Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB<sub>2</sub>-AcpM<sub>2</sub>

doi:10.1007/s13238-020-00726-6

Protein Cell

2020, Vol. 11

Issue (7) : 505-517 https://doi.org/10.1007/s13238-020-00726-6

RESEARCH ARTICLE

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB₂-AcpM₂

Lu Zhang¹, Yao Zhao^2,^3,⁴, Ruogu Gao^4,⁵, Jun Li², Xiuna Yang², Yan Gao⁶, Wei Zhao¹, Sudagar S. Gurcha⁷, Natacha Veerapen⁷, Sarah M. Batt⁷, Kajelle Kaur Besra⁷, Wenqing Xu², Lijun Bi⁵, Xian’en Zhang⁵, Luke W. Guddat8⁸, Haitao Yang², Quan Wang^2,⁵(

), Gurdyal S. Besra⁷(

), Zihe Rao^1,^2,^5,⁶(

)

¹. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and College of Pharmacy, Nankai University, Tianjin 300353, China
². Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
³. CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (CAS), Shanghai 200031, China
⁴. University of Chinese Academy of Sciences, Beijing 100101, China
⁵. National Laboratory of Biomacromolecules and Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, CAS, Beijing 100101, China
⁶. 6Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China
⁷. School of Biosciences, Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK
⁸. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia

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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

Corresponding Author(s): Quan Wang,Gurdyal S. Besra,Zihe Rao

Issue Date: 31 July 2020

Cite this article:

Lu Zhang,Yao Zhao,Ruogu Gao, et al. Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB₂-AcpM₂[J]. Protein Cell, 2020, 11(7): 505-517.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-020-00726-6
https://academic.hep.com.cn/pac/EN/Y2020/V11/I7/505

1	F Alcaide, GE Pfyffer, A Telenti (1997) Role of embB in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob Agents Chemother 41:2270–2273 https://doi.org/10.1128/AAC.41.10.2270
2	LJ Alderwick, GS Lloyd, H Ghadbane, JW May, A Bhatt, L Eggeling, K Futterer, GS Besra (2011) The C-terminal domain of the arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module. PLoS Pathog 7: e1001299 https://doi.org/10.1371/journal.ppat.1001299
3	T Alliance (2008) Ethambutol. Tuberculosis 88:102–105 https://doi.org/10.1016/S1472-9792(08)70008-8
4	S Berg, J Starbuck, JB Torrelles, VD Vissa, DC Crick, D Chatterjee, PJ Brennan (2005) Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan. J Biol Chem 280:5651–5663 https://doi.org/10.1074/jbc.M411418200
5	S Berg, D Kaur, M Jackson, PJ Brennan (2007) The glycosyltransferases of Mycobacterium tuberculosis—roles in the synthesis of arabinogalaetan, lipoarahinolrnlannan, and other glyeoeonjugates. Glycobiology 17:35r–56r https://doi.org/10.1093/glycob/cwm010
6	C Breton, S Fournel-Gigleux, MM Palcic (2012) Recent structures, evolution and mechanisms of glycosyltransferases. Curr Opin Struct Biol 22:540–549 https://doi.org/10.1016/j.sbi.2012.06.007
7	E Dai, H Zhang, X Zhou, Q Song, D Li, L Luo, X Xu, W Jiang, H Ling (2019) MycoResistance: a curated resource of drug resistance molecules in Mycobacteria. Database. https://doi.org/10.1093/database/baz074
8	VE Escuyer, MA Lety, JB Torrelles, KH Khoo, JB Tang, CD Rithner, C Frehel, MR McNeil, PJ Brennan, D Chatterjee (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 https://doi.org/10.1074/jbc.M102272200
9	R Goude, AG Amin, D Chatterjee, T Parish (2008) The critical role of embC in Mycobacterium tuberculosis. J Bacteriol 190:4335–4341 https://doi.org/10.1128/JB.01825-07
10	M Jankute, JA Cox, J Harrison, GS Besra (2015) Assembly of the mycobacterial cell wall. Annu Rev Microbiol 69:405–423 https://doi.org/10.1146/annurev-micro-091014-104121
11	LL Lairson, B Henrissat, GJ Davies, SG Withers (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77:521–555 https://doi.org/10.1146/annurev.biochem.76.061005.092322
12	RE Lee, K Mikusova, PJ Brennan, GS Besra (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 https://doi.org/10.1021/ja00153a002
13	RE Lee, PJ Brennan, GS Besra (1997) Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay. Glycobiology 7:1121–1128 https://doi.org/10.1093/glycob/7.8.1121
14	HY Lee, HJ Myoung, HE Bang, GH Bai, SJ Kim, JD Kim, SN Cho (2002) Mutations in the embB locus among Korean clinical isolates of Mycobacterium tuberculosis resistant to ethambutol. Yonsei Med J 43:59–64 https://doi.org/10.3349/ymj.2002.43.1.59
15	RE Lee, W Li, D Chatterjee, RE Lee (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 https://doi.org/10.1093/glycob/cwh150
16	MA Lety, S Nair, P Berche, V Escuyer (1997) A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 41:2629–2633 https://doi.org/10.1128/AAC.41.12.2629
17	C Lizak, S Gerber, S Numao, M Aebi, KP Locher (2011) X-ray structure of a bacterial oligosaccharyltransferase. Nature 474:350–355 https://doi.org/10.1038/nature10151
18	V Makarov, G Manina, K Mikusova, U Mollmann, O Ryabova, B Saint-Joanis, N Dhar, MR Pasca, S Buroni, AP Lucarelliet al. (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking Arabinan synthesis. Science 324:801–804 https://doi.org/10.1126/science.1171583
19	K Mikusova, RA Slayden, GS Besra, PJ Brennan (1995) Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob Agents Chemother 39:2484–2489 https://doi.org/10.1128/AAC.39.11.2484
20	M Napiorkowska, J Boilevin, T Sovdat, T Darbre, JL Reymond, M Aebi, KP Locher (2017) Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nat Struct Mol Biol 24:1100–1106 https://doi.org/10.1038/nsmb.3491
21	KD Parris, L Lin, A Tam, R Mathew, J Hixon, M Stahl, CC Fritz, J Seehra, WS Somers (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 https://doi.org/10.1016/S0969-2126(00)00178-7
22	PK Qasba, B Ramakrishnan, E Boeggeman (2005) Substrateinduced conformational changes in glycosyltransferases. Trends Biochem Sci 30:53–62 https://doi.org/10.1016/j.tibs.2004.11.005
23	SV Ramaswamy, AG Amin, S Goksel, CE Stager, SJ Dou, H El Sahly, SL Moghazeh, BN Kreiswirth, JM Musser (2000) Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 44:326–336 https://doi.org/10.1128/AAC.44.2.326-336.2000
24	H Safi, B Sayers, MH Hazbon, D Alland (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 https://doi.org/10.1128/AAC.01486-07
25	CM Sassetti, DH Boyd, EJ Rubin (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84 https://doi.org/10.1046/j.1365-2958.2003.03425.x
26	M Seidel, LJ Alderwick, HL Birch, H Sahm, L Eggeling, GS Besra (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 https://doi.org/10.1074/jbc.M700271200
27	L Shi, S Berg , A Lee, JS Spencer, J Zhang, V Vissa, MR McNeil, KH Khoo, D Chatterjee (2006) The carboxy terminus of EmbC from Mycobacterium smegmatis mediates chain length extension of the arabinan in lipoarabinomannan. J Biol Chem 281:19512–19526 https://doi.org/10.1074/jbc.M513846200
28	SB Snapper, RE Melton, S Mustafa, T Kieser, WR Jacobs (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919 https://doi.org/10.1111/j.1365-2958.1990.tb02040.x
29	Q Sun, TY Xiao, HC Liu, XQ Zhao, ZG Liu, YN Li, H Zeng, LL Zhao, KL Wan (2018) Mutations within embCAB are associated with variable level of ethambutol resistance in Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother 62:e01279–17 https://doi.org/10.1128/AAC.01279-17
30	K Takayama, JO Kilburn (1989) Inhibition of synthesis of arabinogalactanby ethambutol in Mycobacterium smegmatis. Antimicrob Agents Chemother 33:1493–1499 https://doi.org/10.1128/AAC.33.9.1493
31	I Vasileios, CM Herrera, KM Schultz, OB Clarke, J Vendome, D Tomasek, S Banerjee, KR Rajashankar, MB Dufrisne, B Klosset al. (2016) Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Nature 351:6273 https://doi.org/10.1126/science.aad1172
32	WHO (2018) Global tuberculosis report 2018. WHO, Geneva
33	BA Wolucka, MR McNeil, E de Hoffmann, T Chojnacki, PJ Brennan (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	HC Wong, G Liu, YM Zhang, CO Rock, J Zheng (2002) The solution structure of acyl carrier protein from Mycobacterium tuberculosis. J Biol Chem 277:15874–15880 https://doi.org/10.1074/jbc.M112300200
35	L Zhang, Y Zhao, Y Gao, L Wu, R Gao, Q Zhang, Y Wang, C Wu, F Wu, SS Gurchaet al.(2020) Structures of cell wall arabinosyl transferases with the anti-tuberculosis drug ethambutol. Science. https://doi.org/10.1126/science.aba9102

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