WILSON’S DISEASE: A
MOLECULAR FOCUS
PAUL TRUCHE
CABM 010
April 7, 2010
WHAT IS WILSON’S DISEASE?
 Wilson’s
disease is a neurodegenerative
disease caused by a buildup of copper in
the body’s tissues
 Effects 1 in 30,000 according to the Oxford
Textbook of Medicine
 1 in 100,000 posses a single copy of the
mutated gene
CAUSE OF WILSON’S DISEASE
 Mutation
of the gene ATP7B is
responsible for Wilson’s disease
 It
is due to an abnormal gene located on
chromosome 13: an autosomal recessive
inheritable trait
GENETIC BACKGROUND



This gene is expressed predominantly in the liver, kidney
and placenta. It is also expressed in the brain, heart, lungs
and pancreas (Warrell)
The ATP7B gene codes for the protein ATPase, Cu++
transporting, beta polypeptide, also known as ATPase 2 or
ATP7B.
Over 70 mutations have been found that are linked to
Wilson’s disease. Gene deletions, and mutations that cause
the allele to be completely unexpressed cause the worst
forms of Wilson’s disease.
WHY DO WE NEED COPPER?
Copper is an essential
 mediates various cellular processes:
 mitochondrial energy generation (cytochrome c
oxidase)
 iron uptake regulation (ceruloplasmin),
 melanin formation (tyrosinase)
 oxygen-radical scavenging (superoxide
dismutase)
 collagen and elastin cross-linking (lysyl oxidase)

WHEN WE HAVE TOO MUCH COPPER
Excess copper can do many harmful things:
 generates reactive oxygen species,
superoxide radicals and hydroxy radicals,
which can damage cellular components.
 Copper can bind to histidine, cysteine, and
methionine residues of various proteins,
which results in the inactivation of the
proteins
COPPER TRANSPORT THROUGH THE BODY





Ingested copper is first absorbed in the stomach,
duodenum and small intestine.
Copper first binds to albumin, transcuprien or
histidine in the blood.
Copper in the blood moving through the liver is
removed by high affinity copper transport protein
(hCTR1 and hCTR2).
Once inside the liver cells, copper interacts with
various other proteins including HAH1, ATOX1, and
CCS
Within liver cells, ATP7B brings copper across
different membranes and prepares it for transport
back out of the cell when copper needs to be removed
COPPER IN THE BLOOD STREAM
 The
protein ATP7A is responsible with
the transport of ingested copper to cells
within the body.
 The ATP7A protein carries copper
through the membrane of intestinal
enterocytes and then into the portal blood
stream.
 Once in the blood stream, copper is
transported throughout the body.
MOVEMENT OF EXTRACELLULAR COPPER
INTO CELLS
 Outside
the cell, the protein called high
affinity copper transport protein (Ctr1)
first interacts with the copper cation.
 Once inside the cell, ATP7B the vesicular
compartments when the cell needs to
excrete excess copper
 Another protein, Atox1 delivers copper to
the secretory pathway and docks with
either copper-transporting ATPase.
PROTEINS INVOLVED IN COPPER
HOMEOSTASIS
ATP7A
 ATP7B
 Ctr1
 Atox1
 Cox17


Other less understood proteins are hypothesized
to play a role in copper homeostasis, but both
their structure and functional role are still
unknown
ATP7A
ATPase, Cu++ transporting, alpha
polypeptide
 found in most tissues, but absent from the liver
 In the small intestine, the ATP7A protein helps
control the absorption of copper from food.
 In other organs and tissues, the ATP7A protein
has a dual role and shuttles between two
locations within the cell.
STRUCTURE OF ATP7A
Figure above: Proposed mechanistic structure of the
ATP7A protein embedded in the cell membrane. Note the
six copper binding sites that extend into the cytosol. ATP
interacts with the N domain to actively transport copper
through the membrane.
Image from Mutagenetix
Database of Mutations and Phenotypes
Figure below:
Fourth Copper Binding
Domain of the ATP7A protein. Not ever
domain has been structurally determined
and mutation at different domains shows
different structural and functional changes
in the protein suggesting that different
structures exist between different binding
sites
ATP7B: THE WILSON DISEASE PROTEIN
ATPase, Cu++ transporting, beta polypeptide



The full structure of ATP7B has never been completely
rendered
six metal-binding subdomains (MBDs), each coordinating
one copper between 2 Cys residues in an invariant CxxC
motif.
The large cytosolic N-terminal domain of ATP7B binds
copper and plays a key role in regulation of ATP7B.
STRUCTURE OF ATP7B
Figure above: proposed
structure of the ATP7B protein
complex. (below): 3D structure
of metal binding sites 3 and 4
on the ATP7B protein. These
are the actual sites of metal
binding within the protein
structure.
Figure above: N domain of the ATP7B
protein. Helices are highlighted in red.
The uppermost loop is the area
responsible for the majority of mutation
within the protein.
This domain
interacts with ATP.
METAL BINDING DOMAINS
Figure: The metal binding domains of the
ATP7B molecule provide the major mechanistic
location involved in Wilson’s disease. Mutation
in the protein can often cause an inability for
these sites to bind properly to copper. The 3D
structure of the flexible loops that link the
metal binding sites is shown in orange.
Copper
Binding
Domain
3
Copper
Binding
Domain
4
Figure: Copper Binding
site between two cystiene
residues on the metal
binding domain of
ATP7B. Mutations
around these binding
sites cause inability of
the protein to transport
copper. Cystiene
residues responsible for
copper binding are
located in two regions.
Each of these regions is
capable of binding
copper within each
copper binding domain.
Why copper may bind to
one over the other is not
understood.
N DOMAIN
ATP
Interacting
Sites
N-DOMAIN
Figure: Residues E1064, H1069,
G1099, and G1101 have been
implicated as ATP binding sites
within the N-domain.
Figure: The region between
ALA 1114 and THR 1143
has been attributed to many
of the sequence mutations
responsible for Wilson’s
disease. This region
corresponds exactly to the
sequence insert, which is
found in mammalian
copper-ATPases, but absent
in bacteria or lower
eukaryotes.
CTR1
Copper Transport Protein (Ctr1)
 Ctr1 is a homotrimeric protein, conserved from
yeast to humans, that transports Cu across the
plasma membrane with high affinity and
specificity.
ATOX1
Copper transport protein (ATOX1)
 plays a role in copper homeostasis by binding and
transporting cytosolic copper to ATPase proteins
in the trans-Golgi network for later incorporation
to the ceruloplasmin.
STRUCTURE OF ATOX1
ATOX1 copper binding site.
Copper binding occurs
between four cysteines: CYS
12.A, CYS 15.A, CYS 15.B
and CYS 12.B. Two of each
cysteine come from adjacent
chains that make up the
biological assemboly
COX17


Cytochrome c oxidase copper chaperone
An enzyme that may be involved in the
recruitment of copper to the mitochondria for
incorporation into the COX apoenzyme
MUTATIONS OF THE ATP7B PROTEIN




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Numerous mutations in the ATP7B gene have been
implicated in Wilson’s disease.
The exact number of different mutations that can
cause the disease is still unknown, but certain
mutations account for a majority of those seen in
clinical cases.
Mutation of any of the six metal binding domains
within the protein are the primary cause for inactive
ATP7B proteins.
Mutations in different metal binding domains cause
different mutations in the overall protein structure
and show that each binding domain may have specific
mechanisms.
This finding may explain the need for multiple copper
binding sites, but this is still not understood .
MUTATIONS OF THE ATP7B PROTEIN




Mutations in the copper binding sites can lead to an
increase in oxidation with subsequent decrease in
copper binding.
While the need for multiple copper binding sites is not
completely understood, the interdomain
communication between the metal binding sites is
necessary for maintenance of reduced cysteines and
proper copper acquisition.
The metal binding domain interactions may involve a
hydrogen-bonding network that allows copper binding
to be translated into larger structural
rearrangements via the loops between the metal
binding sites.
These rearrangements are likely to play a major role
in regulating ATPase activity as well as mediating
interactions with cellular trafficking of copper.
MUTATIONS IN THE N DOMAIN
The first group of mutations includes 16 residues in
the following regions:
1,061–1,069
1,099–1,106
1,146–1,153
all located in the immediate vicinity of the ATPbinding site.
Mutations in these locations most likely interfere
with ATP binding in the protein.
MUTATIONS NEAR THE ATP BINDING SITE
Figure: Mutations near
the ATP binding site
account for the most
common inactive forms of
the ATP7B protein.
Mutations for any residue
in these locations seems
to effect the ability for the
protein to interact with
ATP. Specific regions
where mutations occur
include:
1,061–1,069 (Red)
1,099–1,106 (Blue)
1,146–1,153 (Green)
MUTATIONS (CONTINUED)
The second group of mutations affects the following
residues
 1,033–1,038 ,which is a part of the hinge region
connecting the N- and P-domains
 1,168–1,176, which in the structure of the fulllength protein would face the P-domain.
These mutations most likely alter the interactions
between the N domain and other domains in the
protein.
There are other mutations that occur in other parts
of the N domain and usually result in disruption
of protein folding and other overall protein
structure differences
OTHER MUTATIONS
Figure: Mutation sites
not near the ATP
binding site include:
1,033–1,038 (Red)
1,168–1,176 (Blue)
Mutations in these
areas most likely
disturb the
interactions between
the N terminus and
the rest of the protein
PHYSIOLOGICAL EFFECTS OF MUTANT
ATP7B
The physiological effects of most mutations to the
ATP7B protein lead to the clinical presentation of
Wilson’s disease.
 Without proper function of the ATP7B protein,
copper is unable to be removed by the
hepatocytes in the liver.

OUTLINE OF THE DISEASE PATHOLOGY
1) Failure of ATP7B to release copper from liver
cells
2) Increase is copper levels in the liver
3) Inability for liver cells to further store copper
4) This excess copper in the blood stream is
deposited in brain tissue
5) Neurologic and Hepatic symptoms begin to arise
due to this build up of copper.
SYMPTOMS OF WILSON’S DISEASE
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The earliest neurological symptoms include:
difficulty speaking
Drooling
clumsiness of the hands
change in personality.
Acute abdominal symptoms include:
 Acute crises of acute hepatic necrosis,
 Coombes negative hemolytic anemia
 acute renal tubular damage may occur
These crises are believed to be related to a massive
release of copper from damaged or dead hepatocytes
TREATMENT OF WILSON’S DISEASE
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Treatment for those diagnosed with Wilson’s disease
focuses on the removal of copper from the body. In cases
where liver damage has progressed to the point of complete
failure, a liver transplant is necessary to replace dead and non
functioning liver cells. Those who have had a liver transplant
have not shown signs of Wilson’s disease after liver
transplantation. For those who do not have full liver failure,
pharmacological treatment is available.
Pharmacological treatment of Wilson’s disease includes
several drugs. Penicillamine binds copper and releases it
through the urine. Trientine binds the copper and also
releases it through the urine. Zinc acetate blocks further
copper from being absorbed into the intestinal tract.
Along with drug therapy, treatment involves avoiding
ingestion of further copper. Common foods that contain
copper are chocolate, dried fruit, liver, mushrooms, nuts,
shellfish.
CONCLUSION
Many of the mechanisms involved in the transport of
copper and those involved in Wilson’s disease are not
well understood.
Understanding the genetics and mechanisms behind
copper transport will allow a better understanding of
the pathology of the disease and allow more effective
treatment options. Although current treatments for
the disease are fairly effective, there is no way to
reverse neurologic damage or completely cure the
disease.
Finding out how to reverse the neurologic damage will
be an important step towards healing damage to the
brain.
DISCUSSION/QUESTIONS
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