Morgan Korinek '12 and Andrea Pohly '12
II. General Structure
III. Crossover Loop
IV. Differences between HsUCHL3 and FsUCHL3
V. Dual Ubiquitin and Nedd8
is one of the most-widespread infectious diseases affecting people
around the globe. Malaria is caused by protists of
the genus Plasmodium and
the blood by mosquitoes. The
Plasmodium parasites in
red blood cells can cause a human host to develop symptoms such as a
headache and/or fever, but can also can have more severe
such as a coma or even death. While there are 5 different
of Plasmodium, the most
of malaria is caused by one species in particular: Plasmodium
PfUCHL3, a ubiquitin
C-terminal hydrolase (UCH), is thought to be essential for Plasmodium
falciparum to survive.
present in all eukaryotes that is composed of a
amino acids. This ubiquitin-proteosome system is crucial to
survival and is involved in
protein degradation as well as cell's maintenance of synthesized and
degraded proteins. When
this balance is out of equilibrium, the cell can run into functional
and can eventually lead to cell death.
In this tutorial, we
examine the structure
of pfUCHL3 and how it differs from Hs, or human, UCHL3. Because pfUCHL3 binds
differently than its human counterpart, this protein is a potential
of a central
in complex with a ubiquitin-based suicide
allows us to show the dual specificity of this
of Ubiquitin has backbone carbonyl and amide groups that
hydrogen bonds to the PfUCHL3 residues. The C-terminus of
Ubiquitin binds to the
narrow groove, lined by the catalytic triad residues: Cys-92, His-164,
residues are all within hydrogen bonding distance
of each other.
additional hydrogen bond to the backbone nitrogen of Ub Leu-73
Ub-binding groove of PfUCHL3.
the hydrogen bond donors and acceptors of Ub C-terminal residues 71-75
important element of the bound complex is the active site crossover
unbound PfUCHL3, the crossover loop is very ordered. This is
due to crystal contacts that hold the loop in a distinct conformation.
No significant structural changes occur upon the binding of
encounters the C-terminal of the
Ubiquitin suicide substrate (UbVME) and is thought to assist in the
substrate. When the substrate is not
present, the crossover loop becomes flexible and cannot be detected in
crystal structure, hence why we cannot see it on this free molecule. However, when ubiquitin is
the crossover loop connects
the two halves of the catalytic center, bringing key residues close
catalyze the binding of ubiquitin. The
crossover loop also
functions as a substrate filter by limiting the types and sizes of
can hydrolyze. While the structure and function of the
loop is not yet well understood, there are a few key features that are
visible in the ubiquitin bound crystal structure.
point to the opposite side of the binding
groove and occupy a hydrophobic binding pocket where the substrate
157 is located in
the crossover loop and forms a salt bridge with the side chain of Arg-74
of Ub to provide stability to the bound complex. This ubiquitin
binding pocket can now be visualized. (1)
Differences between HsUCHL3 and FsUCHL3
key difference between HSUCHL3 and FsUCHL3 is that the
sequence of the
crossover loop is
not conserved. Therefore, these two
enzymes do not interact with Ub in the same pattern and do
the same conformation. While the Ub recognition
and binding mode between
each enzyme and Ub is similar, the Ub
both enzymes have a few key differences.
In PfUCHL3, Ser-12 in the Ub-binding-groove forms an
bond to the backbone nitrogen of Ub Leu-73.
Ub-Arg-74, a highly conserved residue and central to Ub
forms a network of hydrogen bonds with HsUCHL3
However, PfUCHL3 residues have a completely
conformation of hydrogen bonds. One
example of this is the formation of a
salt bridge to Asp-157
PfUCHL3 that is not present in
HsUCHL3. Also, the interface of PfUCHL3 and Ub have
the nonconserved residues of Thr-163
These key differences allow researchers to
target the parasite rather than the human host cells. (1)
Ubiquitin and Nedd8 Specificity
the only deNeddylase present in the parasite's genome.
Nedd8 protein is involved in the control of the G1
to S phase of the cell cycle. Interferring with
pathway can cause the cells to bridge multiple S
phases and fail to
undergo mitosis. By interferring with the enzyme
processes Nedd8, researchers can disrupt the cell
cycle and ultimately
lower the cell's viability. (1)
An interesting feature of
UCHL3 is that it can bind both Ubiquitin as well as Nedd8.
Ubiquitin, which corresponds to Gln-72 of PfNedd8,
readily hydrogen bonds to Glu-11
interactions suggest that both Ubiquitin and PfNedd8 are natural
substrates of PfUCHL3. Further research is still in progress
in determining if Nedd8 could be a possible drug target for malaria
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Comeaux, M.T. Duraisingh, R. Gaudet and H.L. Ploegh.
Structural Studies of the Plasmodium
falciparum Ubiquitin and Nedd8
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Identification by functional
of a deubiquitinating/deNeddylating enzyme in Plasmodium
K.C. Park, S.S. Chung,
O. Bang and C.H. Chung. 2003.
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K. Artavanis-Tsakonas and H.L. Ploegh. 2009.
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FASEB Journal 11(1):