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Strong magnesium binding sites in yeast phenylalanine transfer RNA
539 Biochirnica et Biophysica Acta, © Elsevier/North-Holland
515 (1978) 539--542 Biomedical Press
BBA Report BBA 91468 STRONG MAGNESIUM BINDING SITES IN YEAST PHENYLALANINE TRANSFER RNA
POOJAPPAN NARAYANAN
and F A U S T O R AMIREZ*
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, N.Y. 11794 (U.S.A.)
(Received March 6th, 1978) Summary To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op .... Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m 2 G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it linl~s the dihydrouridine arm with the extra (V) loop.
Yeast phenylalanine transfer RNA has been obtained as orthorhombic [1--3] and monoclinic [3, 4] crystals, and models for the molecule have been proposed [5--7]. Refined atomic coordinates for the monoclinic form have recently been published [7]. One of the techniques that have been utilized for the preparation of both crystalline forms of the tRNA involves diffusion of dioxane vapors into aqueous solutions of the compound containing various magnesium chloride: * T o w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s : m s C. 5 - m e t h y l c y t i d i n e ; m 2 G. N 2 - m e t h y l g u a n o s i n e ; m ] G. N ~ - d i m e t h y l g u a n o s i n e ; m 7 G, 7-methylgt~ano~in~.
540
spermine hydrochloride ratios at pH 7, and it has been shown that the monoclinic crystals are favored by higher magnesium concentrations [3]. More recently, the binding of metals in the monoclinic form of the t R N A [8], and the binding of Mg 2+ in the orthorhombic form [9, 10] have been described. This report shows that in the monoclinic form, in addition to the three strong binding sites for magnesium identified by Jack et al. [8], there are several other possible sites in the molecule which are quite suitable for strong binding to magnesium. This conclusion is based on a recent study [11] of the crystal and molecular structure of pentaaquo(diphenyl phosphato) trimagnesium (II), [(C6 H5 0)2 P(O)O] 6 Mg3 (H2 0 ) 5 . The model magnesium phosphodiester hydrate is formulated as [(ArO)2 P(O)O], Mg(H2 0)2° [(ArO)~ P(O)O]: Mg(H2 O)o [(ArO) 2P(O)O] 2Mg(H20)~.
There are two hexacoordinate and one pentacoordinate magnesium ions in the asymmetric unit, the geometries a b o u t the former are nearly octahedral, while that of the latter is intermediate between trigonal bipyramidal and tetragonal pyramidal. Each magnesium is tightly b o u n d to four oxygen atoms from four different phosphate groups and to one or two water molecules, depending on the magnesium coordination number. The phosphate groups occupy t w o different types of positions on the metal coordination polyhedra: vicinal (cis) and distal (trans), which are reflected in significantly different Op .... Op distances, ~ 3 . 0 A, for vicinal-placement, and ~ 4 . 0 h for distalplacement. The relevant parameters for the magnesium hexa-and pentacoordinations in the magnesium phosphodiester hydrate [ 11] were used to scrutinize possible strong binding sites for Mg 2+ in the tRNA. We calculated all Op...Op distances below 5.5 A in the 76 nucleotide residues of the t R N A utilizing the refined atomic coordinates [7]. The results are shown in Table I. The 5.5 A value is taken as the upper limit for distal Op .... Op distances, and 4.0 A as the upper limit for vicinal Op .... Op distances, that are compatible with the data from the model c o m p o u n d [ 1 1 ] , allowing for the quoted standard deviations in the X-ray diffraction analysis of the t R N A (~0.5 A ). One of the pertinent questions in dealing with the binding of Mg 2+ to the phosphate oxygens of the t R N A relates to the number of oxygen atoms which form part of the inner coordination sphere of the ion, i.e., "tight or strong binding". Tight binding m a y result from one-point or two-point coordination. Although one-point coordination cannot a priori be ruled out, our m e t h o d of calculation gives information only on possible two-point coordination sites. Jack et al. [8] found Mg 2÷ strongly b o u n d to three sites in the monoclinic form. Two of the sites (G20, A21, and G19) are in agreement with the sites reported by H o l b r o o k et. al. [ 9 ] , and b y Quigley et al. [ 1 0 ] , for the orthorhombic form. In the monoclinic form, another Mg 2+ is tightly bound to U8, A9, b u t the interpretation given to the magnesium coordination in this region is quite different in the orthorhombic form [9, 1 0 ] . The notation given by the authors [8] for two of the sites is included in Table I. The Mg 2+ in these t w o sites is replaced by Sm 3÷ in samarium-derivatized crystals. In addition, samarium was found in t w o other sites where no magnesium could be detected. There is little d o u b t that the sites designated as Mg 1 and 2, and
541 TABLE I D I S T A N C E S B E L O W 5 . 5 .~ B E T W E E N P H O S P H O R Y L O X Y G E N S ( O P ) IN Y E A S T P H E N Y L A L A N I N E tRNA COMPUTED FROM THE REFINED ATOMIC COORDINATES OF JACK ET AL. [7] Distance
Op....Op
designation
distance ( A )
I n In IV V VI VII VIII
3.4 3.8 3.S 3.8 4.0 4.4 4.6 4.7
Site description T G20-OR, A21-OL US-OR, A9-OR C2-OR, GS-OL m s C49-OR, US0-OL C56-OL, GS7-OL A14-OR, G57-OR (symmetry related) m 2 GI0-OL, U47-OR U7-OR, A14-OL
Phosphate placement
Site notation
on metal polyhedron
( J a c k et, al.)
Vicinal Vicinal Vicinal Vieinal Vieinal Distal Distal Distal
Mg Mg ---Sm -Sm
I"R, L = r i g h t - , l e f t - h a n d e d O p as p e r c o n v e n t i o n (n = n u c l e o s i d e i n d e x ) .
2, S m 2 I, Sm 1
5 3
5'?n i LO~
Pn~OR I
3'0n-I
Sm 1, 2, 3 and 5, are involved in tight metal-phosphate binding, since the corresponding metal-Op interatomic distances are in the range 2.2 to 2.6 A. The magnesium binding site, no. 3, of Jack et al. [8], is: G19~)R, G20N7, G20-06, U59-O4, and C60-N3. However, the only strong binding at this site is that of G19-OR, since the Mg-Op quoted distance is 1.6 A. Samarium does not displace magnesium from the Mg 3 site. Jack et ah have characterized this Mg 3 site as: "a rather special environment linking single-stranded regions of the dihydrouridine and T~ C loops". They suggested that this site is probably involved in the first stage of melting of the tRNA molecule, and that it may be critical for stabilizing the tertiary structure. Although the Mg 3 site is indeed unusual in that it involves only one phosphoryl oxygen in the binding, the rejection of residues G20, U59 and C60 as members of that site on the grounds of unduly long metal-ligand distances raises questions concerning the above roles for the Mg 3 site. Table I contains four new sites, III-V and VII, which by the criterion of Op .... Op interatomic distances, could give rise to strong magnesium binding. In view of the difficulties associated with the unequivocal location of this metal in the macromolecule by X-ray crystallography, it is conceivable that Mg2÷ may be present in some or in all of these new sites in the native tRNA. Of the four hypothetical strong magnesium binding sites, III-V and VII, the latter could be relevant to the problems of first stage melting and tertiary structure stabilization of the tRNA molecule. Site VII is associated with two regions of the tRNA molecule: (1) The augmented dihydrouridine helix, which comprises the double helical dihydrouridine stem (residues 10--13), the dihydrouridine loop (residues 14--21), plus residue 26. (2) The extra loop, which contains residues 44--48. Four of the residues in the extra loop are hydrogen bonded ( .... ) to residues in the augmented dihydrouridine helix, A44 .... m]G26, G45 .... C25-m 2G10, m 7G46 .... G22-C13, and C48 .... G15, where solid lines denote base-pairing (see Fig. l b in Ref. 7). However the fifth residue of the extra loop, U47, seems to be isolated in the tRNA models [5--7]. The presence of magnesium in site VII could strengthen the tertiary structure by linking the extra loop at U47-OR with the augmented
542 dihydrouridine helix at m 2 G10-OL. It is n o t e w o r t h y that in the other hypothetical magnesium sites, III-V, as well as in the two sites previously identified, Mg 1 and 2, the two Op ligands are assigned to vicinal positions on the coordination polyhedron; however, in site VII the two Op ligands are distal, and this bond system Op-Mg-Op might be the first one to break in the melting of the molecule. In addition to the actual number of strong magnesium binding sites in yeast phenylalanine transfer RNA, consideration should be given to the coordination number of the metal ion in each of the sites, and to the respective coordination geometry. We are grateful to Dr. A. Jack for making available the coordinates utilized in the present calculations. This research was supported by Grant GM 20672 from the National Institute of General Medical Science.
References 1 2 3 4 5 6 7 8 9 10 11
Cramer, F., yon der Haax, F., Holmes, K.C., Saenger, W., Schlimme, E. and Schulz, G.E. (1970) J. Mol. Biol. 51,523--530 Kim, S.H., Quigley, G., Suddath, F.L. and Rich, A. (1971) Proc. Natl. Acad. Sci. U.S. 6 8 , 8 4 1 - 845 Ladner, J.E., Finch, J.T., Klug, A. and Clark, B.F.C. (1972) J. Mol. Biol. 72, 99--101 Ishikawa, T. and Sundaxaltngam, M. (1972) Nat. New Biol. 236,174--175 Kim, S.H,, Suddath, F.L., Quigley, G.J., McPherson, A., Sussmann, J.L., Wang, A.H.J., Seeman, N.C. and Rich, A. (1974) Science 185, 435---440 Ladner, J.E., Jack, A., Robertus, J.D., Brown, R.S., Rhodes, D., Clark, B.F.C. and Klug, A. ( 1 9 7 5 ) Nucl. Acids Res. 2, 1629--1638 Jack, A., Ladner, J.E. and Klug, A. (1976) J. Mol. Biol. 108, 619--649 Jack, A., Ladner, J.E., Rhodes, D., Brown, R.S. and Klug, A. (1977) J. Mol. Biol. 111,315--328 Holbrook, S.R., Sussman, J.L., Warrant, R.W., Church, G.M. and Kim, S.H. (1977) Nucl. Acids Res. 4, 2811--2820 Quigley, G.J., Teeter, M.M. and Rich, A. (1978) Proc. Natl. Acad. Sci. U.S. 75, 64--68 Narayanan, P., Ramirez, F., McCaffrey, T., Chaw, Y.F. and Marecek, J.F. (1978) J. Org. Chem. 43, 24--31
515 (1978) 539--542 Biomedical Press
BBA Report BBA 91468 STRONG MAGNESIUM BINDING SITES IN YEAST PHENYLALANINE TRANSFER RNA
POOJAPPAN NARAYANAN
and F A U S T O R AMIREZ*
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, N.Y. 11794 (U.S.A.)
(Received March 6th, 1978) Summary To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op .... Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m 2 G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it linl~s the dihydrouridine arm with the extra (V) loop.
Yeast phenylalanine transfer RNA has been obtained as orthorhombic [1--3] and monoclinic [3, 4] crystals, and models for the molecule have been proposed [5--7]. Refined atomic coordinates for the monoclinic form have recently been published [7]. One of the techniques that have been utilized for the preparation of both crystalline forms of the tRNA involves diffusion of dioxane vapors into aqueous solutions of the compound containing various magnesium chloride: * T o w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . A b b r e v i a t i o n s : m s C. 5 - m e t h y l c y t i d i n e ; m 2 G. N 2 - m e t h y l g u a n o s i n e ; m ] G. N ~ - d i m e t h y l g u a n o s i n e ; m 7 G, 7-methylgt~ano~in~.
540
spermine hydrochloride ratios at pH 7, and it has been shown that the monoclinic crystals are favored by higher magnesium concentrations [3]. More recently, the binding of metals in the monoclinic form of the t R N A [8], and the binding of Mg 2+ in the orthorhombic form [9, 10] have been described. This report shows that in the monoclinic form, in addition to the three strong binding sites for magnesium identified by Jack et al. [8], there are several other possible sites in the molecule which are quite suitable for strong binding to magnesium. This conclusion is based on a recent study [11] of the crystal and molecular structure of pentaaquo(diphenyl phosphato) trimagnesium (II), [(C6 H5 0)2 P(O)O] 6 Mg3 (H2 0 ) 5 . The model magnesium phosphodiester hydrate is formulated as [(ArO)2 P(O)O], Mg(H2 0)2° [(ArO)~ P(O)O]: Mg(H2 O)o [(ArO) 2P(O)O] 2Mg(H20)~.
There are two hexacoordinate and one pentacoordinate magnesium ions in the asymmetric unit, the geometries a b o u t the former are nearly octahedral, while that of the latter is intermediate between trigonal bipyramidal and tetragonal pyramidal. Each magnesium is tightly b o u n d to four oxygen atoms from four different phosphate groups and to one or two water molecules, depending on the magnesium coordination number. The phosphate groups occupy t w o different types of positions on the metal coordination polyhedra: vicinal (cis) and distal (trans), which are reflected in significantly different Op .... Op distances, ~ 3 . 0 A, for vicinal-placement, and ~ 4 . 0 h for distalplacement. The relevant parameters for the magnesium hexa-and pentacoordinations in the magnesium phosphodiester hydrate [ 11] were used to scrutinize possible strong binding sites for Mg 2+ in the tRNA. We calculated all Op...Op distances below 5.5 A in the 76 nucleotide residues of the t R N A utilizing the refined atomic coordinates [7]. The results are shown in Table I. The 5.5 A value is taken as the upper limit for distal Op .... Op distances, and 4.0 A as the upper limit for vicinal Op .... Op distances, that are compatible with the data from the model c o m p o u n d [ 1 1 ] , allowing for the quoted standard deviations in the X-ray diffraction analysis of the t R N A (~0.5 A ). One of the pertinent questions in dealing with the binding of Mg 2+ to the phosphate oxygens of the t R N A relates to the number of oxygen atoms which form part of the inner coordination sphere of the ion, i.e., "tight or strong binding". Tight binding m a y result from one-point or two-point coordination. Although one-point coordination cannot a priori be ruled out, our m e t h o d of calculation gives information only on possible two-point coordination sites. Jack et al. [8] found Mg 2÷ strongly b o u n d to three sites in the monoclinic form. Two of the sites (G20, A21, and G19) are in agreement with the sites reported by H o l b r o o k et. al. [ 9 ] , and b y Quigley et al. [ 1 0 ] , for the orthorhombic form. In the monoclinic form, another Mg 2+ is tightly bound to U8, A9, b u t the interpretation given to the magnesium coordination in this region is quite different in the orthorhombic form [9, 1 0 ] . The notation given by the authors [8] for two of the sites is included in Table I. The Mg 2+ in these t w o sites is replaced by Sm 3÷ in samarium-derivatized crystals. In addition, samarium was found in t w o other sites where no magnesium could be detected. There is little d o u b t that the sites designated as Mg 1 and 2, and
541 TABLE I D I S T A N C E S B E L O W 5 . 5 .~ B E T W E E N P H O S P H O R Y L O X Y G E N S ( O P ) IN Y E A S T P H E N Y L A L A N I N E tRNA COMPUTED FROM THE REFINED ATOMIC COORDINATES OF JACK ET AL. [7] Distance
Op....Op
designation
distance ( A )
I n In IV V VI VII VIII
3.4 3.8 3.S 3.8 4.0 4.4 4.6 4.7
Site description T G20-OR, A21-OL US-OR, A9-OR C2-OR, GS-OL m s C49-OR, US0-OL C56-OL, GS7-OL A14-OR, G57-OR (symmetry related) m 2 GI0-OL, U47-OR U7-OR, A14-OL
Phosphate placement
Site notation
on metal polyhedron
( J a c k et, al.)
Vicinal Vicinal Vicinal Vieinal Vieinal Distal Distal Distal
Mg Mg ---Sm -Sm
I"R, L = r i g h t - , l e f t - h a n d e d O p as p e r c o n v e n t i o n (n = n u c l e o s i d e i n d e x ) .
2, S m 2 I, Sm 1
5 3
5'?n i LO~
Pn~OR I
3'0n-I
Sm 1, 2, 3 and 5, are involved in tight metal-phosphate binding, since the corresponding metal-Op interatomic distances are in the range 2.2 to 2.6 A. The magnesium binding site, no. 3, of Jack et al. [8], is: G19~)R, G20N7, G20-06, U59-O4, and C60-N3. However, the only strong binding at this site is that of G19-OR, since the Mg-Op quoted distance is 1.6 A. Samarium does not displace magnesium from the Mg 3 site. Jack et ah have characterized this Mg 3 site as: "a rather special environment linking single-stranded regions of the dihydrouridine and T~ C loops". They suggested that this site is probably involved in the first stage of melting of the tRNA molecule, and that it may be critical for stabilizing the tertiary structure. Although the Mg 3 site is indeed unusual in that it involves only one phosphoryl oxygen in the binding, the rejection of residues G20, U59 and C60 as members of that site on the grounds of unduly long metal-ligand distances raises questions concerning the above roles for the Mg 3 site. Table I contains four new sites, III-V and VII, which by the criterion of Op .... Op interatomic distances, could give rise to strong magnesium binding. In view of the difficulties associated with the unequivocal location of this metal in the macromolecule by X-ray crystallography, it is conceivable that Mg2÷ may be present in some or in all of these new sites in the native tRNA. Of the four hypothetical strong magnesium binding sites, III-V and VII, the latter could be relevant to the problems of first stage melting and tertiary structure stabilization of the tRNA molecule. Site VII is associated with two regions of the tRNA molecule: (1) The augmented dihydrouridine helix, which comprises the double helical dihydrouridine stem (residues 10--13), the dihydrouridine loop (residues 14--21), plus residue 26. (2) The extra loop, which contains residues 44--48. Four of the residues in the extra loop are hydrogen bonded ( .... ) to residues in the augmented dihydrouridine helix, A44 .... m]G26, G45 .... C25-m 2G10, m 7G46 .... G22-C13, and C48 .... G15, where solid lines denote base-pairing (see Fig. l b in Ref. 7). However the fifth residue of the extra loop, U47, seems to be isolated in the tRNA models [5--7]. The presence of magnesium in site VII could strengthen the tertiary structure by linking the extra loop at U47-OR with the augmented
542 dihydrouridine helix at m 2 G10-OL. It is n o t e w o r t h y that in the other hypothetical magnesium sites, III-V, as well as in the two sites previously identified, Mg 1 and 2, the two Op ligands are assigned to vicinal positions on the coordination polyhedron; however, in site VII the two Op ligands are distal, and this bond system Op-Mg-Op might be the first one to break in the melting of the molecule. In addition to the actual number of strong magnesium binding sites in yeast phenylalanine transfer RNA, consideration should be given to the coordination number of the metal ion in each of the sites, and to the respective coordination geometry. We are grateful to Dr. A. Jack for making available the coordinates utilized in the present calculations. This research was supported by Grant GM 20672 from the National Institute of General Medical Science.
References 1 2 3 4 5 6 7 8 9 10 11
Cramer, F., yon der Haax, F., Holmes, K.C., Saenger, W., Schlimme, E. and Schulz, G.E. (1970) J. Mol. Biol. 51,523--530 Kim, S.H., Quigley, G., Suddath, F.L. and Rich, A. (1971) Proc. Natl. Acad. Sci. U.S. 6 8 , 8 4 1 - 845 Ladner, J.E., Finch, J.T., Klug, A. and Clark, B.F.C. (1972) J. Mol. Biol. 72, 99--101 Ishikawa, T. and Sundaxaltngam, M. (1972) Nat. New Biol. 236,174--175 Kim, S.H,, Suddath, F.L., Quigley, G.J., McPherson, A., Sussmann, J.L., Wang, A.H.J., Seeman, N.C. and Rich, A. (1974) Science 185, 435---440 Ladner, J.E., Jack, A., Robertus, J.D., Brown, R.S., Rhodes, D., Clark, B.F.C. and Klug, A. ( 1 9 7 5 ) Nucl. Acids Res. 2, 1629--1638 Jack, A., Ladner, J.E. and Klug, A. (1976) J. Mol. Biol. 108, 619--649 Jack, A., Ladner, J.E., Rhodes, D., Brown, R.S. and Klug, A. (1977) J. Mol. Biol. 111,315--328 Holbrook, S.R., Sussman, J.L., Warrant, R.W., Church, G.M. and Kim, S.H. (1977) Nucl. Acids Res. 4, 2811--2820 Quigley, G.J., Teeter, M.M. and Rich, A. (1978) Proc. Natl. Acad. Sci. U.S. 75, 64--68 Narayanan, P., Ramirez, F., McCaffrey, T., Chaw, Y.F. and Marecek, J.F. (1978) J. Org. Chem. 43, 24--31