqueous solutions o f different Cu 2÷ concentrations up to [Cu2÷]/RNAase A] molar ratio 1 : 25. The very downfield region o f the 1H NMR spectra of RNAase A under the effect of increasing Cu 2÷ concentrations is shown in Fig. 1A and B at p2H 5.5 and 7.0, respectively. In Fig. 1A (a) we can observe the four well-resolved C-2 histidine peaks
102
C-2 His
A
C-2 His ' r"T-r"--'q
f
C-4HJ Iosl
A J
b
I 9
i [ 8.5 8 ppm from DSS
I Z5
t
8.5
I
l
8 7.5 ppm from DSS
Fig. 1. F o u r i e r t r a n s f o r m 1H N M R s p e c t r a f r o m 7.5 t o 9.0 p p m o f 2 . 5 2 • 10 -3 M R N A a s e A in 2 H 2 0 , 0 . 3 M NaCI, at 3 4 ° C , in t h e p r e s e n c e of d i f f e r e n t Cu 2+ c o n c e n t r a t i o n s . (A) M e a s u r e m e n t s a t p 2 H 5.5. Mola~ r a t i o s [ C u ~ ' ] / [ R N A a s e A ] : a, 0; b , 1 : 84; c, 1 : 63; d, 1 : 4 2 , a n d e, 1 : 25. (B) M e a s u r e m e n t s a t p 2 H 7.0 w i t h m o l a r r a t i o s as in ( A ) .
assigned in agreement with Refs. 6--10. The C-4 His-105 resonance is also evident. Linewidths Avln and chemical shifts 5 are given in Table I. In the presence of the lowest Cu 2÷ concentration examined ([Cu2÷]/ [RNAase A] 1 : 84) the C-2 H i s - l l 9 resonance is no more observable whereas the C-2 and C-4 His-105 and C-2 His-12 peaks are slightly broadened (Fig. 1A (b)). From a curve analysis the spectrum results in the overlapping o f only three absorption curves possessing Lorentzian shape and centered at the C-2 His-105, His-12 and His-48 resonance positions (See Fig. 2A). Similar 1H NMR spectra decomposition is also possible for the solutions with a higher metal ion to enzyme molar ratio. The resulting linewidths are reported in Table I. As we can observe, a parallel broadening of the C-2 His-105 and His-12 resonances occurs by stepwise increasing of the molar ratio [Cu2÷]/[RNAase A]: it results larger for His-105 than for His-12 (Fig. 1A (c--e) and Table I). The C-4 His-105 absorption band behaves as the C-2 proton of the same residue. On the contrary, no effect has been observed on His-48.
I
A]
* F r o m R e f . 3.
(ppm) K ( H z • M -1 ) r (A)
0 1:84 1:63 1:42 1:25
[ Cu2+] [RNAase
8.74 + 0.02 3.3 - 105 5.6
(119)
8 . 6 2 -+ 0 . 0 2 n.e. n.e.
His-ll9
His-105
HVll 2
8-+1 n.d. n.d. n.d. n.d.
(105)
6-+1 1 7 -+ 1 20- + 2 2 4 -+ 2 4 0 -+ 4
AVl/2
p2H 5.5
8 . 5 4 -+ 0 . 0 2 1.3 • 105 6.5
His-12
8+1 1 2 -+ 2 13-+ 2 1 8 -+ 2 2 0 -+ 4
AVl/2(12)
(48)
8 . 2 7 -+ 0 . 0 2 2.8 • 103 *
His-48
18-+2 1 8 -+ 2 1 8 -+ 2 1 8 -+ 2 1 8 -+ 2
HPl/2
8 . 0 6 -+ 0 . 0 2 7.5 - 105 4.8
His-105
6-+1 2 9 -+ 2 3 7 -+ 4 n.d. n.d.
HVll 2(105)
p2H 7.0
7 . 9 4 -+ 0 . 0 2 4.5 • 105 5.3
His-119
6-+1 2 0 -+ 2 2 5 -+ 4 3 0 -+ 4 n.d.
AVl/2(119)
H a l f - h e i g h t l i n e w i d t h s , c h e m i c a l s h i f t s , r a t e s o f b r o a d e n i n g a n d d i s t a n c e s f r o m t h e C u 2+ o f t h e C - 2 h i s t i d i n e r e s o n a n c e s o f a 2 . 5 2 • 1 0 - 3 M R N A a s e t h e p r e s e n c e o f d i f f e r e n t C u 2+ c o n c e n t r a t i o n s a t 3 4 ° C a n d a t p 2 H 5 . 5 a n d 7 . 0 A P l / 2 is i n H z . n . d . , n o t d e t e c t a b l e ; n . e . ; n o t e v a l u a b l e .
TABLE
in
7 . 8 1 -+ 0 . 0 2 6 • 104 7.5
His-12
6-+1 8 -+ 2 8-+ 2 1 0 -+ 2 1 0 -+ 2
AVl/2(12)
A in 2H20
104
A
i
9
I
l
~
8.5 8 ppm from DSS
l
I
8.5 8 7.5 ppm from DSS
Fig. 2. E x p e r i m e n t a l a n d f i t t e d c u r v e s o f t h e h i s t i d i n e r e s o n a n c e s o f R N A a s e A . T h e f i t t i n g w a s o b t a i n e d w i t h a D u p o n t 3 1 0 c u r v e r e s o l v e r , a s s u m i n g e q u a l a r e a s f o r all p e a k s . (A) p 2 H 5 . 5 a n d [ C u 2 + ] / [ R N A a s e A ] m o l a r r a t i o 1 : 2 5 . (B) p 2 H 7 . 0 a n d m o l a r r a t i o s [ C u 2 + ] / [ R N A a s e A ] : I, 1 : 2 5 ; II, 1 : 4 2 .
At p2H 7.0 the presence of increasing amounts of Cu 2÷ in the RNAase A solution causes different linebroadening of the C-2 histidine peaks as well (Fig. 1B (a--e)). At lower Cu 2÷ concentration a decomposition of the absorption
.
, 9.5
I 9
•
I 8.5
_4
I 8
°
t 7.5
ppm from DSS Fig. 3. F o u r i e r t r a n s f o r m I H N M R s p e c t r a f r o m 7.5 t o 9.5 p p m o f 2 . 5 2 • 10 -3 M R N A a s e A in 2 H 2 0 , 0.3 M NaC1, at 3 4 ° C , in t h e p r e s e n c e o f 1 0 -4 M Cu 2+ a n d o f d i f f e r e n t 2 ' , 3 ' - C M P c o n c e n t r a t i o n s , a t p 2 H 5.5: a, only R N A a s e A; b , [ C u 2 + ] / [ R N A a s e A] m o l a r r a t i o 1 : 25; c, [ C u 2 + ] / [ R N A a s e A ] / [ 2 ' , 3 ' - C M P ] m o l a r r a t i o 1 : 25 : 2 5 , a n d d, 1 : 25 : 50.
105 spectra into three overlapping curves is possible. They are centered at the positions as C-2 His-105 H i s - l l 9 and His-12 peaks and possess the linewidths given in Table I. On the other hand, at higher Cu 2÷ concentrations some of the signals are not more detectable, as is indicated in Table I. In Fig. 2B the fitted curves are shown for the two different situations. The effects of increasing amounts of 2',3'-CMP in the solution with the highest [Cu2÷]/[RNAase A] molar ratio (1 : 25) at p2H 5.5 is shown in Fig. 3. The resonance of His-12 shifts downfield and slightly widens out until it disappears under the envelope of His-105 when the 2',3'-CMP concentration is raised up to 2.52 • 10 -4 M (Fig. 3c). In the presence of 5 • 10 -3 M of nucleotide the same peak is shifted further downfield up to 9.14 ppm. The His-105 remains at the same position as in the absence of both Cu 2÷ and nucleotide and a small sharpening o f the peak is now observable. H i s - l l 9 remains undetectable. No results are available at present on the same ternary system at p2H 7.0. The Cu 2÷ maintains very broad peaks even at high nucleotide concentration.
Discussion The IH NMR spectra shown in Fig. 1A and B and the rates o f broadening of the C-2 histidine protons with Cu 2÷ concentration, K=dAvln/d[Cu2+], reported in Table I, indicate a different behavior of the histidine residues at the two p~H studied. At p2H 5.5 the rates of broadening of the C-2 His-105 and His-12 peaks are comparable to each other, e.g. K(105) = 3.3 • l 0 s Hz • M -1. and K(12) = 1.3 • l 0 s Hz • M -1 . Moreover it is not possible to determine that o f the C-2 H i s - l l 9 prot o n resonance. In fact its corresponding absorption band is not detectable even in the presence of the lowest Cu 2÷ concentration: certainly it should be at least two order o f magnitude larger. As a consequence it results in a very strong interaction site between the Cu 2÷ and the protein which is located on the H i s - l l 9 while two weaker ones are located on the His-105 and His-12. Between the last two the His-105 manifests a somewhat stronger interaction than His-12. Up to 1 • 10-4 M Cu 2÷ concentration no effect has been observed on the C-2 His-48 peak. This last result is in agreement with the small rate o f broadening observed by Inhat [3] working at the same p2H with higher Cu 2÷ concentrations. However, our former findings are somewhat in contrast to the conclusions reached by Joyce and Cohn [5] working on the Cu2÷-RNAase A complex at pH 5.0 and with other results obtained by Inhat [3] and by Roberts and Jardetzky [4] working at p2H 6.0 and 5.5, respectively. In the light of the present findings the observations o f Joyce and Cohn [5] can be interpreted in a different way. In fact the enhancement of the strong site with consequent loss of the weak sites due to an absence or modification o f His-12 could reflect the localization o f the strong site on H i s - l l 9 . On the other hand, the loss of one of the two weaker sites as a consequence of the c a r b o x y m e t h y l a t i o n of H i s - l l 9 could simply imply an increase in affinity towards the Cu 2÷ o f one of the two weaker sites when the original is lost. As far as Inhat's and Roberts and Jardetzky's results are concerned, the dif-
106
ferent assignment of the C-2 proton resonances of H i s - l l 9 and His-12, are a consequence of different solution conditions, and the difference in the working p2H does n o t allow any direct comparison between our and their data. The behavior o f the histidines of the active site of RNAase A in solution is now somewhat in agreement with that observed in the crystal studies, as we will discuss further below. At p2H 7.0 there is no evidence for such a strong interaction site with Cu 2÷ which is comparable to the His-119 at p2H 5.5. Probably this is due to a conformational change of the protein. However, as we can see from the rate of broadening values reported in Table I, in this case His-105 provides a stronger ligand than His-12 and H i s - l l 9 . The binding constant of His-12, also in this case, is the smallest one. Unfortunately, at this p2H, data in solution are n o t available in the literature for a comparison. Binding of the Cu 2÷ to RNAase S was studied in the crystal both at pH 5.5 and 7.0 [11]. In analogy with our results, X-ray data indicate a possibility of interaction of the Cu 2÷ mainly with histidines 119 and 105. In particular, near pH 5.5 both measurements imply the localization of a very strong interaction site on His-119 and of a weaker one on His-105. At both pH His-12 is the less interested in the binding with the metal ion. For the crystal this last result was attributed mainly to the presence of sulfate. However, at pH 7.0 this site of interaction disappears in the crystal while in our study K(12) is greatly reduced. Probably the conformational rearrangement that occurs around pH 7.0 might be the main reason for this behavior. The distance r between the Cu 2÷ and the C-2 protons of the different histidines has also been calculated and given in Table I *. Until now experimental evaluation of these distances have not been reported, therefore we cannot make any comparison. We can observe only that the C-2 His-105 proton reduces its distance from Cu 2÷ less than 1 ]k when the p2H is raised from 5.5 to 7.0. On the other hand, for the same range of p2H H i s - l l 9 and His-12 show more drastic variations. Moreover, it is worth noting that for the C-4 His-105 proton the same distance from the Cu 2÷ has been calculated as for the C-2. This supports the hypothesis that the histidine imidazole N-3 is directly involved in the interaction with the metal ion. Finally, we would like to discuss our preliminary results on the behavior of the histidines when the 2',3'-CMP is added to the Cu2+-RNAase A complex at p2H 5.5. The studies present in the literature suggest inhibition of enzymatic activity due to a competition between inhibitor and Cu 2÷ [5,13,14]. The strongest binding site, though not identified, seems to be involved [14]. In our case His-12 suffers the same shift and broadening than in the absence of Cu 2÷, His105 becomes sharper than in the presence of Cu 2÷ while H i s - l l 9 remains very broad and therefore undetectable even at high inhibitor concentration. This last point clearly needs further investigation. At present we can only suggest that 2',3'-CMP acts on His-12 and His-105 in the same way in the presence and in the absence o f Cu :÷ while it has no effect on His-119. * T h e c a l c u l a t i o n o f r h a s b e e n m a d e f o l l o w i n g R e f . 3, u s i n g for t h e c o r r e l a t i o n t i m e t h e v a l u e 1 • 1 0 - 9 s given b y Ref. 12.
107
Acknowledgement We thank Mr. S. Ioppolo for his help in purifying the materials.
References 1 Breslow, E. (1974) in Metal Ions in Biological Systems (Siegel, H., ed.), Vol. 3: High Molecular Complex, p. 133, M. Dekker, Inc., New York 2 Meadows, D.H., Jardetzsky, O., Epand, R.M., RuterJans, H.H, and Scheraga, H.A. (1968) Proc. Natl, Acad. Sci. U.S. 60, 766--772 3 Inhat, M. (1972) Biochemistry 1 1 , 3 4 8 - - 3 4 9 4 Roberts, G.C.K. and J a r d e t z s k y , O. (1970) Adv~ Protein Chem. 24, 447--545 5 Joyce, B.K. and Cohn, M. (1969) J. Biol. Chem. 244, 811--821 6 0 h e , J., Matsuo, H., Saidyama, F. and Narita, K. (1974) J. Bioehem. (Tokyo) 75, 1 1 9 7 - - 1 2 0 0 7 Bradbury, J.H. and Teh, J.S. (1975) Chem. Commun. 936--937 8 Patel, D.L., Cannel, L.L. and Bavey, F.A. (1975) Biopolymers 1 4 , 9 8 7 - - 9 9 7 9 Markley, J.L. (1975) Acc. Chem. Res. 8, 70--80 10 Shindo, H., Hayes, M.B. and Cohen, J.S. (1976) J. Biol. Chem. 251, 2644--2647 11 Allewell, N.M. and Wyckoff, H.W. (1971) J. Biol. Chem. 246, 4 6 5 7 - - 4 6 6 3 12 Benz, F.W., Roberts, G.C.K., Feeney, J. and Ison, R.R. (1972) Biochim. Biophys. Acta 278, 233--238 13 Alger, T.D. (1970) Biochemistry 9, 3 2 4 8 - - 3 2 5 4 14 Breslwo, E. and Girotti, A.W. (1970) J. Biol. Chem. 245, 1 5 2 7 - - 1 5 3 6
