Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme's preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies

Correction: Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome.

[This corrects the article DOI: 10.1371/journal.pone.0183272.]

The mycobacterial GpgS shows high structural homology to the mannosylglycerate synthase (MgS) from <i>Rhodothermus marinus</i>.

<p>a) Stereo representation of the superposed <i>M. tuberculosis</i> GpgS (yellow) and <i>R. marinus</i> MgS (PDB entry 2BO4, green) three-dimensional structures; the UDP and 3-phosphoglycerate molecules crystallized in complex with GpgS are shown in a ball-and-stick representation (carbon, cyan; oxygen, red; nitrogen, blue; phosphorous, orange). b) Structure based sequence alignment of GpgS (this work), <i>R. xylanophilus</i> MpgS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003748#pone.0003748-SMoura1" target="_blank">[34]</a>, and <i>R. marinus</i> MgS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003748#pone.0003748-Flint1" target="_blank">[43]</a>. For clarity, the sequence of the crystallographic GpgS model (with the missing N-terminal region and 166–183 loop) was also included in the alignment (GpgS-crystal). The secondary structure of MgS is represented above and conserved residues are indicated by stars below the alignment. c) Cartoon representation of the dimer interface of structurally homologous glycosyltransferases. From left to right: GpgS from <i>M. tuberculosis</i>; MgS from <i>R. marinus</i> (PDB entry 2BO4); putative GT from <i>B. fragilis</i> (PDB entry 3BCD).</p

GpgS acceptor and donor sugar binding pockets.

<p>a) Close view of the modeled GpgS complex with GDP-mannose and 3-phosphoglycerate. The GpgS active site is shown as a surface representation, the 3-phosphoglycerate is shown as a ball-and-stick model (colour-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003748#pone-0003748-g004" target="_blank">Fig. 4</a>), and the GDP-glucose is shown in yellow sticks. A dashed circle is drawn around the GDP NH2 group that cannot be accommodated within the nucleotide binding pocket, due to the orientation of the side chain of Ser81 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003748#pone-0003748-g004" target="_blank">Fig. 4</a>). A dashed line between the oxygen of the acceptor 3-phosphoglycerate and the anomeric C1 of the mannose is shown corresponding to a distance of 2.8 Å. b) Detailed stereoview of the GpgS (colour coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003748#pone-0003748-g004" target="_blank">Fig. 4</a>) donor sugar binding site in comparison with MgS (green). Some of the GpgS residues mentioned in the text are labeled, and hydrogen bonds are represented by dashed lines. The mannose moiety is shown at the top of the image in blue sticks and Ser210 is highlighted in salmon. c) View of the acceptor sugar binding pocket indicating that a movement of the region containing the amino acids Leu209 and Ser210 would be necessary to prevent steric clash with the O2 of mannose.</p

<i>M. tuberculosis</i> GpgS structure displays a GT-A fold.

<p>a). Cartoon representation of GpgS three-dimensional structure. Helices are represented as cilinders and β-strands are shown as arrows. The structure is coloured from dark blue (N-terminus) to red (C-terminus). Disordered regions at the protein surface that were not visible in the electron density (Arg167-Gly184 and Leu294-Asp302) are indicated by asterisks. b) Topology diagram of <i>M. tuberculosis</i> GpgS; the N-terminal Rossmann-like subdomain is coloured green, while the C-terminal subdomain is shown in blue; the conserved Asp-Xaa-Asp (DSD) motif, immediately after strand β5, is boxed; loops that are not visible in the electron density maps are shown as dotted arrows.</p

Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome

<div><p>In eukaryotes, ribosome assembly is a highly complex process that involves more than 200 assembly factors that ensure the folding, modification and processing of the different rRNA species as well as the timely association of ribosomal proteins. One of these factors, Mpp10 associates with Imp3 and Imp4 to form a complex that is essential for the normal production of the 18S rRNA. Here we report the crystal structure of a complex between Imp4 and a short helical element of Mpp10 to a resolution of 1.88 Å. Furthermore, we extend the interaction network of Mpp10 and characterize two novel interactions. Mpp10 is able to bind the ribosome biogenesis factor Utp3/Sas10 through two conserved motifs in its N-terminal region. In addition, Mpp10 interacts with the ribosomal protein S5/uS7 using a short stretch within an acidic loop region. Thus, our findings reveal that Mpp10 provides a platform for the simultaneous interaction with multiple proteins in the 90S pre-ribosome.</p></div

Crystal structure of <i>ct</i>Imp4-<i>ct</i>Mpp10 complex.

<p>(A) Overall structure of the <i>ct</i>Imp4-<i>ct</i>Mpp10 complex. The BRIX domain of <i>ct</i>Imp4 (teal) is completed by helix α2 of <i>ct</i>Mpp10 (grey). Helix α1 of <i>ct</i>Mpp10 interacts via Trp500 with <i>ct</i>Imp4 (inset). For the sake of clarity <i>ct</i>Mpp10 helix α1 is omitted in the other panels. (B) Comparison of <i>ct</i>Imp4-<i>ct</i>Mpp10 with <i>an</i>Rpf2-<i>an</i>Rrs1 (PDB-ID: 5BY8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183272#pone.0183272.ref030" target="_blank">30</a>]) The overall fold between the BRIX domain proteins <i>ct</i>Imp4 (teal) and <i>an</i>Rpf2 (light-blue) and their ligands <i>ct</i>Mpp10 (grey) and <i>an</i>Rrs1 (orange) is preserved. Beta-augmentation on the C-terminal sub-domain of the BRIX protein is only observed in the <i>an</i>Rpf2-<i>an</i>Rrs1 complex (red circle). (C) Rigid body docking of the <i>ct</i>Imp4-<i>ct</i>Mpp10 complex into the 5.1 Å map of the yeast 90S particle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183272#pone.0183272.ref008" target="_blank">8</a>]. While the BRIX domain of <i>ct</i>Imp4 (teal) and <i>ct</i>Mpp10 helix α2 (grey) are completely covered by electron density, helix α1 of <i>ct</i>Mpp10 is not. This suggests that in context of the 90S, this helix occupies another location. The tip of helix α1 is occupied by a RNA double helix (purple) in the yeast 90S density. (D) Alternative modeling of the termini of <i>ct</i>Imp4 and <i>ct</i>Mpp10 based on the crystal structure and cryo-EM density. In the crystal structure (grey/blue), the N-terminus (residues 515–530) of our <i>ct</i>Mpp10 construct interacts with <i>ct</i>Imp4, whereas when modeled based on the cryo-EM density it extends away (orange) from <i>ct</i>Imp4. Likewise the C-terminus of <i>ct</i>Imp4 can be extended and modeled (red) into the now remaining cavity between <i>ct</i>Mpp10 helix α1 (grey ribbon) and <i>ct</i>Imp4.</p