Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB₂-AcpM₂

Abstract

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its “resting state” and DPA-bound “active state”. EmbB is a fifteentransmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrierprotein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Keywords

Mycobacterium tuberculosis / EmbB / cryo-EM / ethambutol / cell wall synthesis / arabinoglacatan / arabinosyltransferase / acyl-carrier-protein / drug discovery

References

[1] Alcaide F, Pfyffer GE, Telenti A (1997) Role of embB in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob Agents Chemother 41:2270–2273

[2] Alderwick LJ, Lloyd GS, Ghadbane H, May JW, Bhatt A, Eggeling L, Futterer K, Besra GS (2011) The C-terminal domain of the arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module. PLoS Pathog 7: e1001299

[3] Alliance T (2008) Ethambutol. Tuberculosis 88:102–105

[4] Berg S, Starbuck J, Torrelles JB, Vissa VD, Crick DC, Chatterjee D, Brennan PJ (2005) Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan. J Biol Chem 280:5651–5663

[5] Berg S, Kaur D, Jackson M, Brennan PJ (2007) The glycosyltransferases of Mycobacterium tuberculosis—roles in the synthesis of arabinogalaetan, lipoarahinolrnlannan, and other glyeoeonjugates. Glycobiology 17:35r–56r

[6] Breton C, Fournel-Gigleux S, Palcic MM (2012) Recent structures, evolution and mechanisms of glycosyltransferases. Curr Opin Struct Biol 22:540–549

[7] Dai E, Zhang H, Zhou X, Song Q, Li D, Luo L, Xu X, Jiang W, Ling H (2019) MycoResistance: a curated resource of drug resistance molecules in Mycobacteria. Database. https://doi.org/10.1093/database/baz074

[8] Escuyer VE, Lety MA, Torrelles JB, Khoo KH, Tang JB, Rithner CD, Frehel C, McNeil MR, Brennan PJ, Chatterjee D (2001) The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem 276:48854–48862

[9] Goude R, Amin AG, Chatterjee D, Parish T (2008) The critical role of embC in Mycobacterium tuberculosis. J Bacteriol 190:4335–4341

[10] Jankute M, Cox JA, Harrison J, Besra GS (2015) Assembly of the mycobacterial cell wall. Annu Rev Microbiol 69:405–423

[11] Lairson LL, Henrissat B, Davies GJ, Withers SG (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77:521–555

[12] Lee RE, Mikusova K, Brennan PJ, Besra GS (1995) Synthesis of the mycobacterial arabinose donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol, development of a basic arabinosyltransferase assay, and identification of ethambutol as an arabinosyl transferase inhibitor. J Am Chem Soc 117:11829–11832

[13] Lee RE, Brennan PJ, Besra GS (1997) Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay. Glycobiology 7:1121–1128

[14] Lee HY, Myoung HJ, Bang HE, Bai GH, Kim SJ, Kim JD, Cho SN (2002) Mutations in the embB locus among Korean clinical isolates of Mycobacterium tuberculosis resistant to ethambutol. Yonsei Med J 43:59–64

[15] Lee RE, Li W, Chatterjee D, Lee RE (2005) Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live mycobacterial cells using 2D and 3D HR-MAS NMR: structural changes in the Arabinan due to ethambutol treatment and gene mutation are observed. Glycobiology 15:139–151

[16] Lety MA, Nair S, Berche P, Escuyer V (1997) A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 41:2629–2633

[17] Lizak C, Gerber S, Numao S, Aebi M, Locher KP (2011) X-ray structure of a bacterial oligosaccharyltransferase. Nature 474:350–355

[18] Makarov V, Manina G, Mikusova K, Mollmann U, Ryabova O, Saint-Joanis B, Dhar N, Pasca MR, Buroni S, Lucarelli AP (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking Arabinan synthesis. Science 324:801–804

[19] Mikusova K, Slayden RA, Besra GS, Brennan PJ (1995) Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob Agents Chemother 39:2484–2489

[20] Napiorkowska M, Boilevin J, Sovdat T, Darbre T, Reymond JL, Aebi M, Locher KP (2017) Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nat Struct Mol Biol 24:1100–1106

[21] Parris KD, Lin L, Tam A, Mathew R, Hixon J, Stahl M, Fritz CC, Seehra J, Somers WS (2000) Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8:883–895

[22] Qasba PK, Ramakrishnan B, Boeggeman E (2005) Substrateinduced conformational changes in glycosyltransferases. Trends Biochem Sci 30:53–62

[23] Ramaswamy SV, Amin AG, Goksel S, Stager CE, Dou SJ, El Sahly H, Moghazeh SL, Kreiswirth BN, Musser JM (2000) Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 44:326–336

[24] Safi H, Sayers B, Hazbon MH, Alland D (2008) Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin. Antimicrob Agents Chemother 52:2027–2034

[25] Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84

[26] Seidel M, Alderwick LJ, Birch HL, Sahm H, Eggeling L, Besra GS (2007) Identification of a novel arabinofuranosyltransferase AftB involved in a terminal step of cell wall arabinan biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem 282:14729–14740

[27] Shi L, Berg S, Lee A, Spencer JS, Zhang J, Vissa V, McNeil MR, Khoo KH, Chatterjee D (2006) The carboxy terminus of EmbC from Mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan. J Biol Chem 281:19512–19526

[28] Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919

[29] Sun Q, Xiao TY, Liu HC, Zhao XQ, Liu ZG, Li YN, Zeng H, Zhao LL, Wan KL (2018) Mutations within embCAB are associated with variable level of ethambutol resistance in Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother 62:e01279–17

[30] Takayama K, Kilburn JO (1989) Inhibition of synthesis of arabinogalactanby ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 33:1493–1499

[31] Vasileios I, Herrera CM, Schultz KM, Clarke OB, Vendome J, Tomasek D, Banerjee S, Rajashankar KR, Dufrisne MB, Kloss B (2016) Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Nature 351:6273

[32] WHO (2018) Global tuberculosis report 2018. WHO, Geneva

[33] Wolucka BA, McNeil MR, de Hoffmann E, Chojnacki T, Brennan PJ (1994) Recognition of the lipid intermediate for arabinogalactan/arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria. J Biol Chem 269:23328–23335

[34] Wong HC, Liu G, Zhang YM, Rock CO, Zheng J (2002) The solution structure of acyl carrier protein from Mycobacterium tuberculosis. J Biol Chem 277:15874–15880

[35] Zhang L, Zhao Y, Gao Y, Wu L, Gao R, Zhang Q, Wang Y, Wu C, Wu F, Gurcha SS(2020) Structures of cell wall arabinosyl transferases with the anti-tuberculosis drug ethambutol. Science. https://doi.org/10.1126/science.aba9102

RIGHTS & PERMISSIONS

2020 The Author(s) 2020