Limits...
The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

Alderwick LJ, Lloyd GS, Ghadbane H, May JW, Bhatt A, Eggeling L, Fütterer K, Besra GS - PLoS Pathog. (2011)

Bottom Line: Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively.Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM.The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

Show MeSH

Related in: MedlinePlus

Stereo diagram of EmbCCT and topology of its subdomains.A) Stereo ribbon diagram of EmbCCT with definition of the secondary structure elements. Grey spheres indicate the boundaries of the disordered loops. The Ca2+ ion (yellow sphere), and positions of Trp985 (yellow sticks) and of the Ara(1→5)Ara-O-C8 ligand (magenta) are shown. B) Topology diagrams of subdomains I (top) and II (bottom), illustrating the connectivity of secondary structure elements and the jelly roll topology of subdomain II.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044687&req=5

ppat-1001299-g002: Stereo diagram of EmbCCT and topology of its subdomains.A) Stereo ribbon diagram of EmbCCT with definition of the secondary structure elements. Grey spheres indicate the boundaries of the disordered loops. The Ca2+ ion (yellow sphere), and positions of Trp985 (yellow sticks) and of the Ara(1→5)Ara-O-C8 ligand (magenta) are shown. B) Topology diagrams of subdomains I (top) and II (bottom), illustrating the connectivity of secondary structure elements and the jelly roll topology of subdomain II.

Mentions: EmbCCT crystallised in space group P6522 over a diverse range of reservoir conditions, with one molecule in the crystallographic asymmetric unit. Crystals were generated with or without an Araf acceptor analogue (see below) present in the crystallisation droplet. The experimental density, phased by multi-wavelength anomalous dispersion (2.7 Å, Table 1), was of very good quality (Fig. S2A), defining the structure for residues 735–1067, except for two disordered loops (795–824 and 1016–1037, Fig. 2A). EmbCCT is composed of two distinct subdomains, separated by a deep crevice marked by the disordered loops (residues 795–824 and 1016–1037). Subdomain I, which encompasses residues 746–760 and 967–1067, displays a mixed α/β structure, with a 5-stranded β-sheet forming a semi-barrel (Fig. 2A). The long H6-S13 loop, which forms a minor crystal packing interface, protrudes from the core of subdomain I with a helical half-turn at its tip (Fig. 2A). Subdomain II (residues 761–966) forms an anti-parallel β-sandwich structure, of which the ‘outer’ sheet (S2, S4, S10, S6, S7) faces solvent while the ‘inner’ sheet (S3, S11, S5, S9, S8) packs against the core of the domain (Fig. 2A). The β-sandwich of subdomain II assumes a jellyroll fold (Fig. 2B), a fold typical for polysaccharide binding units in plant lectins and carbohydrate active enzymes [21]. Although not part of the formal jellyroll description, strands S2 and S8 extend the ‘outer’ and ‘inner’ sheet, respectively, while helix H4 forms a boundary to the ‘outer’ sheet. A high-density peak (14σ, anomalous density difference map, Fig. 3A) is embedded between loops S3–S4 and S10–S11. Quasi-octahedral coordination geometry and the distribution of peak-ligand distances from 2.40 to 2.63 Å (Fig. 3A) suggest a bound Ca2+ ion [22]. The metal ion appears shielded from solvent, although including 10 mM EDTA in the cryoprotectant buffer significantly diminished the height of the density peak (Fig. S2B). Substitution of Asp949 by serine in EmbCCT, the only side chain in direct contact with the Ca2+ ion (2.6 Å, bidentate, Fig. 3A), resulted in very poor recombinant expression compared to wild-type and other point mutants probed in this study (see below). Together these observations suggest that the Ca2+ ion is important for the structural integrity of EmbCCT.


The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

Alderwick LJ, Lloyd GS, Ghadbane H, May JW, Bhatt A, Eggeling L, Fütterer K, Besra GS - PLoS Pathog. (2011)

Stereo diagram of EmbCCT and topology of its subdomains.A) Stereo ribbon diagram of EmbCCT with definition of the secondary structure elements. Grey spheres indicate the boundaries of the disordered loops. The Ca2+ ion (yellow sphere), and positions of Trp985 (yellow sticks) and of the Ara(1→5)Ara-O-C8 ligand (magenta) are shown. B) Topology diagrams of subdomains I (top) and II (bottom), illustrating the connectivity of secondary structure elements and the jelly roll topology of subdomain II.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044687&req=5

ppat-1001299-g002: Stereo diagram of EmbCCT and topology of its subdomains.A) Stereo ribbon diagram of EmbCCT with definition of the secondary structure elements. Grey spheres indicate the boundaries of the disordered loops. The Ca2+ ion (yellow sphere), and positions of Trp985 (yellow sticks) and of the Ara(1→5)Ara-O-C8 ligand (magenta) are shown. B) Topology diagrams of subdomains I (top) and II (bottom), illustrating the connectivity of secondary structure elements and the jelly roll topology of subdomain II.
Mentions: EmbCCT crystallised in space group P6522 over a diverse range of reservoir conditions, with one molecule in the crystallographic asymmetric unit. Crystals were generated with or without an Araf acceptor analogue (see below) present in the crystallisation droplet. The experimental density, phased by multi-wavelength anomalous dispersion (2.7 Å, Table 1), was of very good quality (Fig. S2A), defining the structure for residues 735–1067, except for two disordered loops (795–824 and 1016–1037, Fig. 2A). EmbCCT is composed of two distinct subdomains, separated by a deep crevice marked by the disordered loops (residues 795–824 and 1016–1037). Subdomain I, which encompasses residues 746–760 and 967–1067, displays a mixed α/β structure, with a 5-stranded β-sheet forming a semi-barrel (Fig. 2A). The long H6-S13 loop, which forms a minor crystal packing interface, protrudes from the core of subdomain I with a helical half-turn at its tip (Fig. 2A). Subdomain II (residues 761–966) forms an anti-parallel β-sandwich structure, of which the ‘outer’ sheet (S2, S4, S10, S6, S7) faces solvent while the ‘inner’ sheet (S3, S11, S5, S9, S8) packs against the core of the domain (Fig. 2A). The β-sandwich of subdomain II assumes a jellyroll fold (Fig. 2B), a fold typical for polysaccharide binding units in plant lectins and carbohydrate active enzymes [21]. Although not part of the formal jellyroll description, strands S2 and S8 extend the ‘outer’ and ‘inner’ sheet, respectively, while helix H4 forms a boundary to the ‘outer’ sheet. A high-density peak (14σ, anomalous density difference map, Fig. 3A) is embedded between loops S3–S4 and S10–S11. Quasi-octahedral coordination geometry and the distribution of peak-ligand distances from 2.40 to 2.63 Å (Fig. 3A) suggest a bound Ca2+ ion [22]. The metal ion appears shielded from solvent, although including 10 mM EDTA in the cryoprotectant buffer significantly diminished the height of the density peak (Fig. S2B). Substitution of Asp949 by serine in EmbCCT, the only side chain in direct contact with the Ca2+ ion (2.6 Å, bidentate, Fig. 3A), resulted in very poor recombinant expression compared to wild-type and other point mutants probed in this study (see below). Together these observations suggest that the Ca2+ ion is important for the structural integrity of EmbCCT.

Bottom Line: Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively.Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM.The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

Show MeSH
Related in: MedlinePlus