UCHL3
in Plasmodium
falciparum
Morgan Korinek '12 and Andrea Pohly '12
Contents:
I. Introduction
II. General Structure
III. Crossover Loop
IV. Differences between HsUCHL3 and FsUCHL3
V. Dual Ubiquitin and Nedd8
Specificity
VI. References
I.
Introduction
Malaria
is one of the most-widespread infectious diseases affecting people
around the globe. Malaria is caused by protists of
the genus Plasmodium and
is
transmitted through
the blood by mosquitoes. The
replication
of
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
consequences
such as a coma or even death. While there are 5 different
species
of Plasmodium, the most
severe form
of malaria is caused by one species in particular: Plasmodium
falciparum.
PfUCHL3, a ubiquitin
C-terminal hydrolase (UCH), is thought to be essential for Plasmodium
falciparum to survive.
Ubiquitin (Ub)
is
a protein
present in all eukaryotes that is composed of a
well-conserved sequence
of 76
amino acids. This ubiquitin-proteosome system is crucial to
eukaryotic
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
problems
and can eventually lead to cell death.
(3)
In this tutorial, we
examine the structure
of pfUCHL3 and how it differs from Hs, or human, UCHL3. Because pfUCHL3 binds
ubiquitin
differently than its human counterpart, this protein is a potential
drug target
for malarial
patients.
II.
General Structure
PfUCHL3
consists
of a central
six-stranded
anti-parallel β-sheet
surrounded
by seven
α-helices
Viewing
PfUCHL3
in complex with a ubiquitin-based suicide
substrate (UbVME)
allows us to show the dual specificity of this
enzyme.
.
The β
–sheet
conformation
of the
C-terminus
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,
and Asp-179
These three
residues are all within hydrogen bonding distance
of each other.
.
Ser-12
forms
an
additional hydrogen bond to the backbone nitrogen of Ub Leu-73
in the
Ub-binding groove of PfUCHL3.
All
of
the hydrogen bond donors and acceptors of Ub C-terminal residues 71-75
are then
fulfilled. (1)
III.
Crossover Loop
An
important element of the bound complex is the active site crossover
loop. In
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
Ubiquitin. (5)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825479/figure/F4/
This
loop
encounters the C-terminal of the
Ubiquitin suicide substrate (UbVME) and is thought to assist in the
positioning
of the
substrate. When the substrate is not
present, the crossover loop becomes flexible and cannot be detected in
the
crystal structure, hence why we cannot see it on this free molecule. However, when ubiquitin is
present,
the crossover loop connects
the two halves of the catalytic center, bringing key residues close
enough to
catalyze the binding of ubiquitin. The
crossover loop also
functions as a substrate filter by limiting the types and sizes of
substrates the
enzyme
can hydrolyze. While the structure and function of the
cross-over
loop is not yet well understood, there are a few key features that are
visible in the ubiquitin bound crystal structure.
Leu-71
and Leu-73
point to the opposite side of the binding
groove and occupy a hydrophobic binding pocket where the substrate
attaches.
Asp-
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)
IV.
Differences between HsUCHL3 and FsUCHL3
The
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
not share
the same conformation. While the Ub recognition
and binding mode between
each enzyme and Ub is similar, the Ub
interface
of
both enzymes have a few key differences.
In PfUCHL3, Ser-12 in the Ub-binding-groove forms an
additional
hydrogen
bond to the backbone nitrogen of Ub Leu-73.
Ub-Arg-74, a highly conserved residue and central to Ub
recognition,
forms a network of hydrogen bonds with HsUCHL3
residues.
However, PfUCHL3 residues have a completely
different
conformation of hydrogen bonds. One
example of this is the formation of a
salt bridge to Asp-157
in
PfUCHL3 that is not present in
HsUCHL3. Also, the interface of PfUCHL3 and Ub have
the nonconserved residues of Thr-163
and Ser-219
.
These key differences allow researchers to
specifically
target the parasite rather than the human host cells. (1)
V.
Dual
Ubiquitin and Nedd8 Specificity
PfUCHL3
is
the only deNeddylase present in the parasite's genome.
The
Nedd8 protein is involved in the control of the G1
to S phase of the cell cycle. Interferring with
the Nedd8
pathway can cause the cells to bridge multiple S
phases and fail to
undergo mitosis. By interferring with the enzyme
that
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.
Arg-72 in
Ubiquitin, which corresponds to Gln-72 of PfNedd8,
readily hydrogen bonds to Glu-11
and Asn-13
of PfUCHL3.
These
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
as
well. (2)
VI.
References
(1)
Artavanis-Tsakonas,
K.,
W.A. Weihofen, J.M. Antos, B.I. Coleman, C.A.
Comeaux, M.T. Duraisingh, R. Gaudet and H.L. Ploegh.
2010.
Characterization and
Structural Studies of the Plasmodium
falciparum Ubiquitin and Nedd8
Hydrolase
UCHL3. The
Journal of Biological
Chemistry 285(9): 6857-6866.
(2)
Artavanis-Tsakonas,
K.,
S. Misaghi, C.A. Comeaux, A. Catic, E. Spooner,
M.T. Duraisingh and H.L. Ploegh. 2006.
Identification by functional
proteomics
of a deubiquitinating/deNeddylating enzyme in Plasmodium
falciparum. Molecular
Microbiology
61(5):
1187-1195.
(3)
Kim,
J.H.,
K.C. Park, S.S. Chung,
O. Bang and C.H. Chung. 2003.
Deubiquitinating Enzymes as Cellular Regulators. J.
Biochem 134(1): 9-18.
(4)
Pickart,
C.M. and M.J.
Eddins. 2004. Ubiquitin: structures, functions,
mechanisms. Biochimica
et
Biophysica
Acta 1695:
55-72.
(5)
Popp,
M.W.,
K. Artavanis-Tsakonas and H.L. Ploegh. 2009.
Substrate
Filtering by the Active Site Crossover Loop in UCHL3
Revealed by
Sortagging and
Gain-of-function Mutations. The
Journal of Biological Chemistry
284(6):
3593-3602.
(6)
Wilkinson,
K.D.
1997. Regulation of ubiquitin-dependent processes by
deubiquitinating enzymes. The
FASEB Journal 11(1):
1245-1256.
Back
to Top