Crystal structure and conformation of 2-thio-5-methylaminomethyluridine dihydrate

Abstract

CllH21N307 s is orthorhombic, space group P212121, with a = 13.695 (7), b = 11.364 (6), c = 9.748 (5) A, Z = 4. The structure, which was refined to R = 0.063 for 915 counter reflections, exhibits a zwitterionic base with N(3) deprotonated and N(51) protonated. The orientation of the base is anti (X = 15°); the ribosyl moiety shows a C(3')-endo conformation and a gauche-gauche arrangement of C(4')-C(5'). These conformational features are stabilized by the characteristic intramolecular interaction C(6)--H.--O(5'). One water molecule forms an eight-membered ring with the aid of two hydrogen bonds to 0(4) and the amino group. The nucleosides exhibit no base stackiaag. The packing is mainly determined by the interaction with the water molecules leading to a great variety of hydrogen bonds and to water channels parallel to e.Uridine is seldom found in the wobble position of tRNA's but is replaced by 5-substituted derivatives which may, furthermore, contain S instead of the 2-keto O atom. The structure of Phe-tRNAyeast (Kim et al., 1974;Ladner et al., 1975) shows the wobble nucleoside in a stericaUy exposed position. The base-pairing properties between codon and anticodon are certainly influenced by the exact conformation of the anticodon region which depends on the interaction of the wobble nucleoside with its neighbours and the anticodon loop backbone.In our attempt to clarify the function of 5-substitution, we report here the crystal structure of 2-thio-5-methylaminomethyluridine dihydrate (s2mnmSU.2H20). This nucleoside which was first isolated and whose structure was elucidated by Carbon, David & Studier (1968) 0567-7408/79/010122-04501.00 occurs in the wobble position of Gln-Lys-and GlutRNAe, co,, and forms base pairs only with adenine and not with guanine, i.e. the wobble recognition is restricted by the 5-substituent.

The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue.

“…However, the poor electron density for nucleotide U-33 suggests flexibility in this region which adds some uncertainty about the exact backbone conformation. In the modelled 2'-endo conformation for the E.coli tRNALYS complex, the distance S2-02' is 3.3 A, which is slightly longer than the corresponding distance observed for an S2-water hydrogen bond in the crystal structure of free mnm5s2U (Egert et al, 1979). The mnm5 substituent is directed away from the tRNA backbone ( Figure 6C) and clearly does not form a hydrogen bond with the ribose of U-33, as has been proposed in the context of the free tRNA conformation (Hillen et al, 1978).…”

“…This raises the question of whether in fact the N3 of the mnm5s2U-34 is unprotonated in the complex structure (see Discussion), the situation observed in the crystal structure of mnm5s2U dihydrate (Egert et al, 1979). In the current structures, although the resolution of the data is not sufficient to determine sugar puckers directly, the ribose of nucleotide 34 has been modelled most satisfactorally into the electron density as 2'-endo.…”

“…We further hypothesize that part of the role of the s2 modification is, by virtue of its interaction with the 02' of the ribose-34, to lower the pK of the N3 hydrogen, thus favouring deprotonation. The pK of 2-thiouridine is 8.8, compared with 9.2 for uridine, whereas that of mnm5s2U is not reported, although the crystal structure of the dihydrate at neutral pH has N3 deprotonated (Egert et al, 1979). This would make the protonation state of the N3 position of mnm5s2U-34 resemble that of C-34, both then being able to make favourable hydrogen bonds with the main chain amide of Lys115.…”

“…The parameters and topology for the refinement of the protein were from Engh and Huber (1991) and for the tRNA from Parkinson et al (1996). The modified base mnm5s2U-34 was refined using the geometry determined from the crystal structure of mnm5s2U dihydrate which is observed in tRNALYS anticodon recognition by Iysyl-tRNA synthetase zwitterionic form with the N3 deprotonated and the amino N(5 1) of the mnm5 protonated (Egert et al, 1979). The occupancy of the tRNA molecule was set arbitrarily to 0.5-0.7 to avoid unphysically high B-factors.…”

Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone

“…Though mo 5 U was studied ( 10–12 ), 3D structure of the 5-oxyuracil-containing oligonucleotide has never been investigated. To better understand the role of the 5-O-CH 3 modification at the atomic level, we have decided to synthesize an RNA-like system by taking advantage of A-form DNA, which has the same sugar pucker and overall A-form duplex structure as RNA ( 13 , 14 ).…”