Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Abstract

Erythromycin resistance methyltransferases are S-adenosyl methionine dependent Rossmann-fold methyltransferases that convert A2058 of 23S rRNA to m62A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multi-drug resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens while ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24 % sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics and RNA affinity binding assays. We demonstrate that residues in α4, α5 and the α5-α6 linker are essential for methyltransferase function including: an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker which likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.

Crystal structure and functional analysis of mycobacterial erythromycin resistance methyltransferase Erm38 reveals its RNA-binding site

“…While several positively-charged sites proved to be essential for methylation activity, mutation of these sites individually did not cause significant changes in binding affinity for the RNA substrate. Such a disconnect between enzyme activity and RNA binding affinity was observed in ErmE (24) and is analogous to the lack of correlation between binding affinity and specificity for aptamers (25). Maravic et al also described an E. coli strain carrying ErmC gene with a R134A mutation (equivalent to R141A of Erm38) that resulted in complete loss of erythromycin resistance, while mutations R112A and K133A in ErmC (equivalent to R119A and R140A of Erm38) reduced erythromycin resistance (26).…”

Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methyltransferases

“…In an in vitro setting, chimera’s which consisted of loop1 and loop12 of Erm onto an otherwise KsgA scaffold made it capable of methylating Erm mini-RNA substrates . Furthermore, saturation mutagenesis of loop12 also confirmed that positively charged residues in loop12 of Erm are important for substrate recognition . Thus, it appears that these important loop elements are crucial in tunning specificity.…”

“…22 Furthermore, saturation mutagenesis of loop12 also confirmed that positively charged residues in loop12 of Erm are important for substrate recognition. 49 Thus, it appears that these important loop elements are crucial in tunning specificity. The 30S−KsgA cryo-EM structure shows that loop 12 and another loop14 that resides in the C-terminal head domain serving as anchor points via interaction with the peripheral RNA segments provide an additional layer of stringency to the recognition process.…”

Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methytransferases

“…To corroborate the fact that these loops indeed tune specificity, an earlier study showed that chimera’s which consisted of loop1 and loop12 of Erm onto an otherwise KsgA scaffold made it capable of methylating Erm mini-RNA substrates (Bhujbalrao & Anand, 2019). Furthermore, saturation mutagenesis of loop12 also confirmed that positively charged residues in loop12 of Erm are important for substrate recognition (Rowe, Mecaskey et al, 2020).…”