M.
tuberculosis Antigen 85B
Kat Meagley '11 and Suzanne Rohrback '11
Contents:
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-43, Ser-126, Trp-264,
and His-262
residues, primarily through functional groups
.
Additional
hydrogen
bonds, formed by water
molecules, indirectly
connect Ser-263, Asp-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-235, Phe-232, Lys-239,
and Asp-154
,
as
well as indirect
contacts
with the backbones of Asp-154, Ser-326, Ser-235, Asn-231, Gln-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.
<http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/scripting/molmast.htm>.
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