Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

• Published in: Molecular cell Vol. 78; no. 4; pp. 683 - 699.e11
• Main Authors: Tan, Yong Zi, Zhang, Lei, Rodrigues, José, Zheng, Ruixiang Blake, Giacometti, Sabrina I., Rosário, Ana L., Kloss, Brian, Dandey, Venkata P., Wei, Hui, Brunton, Richard, Raczkowski, Ashleigh M., Athayde, Diogo, Catalão, Maria João, Pimentel, Madalena, Clarke, Oliver B., Lowary, Todd L., Archer, Margarida, Niederweis, Michael, Potter, Clinton S., Carragher, Bridget, Mancia, Filippo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.05.2020
• Subjects: acyl carrier protein; Acyl Carrier Protein - genetics; Acyl Carrier Protein - metabolism; arabinofuranose; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Catalytic Domain; Cell Membrane - metabolism; Cell Wall - metabolism; Cryoelectron Microscopy - methods; Galactans - metabolism; glycosyltransferase; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; lipoarabinomannan; lipomannan; Lipopolysaccharides - metabolism; membrane protein; Mutation; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Mycobacterium smegmatis - growth & development; Mycobacterium tuberculosis; nanodisc; Phylogeny; Protein Conformation; single-particle cryo-electron microscopy; Substrate Specificity; arabinofuranose; single-particle cryo-electron microscopy; lipomannan; acyl carrier protein; lipoarabinomannan; Mycobacterium tuberculosis; glycosyltransferase; nanodisc; membrane protein
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2020.04.014

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides—arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function. [Display omitted] •Cryo-EM structures of mycobacterial arabinofuranosyltransferase D (AftD) were solved•AftD has a conserved GT-C glycosyltransferase fold and binds complex arabinose glycans•Acyl carrier protein (ACP) is complexed to AftD, also endogenously•Impairment of ACP binding alters conformation, suggesting ACP plays a regulatory role Tan et al. present the cryo-EM structures of essential wild-type and mutant mycobacterial arabinofuranosyltransferase D (AftD), revealing the putative active site geometry and carbohydrate-binding modules. Acyl carrier protein (ACP) was tightly associated with AftD. Impairing ACP binding blocks AftD’s active site, suggesting that ACP regulates enzyme function.