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Case Study: Ubiquitin
Case Study: Ubiquitin
Eduardo Cruz-Chu and JC Gumbart
1
Introduction
Without a doubt, the most organized and coordinated machine known is
the biological cell. Inside its micrometer-scale diameter, a wide variety of
macromolecules (DNA, proteins, sugars, lipids, etc.) work together in a co-
operative way, balancing energy and matter to keep the cell alive. Within
the cell, proteins are the overachievers. They allow the movement of water
and ions through the cell membrane, help ATP to store energy, assist DNA
during replication, recognize foreign infections, and more. However, all of
these functions dont work independently of each other. To maintain har-
mony and eciency between various functions, most processes have to be
turned on or o according to dierent cellular stages and changes within the
environment.
To this end, together with the mechanisms to assemble functional proteins
and to turn on their functions, there should be counterparts to suppress and
disassemble proteins when they are no longer needed. The cellular machine
depends on assembly and disassembly to regulate the eective concentration
of proteins and their corresponding activities [1]. Furthermore, defective
1 proteins also need to be removed. Consequently, protein degradation, the
process of disposing of proteins, is a fundamental task. Inside the cell, such
a critical function is a cooperative eort that depends on many dierent
proteins, a pathway that involves dierent enzymes and reactions.
In this case study, we are going to focus on ubiquitin, a key player in
eukaryotic intracellular protein degradation. Its relevance has been widely
recognized in the scientic community, and the pioneering researchers, Aaron
Ciechanover, Avram Hershko and Irwin Rose were awarded the Noble Prize
in Chemistry in 2004 for the discovery of ubiquitin-mediated protein degra-
dation.
Here, we will use the following les:
2 Figure 1:
Sequence alignment of ubiquitins from dierent organisms, colored according
to amino acid conservation. In blue are identical residues, in green are conserved substitu-
tions, in light brown are semi-conserved substitutions, and in red there is no conservation
among residues. In the far-right column, protein accession numbers to the NCBI database
are given (http://www.ncbi.nlm.nih.gov/entrez/).
2
Ubiquitin and Evolution
As its name suggests, ubiquitin is found in many life forms. From humans
to yeast, ubiquitin is consistently present throughout all eukaryotes. Re-
markably, its genetic sequence is preserved without almost any modication.
In Figures 1 and 2, we show a comparative analysis of dierent ubiquitins
amino acid sequences that illustrate the high degree of conservation.
Even though proteins are linear polymers, they do not assume a linear
shape. In order to be functional, a protein must fold into a particular, usually
compact geometry. The native conformation found in living cells is mainly
determined by the amino acid sequence. Hence, proteins with similar se-
quences are expected to have similar folded structures. The more similar
sequences are, the more likely it is that they share a common structure.
Ubiquitins sequence conservation becomes obvious when we compare ani-
mals and plants. Humans, mice, pigs, guinea pigs, rabbits, chickens, and fruit
ies have exactly the same ubiquitin sequence. A similar situation is seen
with the soybean, garden pea, oat, wild oat, barley, wheat, maize, common
sunower, tomato, potato, garden asparagus, rice, carrot and turnip - all
3 Figure 2:
Surface representation of ubiquitin using VMD. The views are related by a
180
o
rotation. Colors were assigned according to the amino acid conservation in Figure 1.
ubiquitins in this group share the same sequence. Between both groups, the
dierence in sequence is just two amino acids. That high degree of conser-
vation has been considered indicative of the importance of each amino acid
for the functionality of ubiquitin.
This conservation over millions of years of evolution leads to one conclu-
sion about ubiquitin: its function is so crucial to the survival of any eukaryotic
cell that it was practically perfected before multi-cellular organisms arose. In
fact, it is a key regulatory label for many dierent cellular processes in addi-
tion to the degradation of either unassembled or misfolded proteins. These
functions rely on ubiquitins ability to be covalently linked to other proteins
as well as on its particular structural features.
3
Ubiquitins Prole
Besides its biological relevance, ubiquitins physical and structural features
make it an attractive candidate for experimental and theoretical studies of
proteins. First, it is small, composed of just 76 amino acids and with a
molecular weight of 8433 Da. It is also a high-temperature thermostable
globular protein; it is very soluble and, at neutral pH, its folded structure is
quite stable. To unfold ubiquitin through heating in solution, one needs to
reach temperatures around 100
o
C [3], i.e. the temperature of boiling water!
We can use structures resulting from x-ray crystallography to examine
ubiquitin in more detail. Its compact structure becomes evident as seen in
4 Figure 3:
A structural view of ubiquitin with helices in purple, the -sheet in blue,
and turns and coil in grey [2]. (The gure can be reproduced using the included le
1UBQ.pdb.)
Figure 3. At this resolution, 1.8
A, individual atoms can be seen, with the
exception of hydrogens [2]. Ubiquitins secondary structure also has three
and one-half turns of -helix, a short piece of 3
10
helix (a helix with three
residues per turn instead of 3.6 for -helices), a mixed -sheet that contains
ve strands, and seven reverse turns [2]. Its core is organized in a (2)--
(2) fashion known as the -grasp fold. Many other proteins share this kind
of fold, and due to the popularity of ubiquitin, it has also been called the
ubiquitin-like fold (Figure 4). Look at ubiquitin in VMD and explain why is
it called a -grasp fold?
In Figure 5 we can see an alignment of dierent ubiquitin-like fold pro-
teins. Multiple structural alignments do not include sequence information but
rather just the three dimensional organization of the protein. These compar-
ative analyses of proteins, at the level of sequence and structure, have became
powerful tools. They are used to trace evolutionary relationships as well as
to predict folded structures. However, the accuracy of alignment predictions
relies on sequence and structure databases as well as alignment algorithms.
As previously mentioned, ubiquitin functions by covalently attaching it-
self to other proteins, known as substrate proteins. The process of conju-
5 Figure 4:
The simplicity of the -grasp fold is shared by many dierent proteins. In the
gures, from left to right; on the top : 1FMA.pdb, 1IBX.pdb, 1H8C.pdb, 1I42.pdb; on the
bottom : 1GNU.pdb, 1RFA.pdb and 1UBQ.pdb. Try aligning these using the Multiseq
extension of VMD, then look to Figure 5 to compare (all pdb les are provided).
Figure 5:
Multiple structural alignment using VMD for the structures given in Figure
4. On the left, alignment colored according to structure similarity. Blue means high
similarity, red low. Comparison with ubiquitin (in the center) shows that the (2)--(2)
motif is preserved. On the right, the same alignment is colored according to sequence
similarity. Sequence similiarity is low among the dierent structures.
6 gation between ubiquitin and the substrate protein is called ubiquitination.
The linkage is made using ubiquitins GLY76 carboxyl group and the -amino
group of a Lys residue in the substrate through an isopeptide bond. Once the
bond is made, the attached ubiquitin can use a lysine residue to link another
ubiquitin, producing a poly-ubiquitin chain. The number of ubiquitins and
how they are linked determines the location in the cell where a tagged protein
is going to go. When the substrate protein reaches its target, ubiquitin is
detached and released in order to be used again. The conjugation process is
energy driven by ATP. Ubiquitin is also assisted in the conjugation process
by three other proteins that will be discussed in the next section.
By 1980, it was still not completely clear why proteins would need energy
for degradation inside the cell. Outside the cell, catalyzed protein breakdown
is energy independent. For example, proteins taken with food are degraded
in the intestines before being absorbed. However, this energy requirement to
degrade proteins in the cell is not without purpose.
4
Broad Functionality of Ubiquitin is the Re-
sult of Team Work
Ubiquitin denitely deserves its name; not only for being omnipresent, but
also because it is involved in a diversity of cel