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M. tuberculosis Antigen 85B

Kat Meagley '11 and Suzanne Rohrback '11


I. Introduction

Mycobacterium tuberculosis, the bacteria which causes tuberculosis, kills millions of people worldwide each year.  As drug-resistant strains become increasingly prevalent, the need for new therapeutic targets has become vital.  Antigen 85B has emerged as one such possibility.

M. tuberculosis produces three variations of antigen 85 proteins: A, B, and C.  They are secreted, and found in phagosomal space and on the bacteria’s cell wall.  All variations function as mycolyl transferases, catalyzing  the transfer of mycolic acid from one trehalose 6-monomycolate to another, forming trehalose 6,6’-dimycolate (TDM), a glycolipid located at the external layer of the cell wall (Ronning et al, 2000; Anderson et al, 2001). 

It has been proposed that TDM inhibits phagosome-lysosome fusion, an essential step in the intracellular destruction of microorganisms, possibly through the production of cytokines (Lima et al, 2001).

The antigen 85 enzymes also bind to fibronectin, an extracellular matrix glycoprotein involved in numerous biological processes including immune responses.  This interaction appears to reduce phagocytosis of M. tuberculosis, promoting infection. (
Bentley-Hibbert et al 1999; Ronning et al 2004)

Recently, the structure of antigen 85B bound to its substrate trehalose was solved by Anderson et al (2001), leading to new insights about the function of the protein as well as its high potential as a therapeutic target.

II. General Structure

Antigen 85B is a 30 kDa protein composed of 285 residues.  Structurally, it forms an α-ß hydrolase fold with a core of 8 ß sheets surrounded by α helicies and a catalytic triad of Ser-126, His-262 and Glu-230 . Two molecules of the substrate trehalose are bound, on opposite ends of the active site groove .  (Anderson et al, 2001)

III. Trehalose Binding

Oxygen atoms in both trehalose molecules interact with Antigen 85B via hydrogen bonds.  The more interiorly located trehalose, Tre1, forms direct contacts with surrounding Arg-43Ser-126Trp-264, and His-262 residues, primarily through functional groups . Additional hydrogen bonds, formed by water molecules, indirectly connect Ser-263Asp-40,  and Asn-223 residues to Tre1 as well as binding independently . (Anderson et al, 2001)

The second trehalose molecule, Tre2, forms direct hydrogen bonds with Ser-235Phe-232Lys-239, and Asp-154 , as well as indirect contacts with the backbones of Asp-154Ser-326Ser-235Asn-231Gln-157, and Met-159  . Unlike Tre1, which has protein interactions surrounding the entire molecule, Tre2 is bound unevenly, leaving part of the molecule free  (Anderson et al, 2001).

A tunnel 15 Å long leads away from the binding site of Tre1 . This is hypothesized to be the mycolate α-chain binding site of trehalose 6-monomycolate (Ronning et al, 2000), indicating the longer mycolate ß-chain would be bound to the cell wall (Anderson et al, 2001). It has been proposed that an electron transfer from Glu-230 to His-262 to Ser-126 leads to Ser-126 making a nucleophilic attack, removing the mycolate group bound to the 6-OH of trehalose 6-monomycolate in the Tre1 binding site (Ronning et al, 2000). Following a rotation torsioning Phe-232 out of the active site groove, the mycolate could then be transferred to the 6'-OH of the trehalose 6-monomycolate in the Tre2 binding site, forming TDM (Anderson et al, 2001).

IV. Fibronectin Binding

Although antigen 85B is found primarily in the cell wall of  M. tuberculosis, it also circulates in the blood, in complexes with plasma fibronectin or immunoglobulin G, promoting infection.  The binding sites of  fibronectin and trehalose  are located on different domains of antigen 85B (Bentley-Hibbert et al 1999).  

A fibronectin binding motif appears to be located on an exposed surface loop of α2 and β4   . This motif is conserved across the protein variations.  Specifically, residues 58-68 correspond to the hypothesized binding domain of fibronectin proposed by Naito et al (1998)   . In contrast, Ronning el al (2004) proposed that a much larger surface patch, composed of residues 1-30, 41-68, and 100-113, is involved in this function  .Furthermore, there is a conserved patch emcompassing about 28% of the total accessible surface area of the protein.  It has been proposed that some or all of these residues interact with fibronectin (Ronning et al, 2000).

Despite their importance, none of these proposed fibronectin binding motifs have been characterized before (Anderson et al, 2001; Ronning et al, 2000).  Further studies will be necessary to determine the exact nature of this interaction.

V. Variant Comparisons

Although antigen 85A, B, and C are perform the same functions, their compositions are not identical; almost every surface residue not directly involved in either trehalose binding, fibronectin binding, or maintaining the tertiary structure has been mutated in at least one of the antigen 85 proteins (Anderson et al, 2001; Ronning et al, 2000).  

B and A have a two-residue insertion at residue 30, which adds Asn and Gly residues   (Anderson et al, 2001). B and A also have a disulfide bride between Cys-87 and Cys-92, forming two antiparallel β-sheets  . Antigen 85C  has Gln and Glu residues instead Cys at these locations.  As a result, the spatial positions of the surrounding amino acids differs. (Ronning et al, 2004)   

The composition of the active site of all three variants is highly conserved, including a virtually identical catalytic triad.  However, there are still some minor variations.  Most notably, the active site of antigen 85C contains Phe150 and Trp158, while in antigen 85B these residues are Leu-152 and Gly-160 .   Though not affecting volume or substrate affinity, this deviation results in the active site of antigen 85C having a slightly more closed conformation than B (Anderson et al, 2001; Ronning et al, 2000)

As mentioned above, most variation is seen in residues which are not involved in substrate binding or maintaining tertiary structure.  This commonly results in conserved residues, which face the protein's interior, adjacent to solvent-exposed variable residues, as seen in α10 (Ronning et al, 2004).

The three variants are encoded from separate genes, and are expressed in different concentrations, generally in a 3:2:1 ratio of B:A:C.  However, this ratio varies in response to changes in environment.  Even though B is expressed most highly, C is 8 times more biologically active than B.  It is very likely that possessing three seemingly redundant forms of antigen 85 assists M. tuberculosis in evading the immune response of a host (Ronning et al, 2000; Anderson et al, 2001; Ronning et al, 2004).

VI. Therapeutic Implications

Antigen 85 proteins show great potential as therapeutic targets for tuberculosis treatment.  They are highly accessible, due to their location on the outer portion of the cell wall.  Also, mycolyl transfer does not occur in humans, so disrupting this process is unlikely to affect patients (Anderson et al, 2001).

Anderson et al (2001) proposed a class of drugs composed of two linked trehalose molecules.  This molecule would compete with trehalose 6-monomycolate for access to the active site, and, due to contacts between the linker region and protein, it may be possible to manipulate it into binding much more strongly than the endogenous ligand.   These molecules would affect all variations of the antigen 85 protein, and would prevent construction of the cell wall, making the M. tuberculosis bacteria more vulnerable.

VII. References

Anderson, Daniel, Günter Harth, Marcus Horwitz, and David Eisenberg.  2001.  An Interfacial Mechanism and a Class of Inhibitors Inferred from Two Crystal Structures of the Mycobacterium tuberculosis 30 kDa Major Secretory Protein (Antigen 85B), a Mycolyl Transferase.  J. Mol. Biol. 307: 671-681.

Bentley-Hibbert, Stuart, Quan, Xin, Newman, Thomas, Huygen, Kris, Godfrey, Henry P. 1999. Patyhophysiology of Antigen 85 in Patients with Actibe Tuberculosis: Antigen 85 Circulates as Complexes with Fibronectin and Immunoglobulin G. Infection and Immunity. 67. 2: 581-588.

Lima, Valeria, Vania Bonato, Karla Lima, Sandra Dos Santos, Ruben Dos Santos, Eduardo Goncalves, Lucia Faccioli, Izaira Brandao, Jose Rodrigues-Junior, and Celio Silva.  2001.  Role of Trehalose Dimycolate in Recruitment of Cells and Modulation of Production of Cytokines and NO in Tuberculosis.  Infection and Immunity, 69(9): 5305-5312.

Naito, M., N. Ohara, S. Matsumoto, and T. Yamada.  1998. The novel fibronectin-binding motif and key residues of mycobacteria. J. Biol. Chem. 273, 2905.

Ronning, Donald, Thomas Klabunde, Gurdyal Besra, Baralakshmi Vissa, John Belisle, and James Sacchettini.  2000.  Crystal Structure of the Secreted Form of Antigen 85C Reveals Potential Targets for Mycobacterial Drugs and Vaccines.  Nature Structural Biology 7(2): 141-146.

Ronning, Donald, Varalakshmi Vissa, Grurdyal Besra, John Belisle, and James Sacchettini.  2004.  Mycobacterium tuberculosis Antigen 85A and 85C Structures Confirm Binding Orientation and Conserved Substrate Specificity.  J. Biol. Chem. 279(35): 36771-36777.

Silva, Nathan and David Marcey.  Intro to Jmol Scripting.  <>. 

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