Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

Published online 12 October 2015 Nucleic Acids Research, 2015, Vol. 43, No. 20 10015–10025 doi: 10.1093/nar/gkv975 Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A Tatsuya Kaminishi1,† , Andreas Schedlbauer1,† , Attilio Fabbretti2,† , Letizia Brandi2,† , Borja Ochoa-Lizarralde1 , Cheng-Guang He2 , Pohl Milón3 , Sean R. Connell1,4,* , Claudio O. Gualerzi2,* and Paola Fucini1,4,* 1 Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain, 2 Laboratory of Genetics, Department of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy, 3 School of Medicine, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas - UPC, Lima, L-33, Perú and 4 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain Received March 27, 2015; Revised August 20, 2015; Accepted August 22, 2015 ABSTRACT Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. INTRODUCTION Hygromycin A (HygA) is a natural product of Streptomyces hygroscopicus first isolated in 1953 (1,2). It is endowed with promising biological activities and has a unique structure (Supplementary Figure S1) consisting of a furanose, cinnamic acid and aminocyclitol moiety (3). The biosynthetic pathway of HygA has been elucidated (4) and its total chemical synthesis has also been described (5–7). HygA has a relatively broad antimicrobial spectrum, displaying activity against gram-positive bacteria including mycobacteria and actinomycetes (3). In addition this molecule is also active against Serpulina (Treponema) hyodysenteriae (the agent of swine dysentery), leptospira and endomoeba (1,3). The limited activity of HygA against enteric gram-negative bacteria has been attributed to the efficient AcrA/B efflux pump operating in these organisms (8). The structure and biological activity of HygA are distinct from those of hygromycin B, another antibiotic produced by the same organism, but HygA displays some common features with chloramphenicol (1–3) and orthoformimycin (9). HygA was shown to be a translational inhibitor; or more precisely, HygA was found to bind to the large (50S) ribosomal subunit and to inhibit the peptidyl transferase (PT) activity of the ribosome (10–12). Other translational steps, such as the enzymatic (EF-Tu dependent) binding of aminoacyl-tRNA to the ribosomal A site and the translocation of peptidyl-tRNA from the A to the P site were found to be unaffected by HygA (10). Furthermore, since HygA is a more potent agent than chloramphenicol and inhibits ribosomal binding of chloramphenicol, it was suggested that the binding sites of these two antibiotics are close or partially overlapping (10). A structural similarity has been observed between HygA, A201A and puromycin (4,13–17). More precisely, the 6 -7 dihydroxy-␣-methylcinnamic acid moiety of HygA (Supplementary Figure S1) and the 7 -hydroxy-␣-methylcinnamic acid present in A201A are similar to the tyrosine-derived moiety of puromycin (4). Similar to HygA, both A201A and puromycin are potent inhibitors of protein synthesis and all three antibiotics prevent peptide bond formation (10,13,17–20). Puromycin, the best characterized of the three antibiotics, binds, as do HygA and chloramphenicol, to the A site of the large subunit where it structurally mimics the aminoacyl-tRNA 3 terminus and can serve as an acceptor of the polypeptide chain via the 2 amino group (21,22). Moreover, in its higher * To whom correspondence should be addressed. Tel: +34 946 572 515; Fax: +34 94 657 25 02; Email: Correspondence may also be addressed to Claudio Gualerzi. Tel: +39 07 374 032 40 Fax: +39 07 374 032 90; Email: Correspondence may also be addressed to Sean Connell. Tel: +34 946 572 529; Fax: +34 94 657 25 02; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 10016 Nucleic Acids Research, 2015, Vol. 43, No. 20 affinity form of CC-puromycin, it can induce on the large subunit, the same active conformation of the PTC, as observed on the complete 70S ribosome (22–26). Despite the similarity between the three antibiotics, and in contrast to puromycin, both HygA and A201A do not carry at the 2 position a reactive amino group ((17) and Supplementary Figure S1) and therefore cannot act as acceptor substrates in peptide bond formation. In situ chemical probing showed that macrolides with a mycarose containing disaccharide at position 5 of the lactone ring, such as, carbomycin, tylosin and spiramycin, inhibit or compete with HygA for ribosomal binding. On the other hand, HygA can bind to the ribosome concomitantly with macrolides that have only a monosaccharide extension on the lactone ring and do not inhibit the PT reaction (11). Overall, the available data indicate that although HygA binds to the ribosome in a region that overlaps that of other 50S-targeting antibiotics, its binding mode is clearly distinct. In light of these data and the frequent occurrence of resistance and cross-resistance phenotypes acquired by pathogenic bacteria to 50S-targeting antibiotics, the use of HygA as a pharmacophore for the development of new antiinfectives capable of overcoming existing resistance mechanisms, requires a precise knowledge of the relationship between the binding site of HygA and that of chloramphenicol and other 50S inhibitors, the macrolides in particular. In this study we used a combination of X-ray crystallography and biochemical approaches to address the ribosomal localization of HygA and describe its relationship to other anti-infectives. Taken together the structural and biochemical data presented indicate that HygA binds within the PTC such that it would clash with the aminoacyl-ribosyl moiety at the 3´end of the A-site tRNA. Therefore its primary mode of action involves blocking the accommodation of the Asite tRNA within the PTC. At the (...truncated)