Max Planck scientists have
uncovered the 3D-structure of an important defense-enzyme and have
developed a new cost-effective way of producing it.
Tumor cells or virally infected cells are a danger to our lifes, but
fortunately killer cells of the immune defense system which are armed
with different specialized digestive enzymes, called granzymes,
eradicate these cells in many instances. The granzymes A and B, two of
these proteases, are highly efficient triggers of intracellular cell
death inducing (cytotoxic) cascades. The same beneficial effector
molecules, however, can also turn their powerful energies against
transplanted organs, grafted stem cells and self-tissues in autoimmune
disorders and are then detrimental and life-theatening to the patients.
Among the 120 different serine proteases of the human genome, granzyme
A is a unique double-headed protease (homodimer) with two identical
catalytic domains connected by a covalent disulfide bond. Clara
Hink-Schauer, Eva Estebanez-Perpina, Florian Kurschus, Wolfram Bode
and Dieter E. Jenne from the Max-Planck-Institutes of Biochemistry and
Neurobiology, Martinsried near Munich, Germany, have now uncovered the
secret of this tandem configuration by analyzing the three-dimensional
structure of granzyme A at 2.5 Å resolution (Nature Structural Biology
10, 535-540, July 2003).
Granzyme B specifically recognizes a highly restricted number of
flexible surface loops next to the negatively charged amino acid
residue called aspartate (Asp) found in a group of intracellular
cysteine proteases known as caspases (cysteine protease with aspartate
specificity) and activates this proteolytic starter (to be taken
unliterally) of the cell death machinery. By contrast, the target
sequences that are cleaved by granzyme A after a basic amino acid
residue are highly heterogenous and are predominantly found in protein
complexes containing subunits with long acidic tails. The so-called
SET complex (containing the SET component) is cleaved at multiple
sites and dismantled during killer cell attack. A cellular
Mg2+-dependent DNase, called NM23-H1, is thereby freed from this
complex and degrades the DNA nucleus by cutting the strands of the DNA
double helix at numerous sites. The research team of the
Max-Planck-Society in Martinsried now explains how the two identical
molecules are assembled into a doubled-headed protease for the defense.
The two catalytic centers point in exactly opposite directions (see
figure). Both surfaces on the front and backside of the dimer are
functionally equivalent. Each molecule (subunit) thus fulfills a
concurrent dual function, presenting substrates with their back to the
adjacent partner and cleaving substrates with their catalytically
active front side. Moreover, the scientists presented an elegant low
budget solution to produce this dimeric enzyme first as a stable
dimeric progranzyme and second as a double-active mature protease at
in large quantities.
Fig.: Schematic
representation of the three-dimensional structure of the double-headed
human granzyme A. Two catalytic subunits consisting of the same
polypeptide chain are tied together in the middle to form a dimer. The
catalytic center of the left unit (molecule) points to the back, that
of the right to the viewer. Both catalytic centers are indicated by a
dashed circle. Both protease subunits display the same structure and
are assembled in a way that they present a 180 degree rotational
symmetry. Front and backside of the dimer are thus functionally
equivalent.
Image: Max Planck
Institute of Neurobiology
Granzymes are obvious
therapeutic targets for preventing killer cell-mediated cell damage
with cell-penetrating inhibitors. Easy access to large quantities of
this enzyme and its three-dimensional structure are important
prerequisites for the rapid development and rational improvement of
existing serine protease inhibitors. Inhibitors that save host cells
from the attack by natural killer cells and cytotoxic T cells could
gain great significance as practical therapeutics in graft-versus-host
diseases, chronic viral infections, and autoimmune disorders like
rheumatoid arthritis and multiple sclerosis.
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