Crystal structure of the putative cell-wall lipoglycan biosynthesis protein LmcA from Mycobacterium smegmatis

Abstract

The bacterial genus Mycobacterium includes important pathogens, most notably M. tuberculosis, which infects one-quarter of the entire human population, resulting in around 1.4 million deaths from tuberculosis each year. Mycobacteria, and the closely related corynebacteria, synthesize a class of abundant glycolipids, the phosphatidyl-myo-inositol mannosides (PIMs). PIMs serve as membrane anchors for hyperglycosylated species, lipomannan (LM) and lipoarabinomannan (LAM), which are surface-exposed and modulate the host immune response. Previously, in studies using the model species Corynebacterium glutamicum, NCgl2760 was identified as a novel membrane protein that is required for the synthesis of full-length LM and LAM. Here, the first crystal structure of its ortholog in Mycobacterium smegmatis, MSMEG_0317, is reported at 1.8 Å resolution. The structure revealed an elongated β-barrel fold enclosing two distinct cavities and one α-helix extending away from the β-barrel core, resembling a `cone with a flake' arrangement. Through xenon derivatization and structural comparison with AlphaFold2-derived predictions of the M. tuberculosis homolog Rv0227c, structural elements were identified that may undergo conformational changes to switch from `closed' to `open' conformations, allowing cavity access. An AlphaFold2-derived NCgl2760 model predicted a smaller β-barrel core with an enclosed central cavity, suggesting that all three proteins, which were collectively termed LmcA, may have a common mechanism of ligand binding through these cavities. These findings provide new structural insights into the biosynthetic pathway for a family of surface lipoglycans with important roles in mycobacterial pathogenesis.

Keywords: MSMEG_0317; Mycobacterium smegmatis; Mycobacterium tuberculosis; lipoarabinomannan; lipomannan.

Figure 1

(a) The PIM/LM/LAM biosynthetic pathway of mycobacteria. Early steps of PIM synthesis are performed by the cytoplasmic enzymes PimA (Korduláková et al., 2002 ▸), PimB (Guerin et al., 2009; Lea-Smith et al., 2008 ▸) and PatA (Korduláková et al., 2003 ▸) to produce AcPIM2 from phosphatidylinositol (PI). Further mannosylation yields AcPIM4, which is transported to the periplasm and can be processed by the mannosyltransferase PimE (Morita et al., 2006 ▸) to form AcPIM6, an end product, or channelled into a parallel pathway for LM and LAM synthesis by the lipoprotein LpqW (Crellin et al., 2008; Kovacevic et al., 2006; Marland et al., 2006 ▸). LM/LAM synthesis is catalysed by the PPM-dependent mannosyltransferases MptB, MptA and MptC (Kaur et al., 2006, 2007; Mishra et al., 2007, 2008; Mishra, Krumbach et al., 2011 ▸). A phospholipid-binding protein, LmeA (Rahlwes et al., 2017 ▸), is involved in maintaining MptA under stress conditions (Rahlwes et al., 2020 ▸). The focus of the current study, LmcA (underlined), also functions at the MptA step in C. glutamicum (Cashmore et al., 2017 ▸). (b) The MSMEG_0317 genetic locus. The MSMEG_0317 gene is encoded within a locus that is highly conserved in Corynebacterineae. Likely orthologous genes in the three species are shown using the same colour. Previously studied genes are tmaT (Yamaryo-Botte et al., 2015 ▸) and mtrP (Rainczuk et al., 2020 ▸), both with roles in cell-wall mycolic acid transport, and the LM/LAM biosynthesis gene NCgl2760 (Cashmore et al., 2017 ▸), while the remaining genes are uncharacterized. The focus of the current study is boxed. (c) Predicted membrane topology of MSMEG_0317. Following cleavage of the putative signal peptide (red), the mature protein is proposed to comprise a large periplasmic N-­terminal domain, a single transmembrane domain and a small cytoplasmic tail. (d) The elution profile of MSMEG_0317Δ on a HiLoad 16/60 Superdex 75 gel-filtration column suggesting a monomeric protein (top) and SDS–PAGE analysis of the eluted MSMEG_0317Δ (∼34 kDa) (bottom). The molecular-weight markers used for calibration are bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa) and equine myoglobin (17 kDa). See also Supplementary Fig. S1.

Figure 2

The amino-acid sequence and the crystal structure of the periplasmic domain of MSMEG_0317Δ. (a) The sequence of MSMEG_0317Δ showing secondary-structure elements derived from the crystal structure of MSMEG_0317Δ. (b) The crystal structure of MSMEG_0317Δ in different views. The secondary-structure elements are colour-coded. The disordered loop 6 is shown by dotted lines. See also Supplementary Figs. S2–S4.

Figure 3

Structural homology and surface representation of the enclosed cavities in MSMEG_0317Δ. (a) Comparison of the MSMEG_0317Δ fold with the CD36 superfamily of scavenger receptor proteins, including the human lysosomal integral membrane protein 2 (LIMP-2) and CD36, a fatty-acid transporter, which show an extended asymmetric β-barrel core. (b) Comparison of the MSMEG_0317Δ enclosed cavities with the CD36 cavity which binds palmitic acid. (c) Close-up of MSMEG_0317Δ cavity 1 entrance 1. (d) Close-up of MSMEG_0317Δ cavity 1 entrance 2. (e) Close-up of MSMEG_0317Δ cavity 2. Hydrogen-bond and salt-bridge interactions are shown as black dashed lines. See also Supplementary Fig. S5.

Figure 4

The crystal structure of xenon-derivatized MSMEG_0317Δ (referred to as MSMEG_0317Δ-Xe). (a) Comparison of the crystal structures of monomer 1 and monomer 2 of MSMEG_0317Δ with MSMEG_0317Δ-Xe. The positions of the five Xe atoms (Xe 1 to Xe 5) in monomers 1 and 2 of MSMEG_0317Δ-Xe are highlighted. The disordered loops 6 and 9 are shown as dotted lines. (b) Overlay of the crystal structure of MSMEG_0317Δ with MSMEG_0317Δ-Xe and close-up view of the base of the β-barrel core to highlight conformational flexibility near the region of loop 3, the α2 turn, loop 11 and loop 9. Loop 9 adopts a closed conformation in MSMEG_0317Δ, while in MSMEG_0317Δ-Xe loop 9 is disordered (dotted line). Hydrogen-bond interactions are shown as black dashed lines and van der Waals interactions are shown as red dashed lines. See also Supplementary Fig. S6.

Figure 5

AlphaFold2-derived predictions of MSMEG_0317 (AF MSMEG_0317) and Rv0227c (AF Rv0227c). (a) Structure of AF Rv0227c. (b) Structure of AF MSMEG_0317Δ. (c) Overlay of the crystal structure of MSMEG_0317Δ with AF Rv0227c and a close-up view of the base of the β-barrel core. Loop 6 in AF Rv0227c is resolved and this loop contains two additional β-strands. Loop 9 in AF Rv0227c adopts an ‘out’ or ‘open’ conformation, in contrast to loop 9 in the MSMEG_0317Δ crystal structure, which adopts an ‘in’ or ‘closed’ conformation. (d) Overlay of the crystal structure of MSMEG_0317Δ with AF MSMEG_0317 and a close-up view of the base of the β-barrel core. Loop 6 in AF MSMEG_0317 is resolved in a similar position as in AF Rv0227c, including the additional two β-strands. Like AF Rv0227c, loop 9 in AF MSMEG_0317 adopts an ‘out’ or ‘open’ conformation. (e) Comparison of the enclosed cavities of the MSMEG_0317Δ, AF Rv0227c and AF MSMEG_0317 models. See also Supplementary Fig. S7.

Figure 6

AlphaFold2-derived prediction of NCgl2760 (AF NCgl2760). AF NCgl2760 adopts a smaller β-­barrel core compared with MSMEG_0317 and Rv0227c; however, the central cavity is still a conserved feature. Note that the N-terminal helix may represent a signal peptide. See also Supplementary Figs. S8 and S9.