25 24 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Macromolecular Crystallography Group Beamline I04-1 Crystal structure of an anti-TB drug target Related publication: 1. Zhang L., ZhaoY., GaoY.,Wu L., Gao R., Zhang Q.,WangY.,Wu C.,Wu F., Gurcha S. S.,Veerapen N., Batt S. M., ZhaoW., Qin L.,Yang X.,WangM., ZhuY., Zhang B., Bi L., Zhang X.,Yang H., Guddat L.W., XuW.,Wang Q., Li J., Besra G. S. & Rao Z. Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol. Science (80-. ). 368, 1211–1219 (2020). DOI: 10.1126/science.aba9102 Publication keywords: Mycobacterium tuberculosis ; CellWall; Structure; Arabinosyltransferase; Ethambutol; Drug target T uberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), is a life-threatening disease that has plagued humanity for thousands of years. Mycobacteria, including Mtb , synthesise a complex cell wall to support and protect the bacterial cells. The front-line anti-TB drug ethambutol inhibits the synthesis of themycobacterial cell wall by targeting the Embproteins (EmbA, EmbBand EmbC). However, although the drug has been in use for fifty years, its mode of action remains unclear. In Mtb ,EmbCisrequiredforthesynthesisoflipoarabinomannan(LAM),animportantvirulencefactorthataddstothepathogen’seffectiveness and helps it infect host cells. LAM also plays a crucial role in host-pathogen interactions and modulating the host immune response during infection. However, the three-dimensional structure of the Emb proteins had yet to be determined. To address the lack of structural information and functional analysis, an international team of researchers determined the crystal structure of EmbC from Mycobacterium smegmatis in complex with a sugar substrate, using Macromolecular Crystallography (MX) on Diamond Light Source's I04-1 beamline. Unexpectedly, they found that the mycobacterial acyl-carrier-protein (AcpM) is bound to EmbC to form a stable EmbC 2 -AcpM 2 complex. A combination of the MX data with a cryo-EM structure of the ethambutol-bound EmbC 2 -AcpM 2 complex suggests that ethambutol works by binding to the same sites as both donor and acceptor substrates of EmbC. These results provide a structural basis for understanding the biochemical function and inhibition of EmbC and the development of new anti-tuberculosis agents. Tuberculosis (TB) caused by Mycobacterium tuberculosis ( Mtb ) is a life- threatening disease that accounts for more than 1.4 million deaths per annum 1 . Ethambutol is one of the five first line anti-TB drugs that are currently in clinical use to treat TB. The membrane-embedded Emb proteins EmbA, EmbB and EmbC, which are involved in cell wall biosynthesis, are regarded as the targets of ethambutol. In Mtb , EmbA and EmbB are responsible for the synthesis of arabinogalactan (AG), while EmbC is required for the synthesis of lipoarabinomannan (LAM) 2 . In particular, LAM is an important glycolipidwhich plays a key role in host–pathogen interactions, as well as in modulating the host immune response during infection 3 . EmbC, like other Emb proteins, is a GT-C family arabinofuranosyl- transferase. Its specific role is to transfer an arabinofuranose residue from decaprenyl-phosphate-arabinose (DPA) to its acceptor, a LAM precursor, leading to the elongation of the arabinan chain in LAM biosynthesis (Fig. 1A). To address the lack of structural information and paucity of functional analysis of any Emb proteins, EmbC from Mycobacterium smegmatis ( Msm ) was selected for structural and functional study. Purified EmbC Msm protein was shown functional as an a (1 → 5) arabinosyltransferase in our established cell-free activity assay, whose activity was inhibited by ethambutol, thus confirming that ethambutol targets EmbC protein. The crystal structure of EmbC Msm was then determined in complex with a di-arabinose substrate at 3.3 Å resolution. The phase problem was solved by single-wavelength anomalous dispersion (SAD). The final model has R work / R free values of 0.232 and 0.265, respectively. Most of the polypeptide could be built into the electron density maps except for residues 1-9, 780-810 of EmbC. The structure has shown a homo-dimeric assembly of EmbC associated with an unexpected AcpM protein at the cytoplasmic surface of each EmbC monomer (Fig. 1B). Analysis of the active site and location of bound sugar substrate and ligands also provides valuable insights into the catalytic basis for arabinose transfer by EmbC. Within the asymmetric unit there are two EmbCs which form a dimer at the transmembrane interface. The mode of dimerisation is characterised by forming hydrophobic clusters between transmembrane (TM) domains close to the cytoplasmic side and periplasmic side. The overall structure of EmbC Msm is composed of a 15-helix transmembrane (TM) domain and N-/C- terminal periplasmic domains (identified as PN and PC). The PN domain (also periplasmic domain linking TM1 and TM2) adopts a jelly-roll-fold (Fig. 1C), which is typical for polysaccharide binding units 4 .The PC domain can be divided into two subdomains, with subdomain-I displaying a mixed a /β structure and subdomain-II exhibiting a jelly-roll-fold (Fig. 1C). The active site is located in a pocket at the junction between the TM domain and the periplasmic domains, composed by PL2-6, helix a 6 in PC domain and residue Trp965 in PC domain. The Msm AcpM has a four-helix topology arranged in a right-handed bundle, similar to that of Mtb AcpM (PDB code: 1KLP). AcpM binds to each EmbC protomer through extensive electrostatic interactions. Helix a 2 of AcpM and the connecting loops at its N-/C-terminus are intimately engaged with the CLs of the EmbC protein (Fig. 1D). This is consistent with the known role of a 2 in AcpM as a contact site with its target proteins ( e.g. AcpS). Mutagenesis and functional studies showed AcpM plays a role in modulating LAM synthesis in vivo by forming complex with EmbC 2 . The Emb proteins have two substrates, an arabinose donor DPA and an acceptor arabinan. The structure shows that there are two substrate entrances leading to the active site, henceforth denoted as the donor entrance and the acceptor entrance (Fig. 2A). In the crystal structure of di-arabinose-bound EmbC 2 , an endogenous phosphate ion appears to be trapped in the active site by the di-arabinose and maltose (part of detergent DDM used for purification) (Fig. 2B-C). It is bound to a positively charged region that includes Arg383, His574 and His575 and is near Thr570, Trp572 and the catalytic Asp279 (Fig. 2B-C). It is proposed that this phosphate represents the phosphate group of DPA as K d values of DPA for any single mutant EmbC proteins were greatly reduced compared with the wildtype. Furthermore, enzymatic activity was also shown completely lost for these mutants of the phosphate binding site. The di-arabinoside group, which is identified as part of the substrate analogue Ara 2 OC8 soaked with EmbC during crystallisation, binds between the catalytic site Asp279 and the phosphate (Fig. 2C). Considering that the two arabinoside groups are located on different sites of Asp279, for clarity, the positions of the two-arabinofuranose rings were denoted as D site (arabinose from donor) and A 0 site (arabinose from the terminal residue of acceptor). The arabinofuranose in the D-site is most likely mimicking the one originated from donor DPA, while the other in the A 0 -site resembles an arabinose from the terminal residue in the acceptor. When superimposing the crystal structure of EmbC in complex with disaccharide to the cryo-EM structure of the ethambutol-bound EmbC, we found that ethambutol overlapped with the disaccharide in a high degree of similarity (Fig. 2D). Since the di-saccharide represents the arabinose groups from both donor and acceptor, we infer that ethambutol inhibits the arabinose transfer reaction by competing with the binding of both substrates in the active site, in accordance with the hypothesis that ethambutol interferes with transfer of arabinose as evidenced by the rapid accumulation of DPA in ethambutol treated Msm cells. In summary, with the support frombeamline I04-1 at Diamond, the crystal structure of a mycobacterial EmbC 2 -AcpM 2 complex has been determined. The di-arabinose bound structure of the EmbC 2 -AcpM 2 complex allows us to understand the structural features required for catalysis. By structural comparison it is suggested ethambutol functions by competing with the substrates for binding to EmbC.The structural informationwill greatly facilitate the development of new anti-tuberculosis agents. References: 1. Global Tuberculosis Report 2020. https://www.who.int/publications/i/ item/9789240013131 2. Jankute M. et al. Assembly of the Mycobacterial Cell Wall. Annu. Rev. Microbiol . 69 , 405–423 (2015). DOI: 10.1146/annurev- micro-091014-104121 3. Korkegian A. et al. Mutations in the Essential Arabinosyltransferase EmbC Lead to Alterations in Mycobacterium tuberculosis Lipoarabinomannan*. J. Biol. Chem. 289 , 35172–35181 (2014). DOI: 10.1074/jbc.M114.583112 4. Boraston A. B. et al. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem. J. 382 , 769–781 (2004). DOI: 10.1042/BJ20040892 Funding acknowledgement: This work was supported by grants from the National Key R&D Program of China (2017YFC0840300) and Project of International Cooperation and Exchanges NSFC (81520108019) to Z.R. Corresponding authors: Dr Lu Zhang, ShanghaiTech University,
[email protected] ; Dr Zihe Rao, ShanghaiTech University; Tsinghua University; Institute of Biophysics, Chinese Academy of Sciences; Nankai University,
[email protected] Figure 1: Function and structure of the EmbC 2 -AcpM 2 complex. (A) Schematic representation of the catalytic reaction of EmbC 2 -AcpM 2 . (B) Overall structure of EmbC 2 -AcpM 2 . (C) Structural details of (left) PN and (right) PC domain. (D) Binding interface of EmbC and AcpM. Figure 2: Substrates binding in the active site of the EmbC 2 -AcpM 2 complex. (A) Overview of the substrate entrances to the active site. (B) Phosphate binding sites of EmbC. (C) Clipped view of the active site with acceptor entrance and donor entrance. Ara 2 , maltose (part of detergent DDM), Pi (phosphate ion), and the catalytic residue (Asp279) are shown as sticks. (D) Structural superposition of di-arabinose bound EmbC in this paper and cryo-EM structure of ethambutol bound EmbC (PDB entry: 7BVE).