Sandbox Reserved 1053

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Revision as of 20:32, 27 April 2015

This Sandbox is Reserved from 02/09/2015, through 05/31/2016 for use in the course "CH462: Biochemistry 2" taught by Geoffrey C. Hoops at the Butler University. This reservation includes Sandbox Reserved 1051 through Sandbox Reserved 1080.

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Background

The antigen 85 (ag85) complex in Mycobacterium tuberculosis, which is responsible for causing the disease Tuberculosis, is composed of three intracellular membrane proteins: Ag85A, B, and C. The Ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. ^[1] The cell wall of Mycobacterium tuberculosis is composed of three primary molecules: peptidoglycans, arbinogalactans, and mycolic acids. Ag85C is of particular interest due to its transfer of mycolic acids, which is one of the major components in determining cell wall integrity. The mycolic acids are responsible for forming the outermost layer of the cell wall. Mycolic acids have a long fatty acid chain and exhibit extreme hydrophobicity, which effectively creates a hydrophobic envelope surrounding the bacterium. The hydrophobic envelope created by they mycolic acids creates a barrier against small hydrophilic molecules, such as Tuberculosis antibiotics. By targeting this mycoloyltransferase activity, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant Mycotaberia tuberculosis. ^[2]

Structure

1 General Structure
2 Enzymatic Activity
3 Methods of Inhibition
- 3.1 Inhibitors

General Structure

Figure 1: Octylthioglucoside, a substrate analog, shown in the binding pocket of Ag85C

Antigen 85C was crystallized in its dimeric form.^[3] The shown in the monomeric form is composed of helices, shown in pink, with one interwoven beta sheet, shown in yellow. The confrontation of a central β-sheet bordered by α–helices creates an a α/β hydrolase fold in Ag85C, and this tertiary conformation is highly conserved across enzymes that function in this capacity. ^[4] The substrate binding pocket of Ag85C is composed of two separate but equally important components; there is carbohydrate binding pocket for the trehalose, and there is a fatty acid binding pocket for the mycolic acid. As a result, trehalose monomycolate can effectively bind to the Ag85C binding pocket. This binding pocket is shown in the left image with a substrate mimic.

Enzymatic Activity

Figure 2: Relation of the catalytic triad to the octylthioglucoside analog in Ag85C

Mutagenesis studies have confirmed the Ag85C functions through a Glu-His-Ser , similar to that of chymotrypsin. By modifying each of the catalytic residues separately testing the enzyme’s relative activity, it has been shown that mutation of any one of these residues dramatically reduces activity. The S124 alcohol’s nucleophilicity is inductively strengthened through H260 and E224, which allows the S124 residue to catalyze a reaction that involves trehalose 6, 6’-dimycolate. The formation of the functional catalytic triad relies on upon Van der Waals interaction between C209 and the peptide bond between L232 and T231. This interaction results in a kinked conformation of the α9 helix, which promotes that activity of the catalytic triad. As a result, Ag85C, a mycolyl transferase, can facilitate the modification of trehalose monomycolates to trehalose dimycolates, which are then transported to the bacterial cell wall. This reaction is shown in Figure 3 below.

Figure 3: General reaction catalyzed by Antigen 85C

Methods of Inhibition

Figure 4: Cys209 stabilizing kinked formation of alpha-9 helix in the native Ag85C enzyme

Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the Mycobacteria tuberculosis cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between , which can be seen in both the scene and Figure 4. The C209 facilitated interaction causes the to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.

Figure 5: Ebselen inhibition relaxing the alpha-9 helix in Ag85C-Ebselen

Ag85C can be inhibited by ebselen, which covalently bounds to the sulfur in C209. Ebselen is a thiol-modifying agent that serves as an electrophile for a C209 nucleophilic attack that results in sulfur-selenium bond formation. Co-crystallization of ebselen with Ag85C provides an explanation for the mechanism of ebselen-based inhibition. The addition of ebselen increases the distance between C209 and L232-T231, which effectively disrupts the interaction that holds the α9 helix in the active conformation. The disruption of this interaction causes the α9 helix to . Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues, which can be seen in Figure 5. The pink helix represents the native enzyme, and the tan helix represents Ag85C covalently bound to ebselen, which is shown in green. Relaxation of the α9 helix due to ebselen removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues destroys the charge relay mechanism, and as a result, the nucleophilicity of the S124 alcohol is not longer strengthened, which decreases serine hydrolytic activity.

Inhibitors

Additional thiol-modifying agents, p-chloromercuribenzoic acid and iodoacetamide, were crystalized with Ag85C. The structures show that each of these thiol-reactive inhibitors covalently bound to C209 and caused a relaxation of the α9 helix in a similar fashion to ebselen.

Figure 6: Known thiol-reactive inhibitors of Ag85C

References

↑ Ronning DR, Klabunde T, Besra GS, Vissa VD, Belisle JT, Sacchettini JC. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nat Struct Biol. 2000 Feb;7(2):141-6. PMID:10655617 doi:10.1038/72413
↑ Jackson M, Raynaud C, Laneelle MA, Guilhot C, Laurent-Winter C, Ensergueix D, Gicquel B, Daffe M. Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol. 1999 Mar;31(5):1573-87. PMID:10200974
↑ Favrot L, Lajiness DH, Ronning DR. Inactivation of the Mycobacterium tuberculosis Antigen 85 complex by covalent, allosteric inhibitors. J Biol Chem. 2014 Jul 14. pii: jbc.M114.582445. PMID:25028518 doi:http://dx.doi.org/10.1074/jbc.M114.582445
↑ Ronning DR, Klabunde T, Besra GS, Vissa VD, Belisle JT, Sacchettini JC. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nat Struct Biol. 2000 Feb;7(2):141-6. PMID:10655617 doi:10.1038/72413

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Revision as of 20:09, 27 April 2015 (edit) Ben Zercher (Talk \| contribs) ← Previous diff		Revision as of 20:32, 27 April 2015 (edit) (undo) Ben Zercher (Talk \| contribs) Next diff →
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	[[Image:C209.jpeg\|200 px\|left\|thumb\|'''Figure 4:''' Cys209 stabilizing kinked formation of alpha-9 helix in the native [http://www.rcsb.org/pdb/explore/explore.do?structureId=1dqz Ag85C] enzyme ]]		[[Image:C209.jpeg\|200 px\|left\|thumb\|'''Figure 4:''' Cys209 stabilizing kinked formation of alpha-9 helix in the native [http://www.rcsb.org/pdb/explore/explore.do?structureId=1dqz Ag85C] enzyme ]]

-	Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the ''Mycobacteria tuberculosis'' cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between C209 and L232-T 23, which can be seen in '''Figure 4'''. The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>α9 helix</scene> to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.	+	Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the ''Mycobacteria tuberculosis'' cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between <scene name='69/694220/C209/1'>C209 and L232-T 23</scene>, which can be seen in both the scene and '''Figure 4'''. The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>α9 helix</scene> to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.

Sandbox Reserved 1053

From Proteopedia

Revision as of 20:32, 27 April 2015

Background

Structure

Contents

General Structure

Enzymatic Activity

Methods of Inhibition

Inhibitors

References

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