Plausible Minimal Substrate for Erm Protein

Abstract

Erm proteins methylate a specific adenine residue (A2058, Escherichia coli coordinates) conferring macrolide-lincosamide-streptogramin B (MLS_B) antibiotic resistance on a variety of microorganisms, ranging from antibiotic producers to pathogens. To identify the minimal motif required to be recognized and methylated by the Erm protein, various RNA substrates from 23S rRNA were constructed, and the substrate activity of these constructs was studied using three Erm proteins, namely, ErmB from Firmicutes and ErmE and ErmS from Actinobacteria The shortest motif of 15 nucleotides (nt) could be recognized and methylated by ErmS, consisting of A2051 to the methylatable adenine (A2058) and its base-pairing counterpart strand, presumably assuming a quite similar structure to that in 23S rRNA, an unpaired target adenine immediately followed by an irregular double-stranded RNA region. This observation confirms the ultimate end of each side in helix 73 for methylation, determined by the approaches described above, and could reveal the mechanism behind the binding, recognition, induced fit, methylation, and conformational change for product release in the minimal context of substrate, presumably with the help of structural determination of the protein-RNA complex. In the course of determining the minimal portion of substrate from domain V, protein-specific features could be observed among the Erm proteins in terms of the methylation of RNA substrate and cooperativity and/or allostery between the region in helix 73 furthest away from the target adenine and the large portion of domain V above the methylatable adenine.

Keywords: Erm protein; antibiotic resistance; methylation; minimal substrate.

FIG 1

Determining the end nucleotide or base pair through the deletion of helix 73 in domain V to be methylated by Erm proteins. (a) Domain V (gg583 nt) used in this study contains only the exact sequence of helix 73, without any further extension on either side, but extra GG dinucleotides were attached at the 5′ end for successful in vitro transcription. Three base pairs (577 nt), five base pairs (567 nt) and A2051 (566 nt) were deleted stepwise from domain V to identify the end nucleotide or base pair to be methylated by Erm proteins. To assist in finding out whether the deletion of A2051 could induce structural alteration, the A2051U and A2051C mutants of 567 nt were constructed, and their substrate activities were measured. The methylatable adenine is circled. (b) Substrate activity of domain V-related constructs associated with the used Erm proteins. Each value represents the mean of at least three independent experiments obtained after 1 h of incubation in the presence of cofactor SAM. Refer to the Results. Mutant substrates in which mutations were introduced at A2058 could not be methylated by Erm proteins to confirm the specific methylation at A2058. Also the 567-nt A2051U and A2051C mutants could not show any substrate activity at all, suggesting that the deletion of A2051 from 567 nt might not induce any structural perturbation. + and – represent positive and negative substrate activity, respectively. (c) To ensure the negative or positive substrate activity of 567 nt, 566 nt, and 567 ntA2051U and 567 ntA2051C mutants, methyl group-accepting activity was monitored over time and in the presence of double enzyme concentrations, together with a control lacking protein because of their quite low substrate activity, if it has substrate activity. Each value represents the mean of at least three independent experiments with standard deviation. 2X denotes monitoring substrate activity with double the concentration of the enzyme.

FIG 2

Confirming the ultimate end on the other side of the methylatable adenine in helix 73 to be methylated by Erm proteins in the context of minimal structure. (a) Loop sequence (7 nt) tethering on the methylatable adenine side contains the natural sequence AAAGA (bold letter denotes the methylatable adenine) connecting helix 73 and 74 and extra CC nucleotides. The loop capping the opposite side of the methylatable adenine in helix 73 was the UUCG tetraloop, which appeared to enhance the substrate activity, as shown in (b), presumably by providing stronger binding affinity to the enzymes than A2051. With the UUCG tetraloop, the other side could be unleashed. Circled is the methylatable adenine. Extra GG and G were attached to the 5′ end of gg37 nt and g21 nt and its mutants (g21 ntA2051U and ntA2051C) to produce more RNA by in vitro transcription. (b) The methyl group-accepting activity of tentatively minimized constructs for determining the ultimate opposite end of the methylatable adenine. The g21 ntA2051U and g21 ntA2051C mutants were employed to assist in finding out whether the deletion of A2051 might induce the structural disturbance from g21 nt. The results are the average value of three independent experiments, which were obtained after 1 h of incubation in the presence of cofactor SAM. Refer to the Results. + and – in front of each value represent positive and negative substrate activity, respectively. (c) To confirm the negative substrate activity of g21 nt to ErmB and ErmE and 20 nt-I, (d) g21 ntA2051U, and g21 ntA2051C mutants to ErmS, methyl group-accepting activity was monitored over time and in the presence of double enzyme concentrations because of their quite low substrate activity, if it has substrate activity. Each value represents the mean of at least three independent experiments with standard deviations. 2X denotes monitoring substrate activity with double the enzyme concentration. g21 nt, which is equipped with A2051 and a 7-nt loop sequence, could be methylated by ErmS, but not by ErmB and ErmE. 20 nt-I, where one base pair was deleted from g21 nt and g21 nt A2051U and A2051C mutants, could not be methylated even with ErmS.

FIG 3

Determining the ultimate end on the side of the methylatable adenine for accepting a methyl group by Erm proteins. (a) The UUCG tetraloop in place of A2051 could support the methyl-transferring activity of all the Erm proteins employed in this study (ErmB, ErmE, and ErmS) to the substrate retaining unleashed AAAGA loop sequence connecting helix 73 to 74. The methylatable adenine is circled. (b) While ErmB and ErmE could not methylate the substrate (23 nt, −) in which just one nucleotide was truncated from 24 nt, ErmS could methylate all the substrates (+) including the one that retained the methylatable adenine alone. Substrate activity of all the substrates from 24 nt to 20 nt increased gradually with time by ErmS, confirming that the ultimate end is the methylatable adenine (c). For ErmB (d) and ErmE (e), no substrate activity increase could be observed over time just after one nucleotide was deleted from 24 nt. 2X denotes monitoring substrate activity with double the enzyme concentration.

FIG 4

Methyl group-accepting activity of the substrate by ErmS, made up of two annealed RNA strands containing only a natural sequence. (a) Structure of annealed substrate (15 nt). Circled is the methylatable adenine. (b) Methylation was carried out with a substrate obtained by preincubating for 5 min at 50°C and annealing for 90 min at 10°C. Reaction mixture was incubated at the designated temperature in the presence of ErmS and SAM (refer to Results). At 18°C, the annealed substrate showed the methyl group-accepting activity with a concomitant increase of activity over time under both normal assay conditions and reaction conditions, where the levels of all of the components (enzyme, SAM, and substrate) were doubled (2X). However, at lower (10°C) and higher (24°C) temperatures alike, ErmS could not methylate it.