Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants

Abstract

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.

Figure 1

Sequence of E.coli microhelix^Ala examined in this study with the A73 discriminator base position circled and the critical 3:70 position required for aminoacylation boxed.

Figure 2

Chemical structures of N73 microhelix^Ala variants and their respective aminoacylation activities (12; this study) given in ΔΔG^‡ (kcal/mol), which is defined as –RTln[(k_cat/K_M)^variant/(k_cat/K_M)^wild-type]. Aminoacylation assays were carried out with the 2′-deoxynucleotide version of the base analogs M, 2AA and 7DAA. ND indicates that the aminoacylation activity has not yet been determined.

Figure 3

RMSDs (Å) from average structures taken over the production run portion of each trajectory. Values have an arbitrary 0 and are calculated excluding the single-stranded region of the microhelix.

Figure 4

Two-dimensional plots of N73 stacking over the G1:C72 base pair. The quadrants indicated by the intersecting solid lines are defined in the subsection of the text entitled 2D stacking analysis. In the above plots the abscissa is the distance (Å) between the COMs of the six-membered rings of the N73 and G1 bases. The ordinate is defined as the distance between the COM of the five-membered ring of N73 and the COM of the six-membered ring of C72.

Figure 5

Percentage of total simulation time that a particular N73 base was found in each of the four stacking quadrants (I–IV) as defined in Figure 4 and in the text.