ARTICLE https://doi.org/10.1038/s41467-020-17202-8 OPEN Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis Yong Zi Tan 1,2, José Rodrigues 3, James E. Keener4, Ruixiang Blake Zheng5, Richard Brunton5, Brian Kloss 6, Sabrina I. Giacometti1, Ana L. Rosário3, Lei Zhang7, Michael Niederweis 7, Oliver B. Clarke 1,8, Todd L. Lowary 5,9, Michael T. Marty 4,10, Margarida Archer 3, Clinton S. Potter2,11,12, ✉ ✉ Bridget Carragher 2,11,12 & Filippo Mancia1 1234567890():,; Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyl- transferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single- particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding. 1 Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA. 2 National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA. 3 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal. 4 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA. 5 Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada. 6 Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY 10027, USA. 7 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 8 Department of Anesthesiology, Columbia University, New York, NY 10032, USA. 9 Institute of Biological Chemistry, Academia Sinica, Academia Road, Section 2, #128, Nangang, Taipei 11529, Taiwan. 10 Bio5 Institute, University of Arizona, Tucson, AZ 85721, USA. 11 Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA. 12 Department of Biochemistry and Molecular ✉ Biophysics, Columbia University, New York, NY 10032, USA. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2020) 11:3396 | https://doi.org/10.1038/s41467-020-17202-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17202-8 he cell envelope is crucial for growth and virulence of acids (Fig. 1a). Another major component is the lipidated hetero- pathogenic mycobacteria like M. tuberculosis1 and is a polysaccharide lipoarabinomannan (LAM)3. Of the enzymes T 2 major contributor to resistance against common antibiotics . involved in mycobacterial cell wall biosynthesis, arabinofuranosyl- Its main component is the mycolyl-arabinogalactan-peptidoglycan transferases are responsible for the addition of D-arabinofuranose complex, which consists of peptidoglycan, a branched hetero- sugar moieties to AG and LAM2. These transmembrane (TM) polysaccharide arabinogalactan (AG) and long chain mycolic enzymes utilize decaprenylphosphoryl-D-arabinofuranose (DPA) to a b LAM capping differs Decaprenol O Decaprenol between M. tuberculosis DPA O P and M. smegmatis O- OH HO O O O P Outer O- O membrane + AG/LAM HO OH HO O Peptidoglycan AG Arabinogalactan EmbB O + O O peptidoglycan complex AG/LAM HO OH HO OH HO EmbB LAM LM PIM O O Ethambutol α-(1→5) linkage Mycobacterium Inner membrane species HO OH c 86 Å 64 Å C 90° C 100 Å Periplasm Inner d membrane N Cytoplasm N 76 Å 45 Å de C-CBM 1053 2 1 α7 N-CBM α3 α1 20 β 14 11 β 8 β 21 9 β 2 7 6 β 3 16 13 12 β β β 15 β 4 β β β β α6 9 β 19 β 1063 β 18 α 5 7 5 β β 17 β Ca2+ α2 α4 1 8 β α8 α9 α10 732 22 β Ca2+ 5 6 11 10 501 β JM5 728 JM2 12 JM1 218 49 JM4 JM3 220 3 4 Periplasm 525 46 C 10 TM TM TM 15b 12a 8a 15 Inner TM TM TM TM TM TM TM TM TM TM TM TM 13 membrane 14 13 11 10 9 7 6 5 4 3 2 1 TM TM TM 14 15a 12b 8b Cytoplasm IL1 N 31 Å 21 Fig. 1 Cryo-EM structure of EmbB. a Model of the cell envelope of mycobacterial cell envelope based on Dulberger et al.75. Red boxes highlight arabinogalactan (AG) and lipoarabinomannan (LAM) components, synthesized by EmbA/EmbB (red) and EmbC, respectively. LAM for M. tuberculosis is capped by α-mannose glycans while in M. smegmatis it is capped by phosphoinositol instead76. b Reaction catalyzed by EmbB, which is inhibited by ethambutol. c Single-particle cryo-EM structure of EmbB, rendered in cartoon form and colored in rainbow from N terminus (blue) to C terminus (red). ACa2+ ion is shown as a green sphere. The bound lipids are represented as ball-and-sticks, colored in brown. Two orthogonal views that are perpendicular to the plane of the membrane are shown. Membrane boundaries were derived from the interface between the nanodisc lipids and solvent. d Arrangement of the TM helices of EmbB, viewed as a slice in the plane of the membrane, as indicated in (b) and magnified. e Two-dimensional topological diagram of EmbB. The two CBMs are enclosed in separate gray boxes. The topology diagram is rainbow colored from N terminus (blue) to C terminus (red). Unbuilt parts of the model, due to poor map density, are indicated by dotted lines. Bound Ca2+ atoms are shown as green circles. 2 NATURE COMMUNICATIONS | (2020) 11:3396 | https://doi.org/10.1038/s41467-020-17202-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17202-8 ARTICLE transfer an arabinofuranose unit to the growing lipidated poly- Table 1 Cryo-EM data collection and modeling 4 saccharides of the cell envelope . statistics EmbB. Arabinosyltransferase B (EmbB), a 117 kDa integral membrane enzyme involved in the α-(1→5)-linked extension of the AG arabinan chain (Fig. 1b), is one of the best characterized members Session 1 Session 2 of the aforementioned family5,6. Its gene belongs to an operon Microscope FEI Titan Krios (same microscope) coding for two other homologous arabinosyltransferases EmbA EM data collection/processing (40% identity between M. smegmatis EmbB and EmbA, 42% Magnification 120,000 37,000 identity in M. tuberculosis, acts also on AG) and EmbC (46% Voltage (kV) 300 identity between M. smegmatis EmbB and EmbC, 44% identical Camera Falcon III Gatan K2 Summit in M. tuberculosis, acts on LAM) (Supplementary Figs. 1 and 2). Mode Counting Counting Set defocus range (μm) 0.5–2.5 0.3–2.9 The operon was named due to the sensitivity of these gene pro- μ fi 7 Defocus mean ± std ( m) 1.8 ± 0.29 1.9 ± 0.27 ducts to ethambutol, a rst-line antibiotic against tuberculosis Exposure time (s) 86.4 8 8 and nontuberculous mycobacterial (NTM) disease . EmbB and Number of frames 80 80 9 EmbC have mutations known to lead to ethambutol resistance , Dose rate (e−/pixel/s) 0.4 4.3 while a clear effect of the drug on EmbA activity has not been Total dose (e−/Å2) 78.02 77.53 established10. The dearth of atomic models of these proteins— Pixel size (Å) 0.665 0.667 only a high resolution structure of the C-terminal soluble Number of micrographs 2158 7833 domain of EmbC is available11—has thus far hindered our Number of particles (after initial 162,271 700,201 understanding of the catalytic action and drug resistance cleanup) mechanisms of these proteins, although this situation has been Number of particles (in 57,970 fi recently remedied to a certain extent by eludication of struc- nal map) tures of the EmbA–EmbB and EmbC–EmbC dimer com- Symmetry C1 12 Resolution (global) (Å) 3.3 plexes . Here, we report the structure of M. smegmatis EmbB Local resolution range 2.8–16.0 to 3.3-Å resolution, providing insights on substrate binding and Directional resolution range 3.0–3.4 reaction mechanism. Mutations that confer ethambutol resis- Sphericity of 3DFSC 0.99 tance map mostly around the putative active site, suggesting SCF valuea 0.98 this to be the location of drug binding. Map sharpening b-factor (Å2) −72.5 Model statistics Results and discussion Initial model used (PDB code) 3PTY Map-to-model resolution (Å) 3.4 Cryo-EM structure of M. smegmatis EmbB. To better under- Model composition stand the function of EmbB at a molecular level, we adopted a Non-hydrogen atoms 15,844 structural genomics approach that identified the M. smegmatis Residue range 21–500, 526–1082 ortholog (68% identical in M. tuberculosis), out of 14 screened, as Ligands 4 a suitable candidate for structural studies based on expression Map CC 0.743 levels in E. coli and stability in detergents compatible with RMSD [bonds] (Å) 0.0065 structure determination13. Using single-particle cryogenic elec- RMSD [angles] (Å) 1.21 tron microscopy (cryo-EM), we determined the structure of All-atom clashscore 2.67 2 EmbB from M. smegmatis expressed in E. coli and reconstituted B factors (Å ) in lipid-filled nanodiscs to 3.3 Å resolution (Supplementary Protein 44.96 Figs. 3–5 and 6a, and Table 1). Here, EmbB appears as a Ligands 44.63 Ramachandran plot monomer, consisting of 15 TM helices and two distinct peri- Favored (%) 94.87 plasmic carbohydrate binding modules (CBMs) (Fig. 1c–e). fi Allowed (%) 5.05 The rst two TM helices are not found in other glycosyl- Outliers (%) 0.08 transferase structures solved to date, and seem to serve to anchor Rotamer outliers 0.00 the N-terminal CBM (N-CBM) to the membrane.