Solution structure of an rRNA methyltransferase (ErmAM) that confers macrolide-lincosamide-streptogramin antibiotic resistance

Abstract

The Erm family of methyltransferases is responsible for the development of resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics. These enzymes methylate an adenine of 23S ribosomal RNA that prevents the MLS antibiotics from binding to the ribosome and exhibiting their antibacterial activity. Here we describe the three-dimensional structure of an Erm family member, ErmAM, as determined by NMR spectroscopy. The catalytic domain of ErmAM is structurally similar to that found in other methyltransferases and consists of a seven-stranded beta-sheet flanked by alpha-helices and a small two-stranded beta-sheet. In contrast to the catalytic domain, the substrate binding domain is different from other methyltransferases and adopts a novel fold that consists of four alpha-helices.

Solution NMR-derived global fold of a monomeric 82-kDa enzyme

“…Note that these values differ slightly from those reported in our previous study of domain orientation in MSG [D a ϭ Ϫ17 Hz and R ϭ 0.45 (20)], in which the x-ray coordinates of glyoxylate-bound MSG (17) were used to determine the alignment tensor parameters (order parameters and orientation) of individual domains. Values of xx ϭ Ϫ74.7, yy ϭ Ϫ11.8, and zz ϭ 86.5 ppm were used for the 13 CO chemical-shielding tensor in all calculations.…”

“…Dihedral-backbone-angle (,) predictions were made by using the backbone 15 N, 13 C␣, and 13 CO chemical shifts of MSG with the program TALOS (28) after the chemical shifts of MSG were removed from the database and the chemical shifts were corrected for 2 H isotope effects. Restraints consisting of the average , values Ϯ 2 SDs (or at least Ϯ20°from the average predicted value) were used for 533 residues of MSG.…”

“…The resulting orientational restraints, such as dipolar couplings and changes in chemical shifts that are generated by such alignment, are extremely valuable in structural studies, in particular for large proteins in which the number of restraints per residue is significantly less than what is normally obtained in studies of small (Յ30-kDa) proteins. A third important contribution has been the development of isotopic-labeling approaches, such as those involving uniform 15 N, 13 C labeling along with high levels of deuteration, which maximize the lifetimes of NMR signals and optimize the HON TROSY effect. NMR experiments that have emerged to exploit these labeling schemes are equally important.…”

“…In a number of cases, solution NMR structures of ␤-barrel membrane-spanning proteins have been described in which the overall aggregate molecular mass of the protein-lipid complex is on the order of 50-60 kDa and the protein component is Ͻ200 residues (10)(11)(12). Solution structures of proteins have been limited to molecules with Ϸ400 residues or less (13)(14)(15).…”

Site-directed Mutagenesis Analysis Elucidates the Role of 223/227 Arginine in 23S rRNA Methylation, Which Is in 'Target Adenine Binding Loop' Region of ErmSF

“…따라서 치환된 각 잔기들은 효소 활성에서 결정적인 기능을 수 행하지는 않지만, 그 것들이 위치하는 부위를 고려하였을 때 이 들이 가지고 있는 양전하가 기질인 RNA와의 상호작용할 수 있 는 환경을 제공하고 있다고 믿어지며, 또한 R223이 좀 더 중요 하게 작용하고 있음을 확인하였다. et al, 1997;Seppala et al, 1998), 이들 단백질은 아미노산 서열 상에서의 유사성이 높아서 하나의 조상 단백질로부터 유래 되었 을 것으로 추정되고 있으며 (Park et al, 2010), 그 작용 기작이 동일하여 이들 단백질은 그 구조가 거의 유사할 것으로 사료된 다. Erm 단백질 중 ErmAM (Yu et al, 1997)과 ErmC′의 구조 그리고 특히 ErmC′의 경우에는 cofactor와의 결합체 구조도 밝 혀졌다 (Bussiere et al, 1998;Schluckbier et al, 1999). (Bujnicki, 1999;Fauman et al, 1999)과 이미 알려져 있는 다른 단백질의 RNA 결합 domain (Burd and Dreyfuss, 1994;Draper and Reynaldo, 1999)과는 다른 몇 개의 α-helix로만 구성된 작은 C-말단 domain으로 구성되어 있으며 이 domain은 RNA 결합 domain으로 제안 되었다 (Yu et al, 1997).…”