Plausible Minimal Substrate for Erm Protein

Abstract

Erm proteins methylate a specific adenine residue (A2058, Escherichia coli coordinates) conferring MLSB (macrolide–lincosamide–streptogramin 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 Erm protein, various RNA substrates from 23S rRNA were constructed, and the substrate activity of these constructs was studied using three Erm proteins: ErmB from Firmicutes and ErmE and ErmS from Actinobacteria. The shortest motif of 15 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 stem 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 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 stem 73 furthest away from the target adenine and the large portion of domain V above the methylatable adenine.

Characteristics of the ErmK Protein of Bacillus halodurans C-125

“…Therefore, the low activity of ErmK compared to ErmC′ could be another measure developed by this extremophile to avoid the unnecessary fitness cost caused by the dimethylation of A2058, presumably due to the relatively low probability of encountering the antibiotics in the favorable growth conditions and retarded growth in neutral environment. Similar but more detailed observations have been reported previously ( 27 ): activity among Erm proteins is varied, that is Erm protein activity adapts to the needs posed by stimuli from the environment with distinct and specific features; for example, in the recognition of the minimal part of domain V for methylation and the decrease in substrate activity upon the same deletion from a certain substrate of RNA resulting in different methylation activity to a defined substrate RNA. Furthermore, in addition to its laggardly activity, the regulatory region of ErmK seems to exhibit less efficient inducibility of ermK compared to ermC′ (S. K. Kim and H. J. Jin, unpublished results).…”

“…The 72 nt RNA substrate composed of helices 73 and 74 (in which the end of each helix was capped with the tetraloop, UUCG) exhibited 60% activity compared to that obtained with domain V with ErmS. In the previous report ( 27 ), ErmS was shown to recognize the shortest motif of the RNA substrate to methylate and exhibit the highest activity with the substrate equipped with helix 73 and its surrounding region that it might reflect the potential for substrate activity of each participating nucleotide more clearly. In this sense, 72 nt RNA might be good enough to play a distinguishing role in comparing Erm proteins for their methylation activity, despite its relatively small size.…”

“…Each Erm protein was expressed and purified from E. coli BL21(DE3) harboring plasmids pHJJ502 (ErmC′) and pHJJ602 (ErmK) as described previously ( 27 , 34 ) with slight modifications. Briefly, transformed E. coli BL21(DE3) cells with the ermC′ or ermK genes in the pET23b vector ( E. coli HJJ502 and E. coli HJJ602) were grown overnight at 37°C in LB medium supplemented with 100 μg/mL of ampicillin.…”

Potential Target Site for Inhibitors in MLSB Antibiotic Resistance

“…Therefore, the Erm protein might assume a distinct local structure with bound substrate RNA in this region and provide the target site to develop the unique inhibitor(s) for enhanced selectivity and presumably with high potency. Furthermore, there is no other perceived homolog, except for KsgA/Dim family proteins [ 16 ] that utilize somewhat different substrates with unique structure and topology for recognition and methylation [ 44 , 45 , 46 ], and Erm proteins harbor distinct sequence and length of the NTER. Therefore, there could not be cross-reactivity to induce toxicity when inhibitors to be developed are administered, although the KsgA/Dim family appears to exhibit some sequence conservation around the shortest motif X with the Erm protein family.…”

“…In order to induce the expression, IPTG (isopropyl-β- d -thiogalactopyranoside) was added to the final concentration of 1 mM and incubation continued for another 18 h at 22 °C. Purification was performed using previously described procedure [ 45 ]. Briefly, cells from 100 mL of culture were collected by centrifugation and resuspended in buffer A (20 mM Tris-HCl [pH 7.0], 500 mM KCl, and 5 mM imidazole).…”