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Crystal structures of Mycobacterium tuberculosis GlgE and complexes with non-covalent inhibitors.

Lindenberger JJ, Veleti SK, Wilson BN, Sucheck SJ, Ronning DR - Sci Rep (2015)

Bottom Line: The maltohexaose structure reveals a dominant site for α-glucan binding.To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site.These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Toledo, 2801 W. Bancroft St. Ms602, Toledo, OH, United States.

ABSTRACT
GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

No MeSH data available.


Related in: MedlinePlus

The high-affinity maltohexaose binding site of the Mtb GlgE.(A) Fo-Fc omit map calculated while omitting maltohexaose is contoured at 3σ showing the binding of maltohexaose (M6) to the Mtb GlgE. Binding is mediated by backbone carbonyls of T474 of the B domain, N512 and L628 of the A domain, and the carbonyl of the sidechain carboxamide group of N629 of the A domain. F631 of the A domain interacts with the fourth sugar via van der Waals interactions. (B) Sequence alignment between Sco GlgEI and Mtb GlgE. Areas defined by the magenta boxes and the M6 symbol indicate the residues forming the maltohexaose binding site observed in Mtb GlgE, while the teal circle indicates the cyclodextrin binding surface observed in Sco GlgEI. The orange box indicates residues important for binding cyclodextrin in Sco GlgEI but differ in Mtb GlgE. (C) Surface conservation comparison of the Mtb and Sco GlgE enzymes. Blue surface indicates identical residues, light-blue semi-conserved changes, and white no sequence similarities. The second subunit of the GlgE dimer uses grey to indicate lack of sequence similarity. The orange surface represents changes observed at the cyclodextrin binding pocket. Maltohexaose is shown as yellow spheres and α-cyclodextrin is shown with cyan bonds. Sequence alignment was performed using ESPript 3.0 (http://espript.ibcp.fr)32.
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f3: The high-affinity maltohexaose binding site of the Mtb GlgE.(A) Fo-Fc omit map calculated while omitting maltohexaose is contoured at 3σ showing the binding of maltohexaose (M6) to the Mtb GlgE. Binding is mediated by backbone carbonyls of T474 of the B domain, N512 and L628 of the A domain, and the carbonyl of the sidechain carboxamide group of N629 of the A domain. F631 of the A domain interacts with the fourth sugar via van der Waals interactions. (B) Sequence alignment between Sco GlgEI and Mtb GlgE. Areas defined by the magenta boxes and the M6 symbol indicate the residues forming the maltohexaose binding site observed in Mtb GlgE, while the teal circle indicates the cyclodextrin binding surface observed in Sco GlgEI. The orange box indicates residues important for binding cyclodextrin in Sco GlgEI but differ in Mtb GlgE. (C) Surface conservation comparison of the Mtb and Sco GlgE enzymes. Blue surface indicates identical residues, light-blue semi-conserved changes, and white no sequence similarities. The second subunit of the GlgE dimer uses grey to indicate lack of sequence similarity. The orange surface represents changes observed at the cyclodextrin binding pocket. Maltohexaose is shown as yellow spheres and α-cyclodextrin is shown with cyan bonds. Sequence alignment was performed using ESPript 3.0 (http://espript.ibcp.fr)32.

Mentions: GlgE utilizes an α-1,6-branched, α-1,4 glucan as a maltosyl-acceptor molecule and thereby extends the linear portion of the glucan using M1P as the maltosyl donor. To further characterize the maltosyl acceptor binding site and the mode of GlgE binding to linear polysaccharides, we soaked Mtb GlgE-MAL co-crystals with maltohexaose immediately prior to performing X-ray diffraction experiments. A crystal structure of a ternary complex with Mtb GlgE bound to maltose and maltohexaose (GlgE-M6) was solved to 4.0 Å resolution (Table 1). Initial inspection of the Fo-Fc maps calculated following rigid body refinement revealed a horseshoe-shaped density that was more than six times above background1112. This density, when fit with the maltohexaose ligand, corresponded to a single molecule of maltohexaose (Fig. 3A).


Crystal structures of Mycobacterium tuberculosis GlgE and complexes with non-covalent inhibitors.

Lindenberger JJ, Veleti SK, Wilson BN, Sucheck SJ, Ronning DR - Sci Rep (2015)

The high-affinity maltohexaose binding site of the Mtb GlgE.(A) Fo-Fc omit map calculated while omitting maltohexaose is contoured at 3σ showing the binding of maltohexaose (M6) to the Mtb GlgE. Binding is mediated by backbone carbonyls of T474 of the B domain, N512 and L628 of the A domain, and the carbonyl of the sidechain carboxamide group of N629 of the A domain. F631 of the A domain interacts with the fourth sugar via van der Waals interactions. (B) Sequence alignment between Sco GlgEI and Mtb GlgE. Areas defined by the magenta boxes and the M6 symbol indicate the residues forming the maltohexaose binding site observed in Mtb GlgE, while the teal circle indicates the cyclodextrin binding surface observed in Sco GlgEI. The orange box indicates residues important for binding cyclodextrin in Sco GlgEI but differ in Mtb GlgE. (C) Surface conservation comparison of the Mtb and Sco GlgE enzymes. Blue surface indicates identical residues, light-blue semi-conserved changes, and white no sequence similarities. The second subunit of the GlgE dimer uses grey to indicate lack of sequence similarity. The orange surface represents changes observed at the cyclodextrin binding pocket. Maltohexaose is shown as yellow spheres and α-cyclodextrin is shown with cyan bonds. Sequence alignment was performed using ESPript 3.0 (http://espript.ibcp.fr)32.
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f3: The high-affinity maltohexaose binding site of the Mtb GlgE.(A) Fo-Fc omit map calculated while omitting maltohexaose is contoured at 3σ showing the binding of maltohexaose (M6) to the Mtb GlgE. Binding is mediated by backbone carbonyls of T474 of the B domain, N512 and L628 of the A domain, and the carbonyl of the sidechain carboxamide group of N629 of the A domain. F631 of the A domain interacts with the fourth sugar via van der Waals interactions. (B) Sequence alignment between Sco GlgEI and Mtb GlgE. Areas defined by the magenta boxes and the M6 symbol indicate the residues forming the maltohexaose binding site observed in Mtb GlgE, while the teal circle indicates the cyclodextrin binding surface observed in Sco GlgEI. The orange box indicates residues important for binding cyclodextrin in Sco GlgEI but differ in Mtb GlgE. (C) Surface conservation comparison of the Mtb and Sco GlgE enzymes. Blue surface indicates identical residues, light-blue semi-conserved changes, and white no sequence similarities. The second subunit of the GlgE dimer uses grey to indicate lack of sequence similarity. The orange surface represents changes observed at the cyclodextrin binding pocket. Maltohexaose is shown as yellow spheres and α-cyclodextrin is shown with cyan bonds. Sequence alignment was performed using ESPript 3.0 (http://espript.ibcp.fr)32.
Mentions: GlgE utilizes an α-1,6-branched, α-1,4 glucan as a maltosyl-acceptor molecule and thereby extends the linear portion of the glucan using M1P as the maltosyl donor. To further characterize the maltosyl acceptor binding site and the mode of GlgE binding to linear polysaccharides, we soaked Mtb GlgE-MAL co-crystals with maltohexaose immediately prior to performing X-ray diffraction experiments. A crystal structure of a ternary complex with Mtb GlgE bound to maltose and maltohexaose (GlgE-M6) was solved to 4.0 Å resolution (Table 1). Initial inspection of the Fo-Fc maps calculated following rigid body refinement revealed a horseshoe-shaped density that was more than six times above background1112. This density, when fit with the maltohexaose ligand, corresponded to a single molecule of maltohexaose (Fig. 3A).

Bottom Line: The maltohexaose structure reveals a dominant site for α-glucan binding.To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site.These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Toledo, 2801 W. Bancroft St. Ms602, Toledo, OH, United States.

ABSTRACT
GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.

No MeSH data available.


Related in: MedlinePlus