Human Tissue Kallikrein 4
Anna Frutiger '09 and Paige Roberts '09
Contents:
I. Introduction
Human tissue kallikrein 4 (hK4) is a member of a highly
conserved gene family which contains 15 human enzymes composed of
closely related serine proteases. Click here
to see a typical serine protease. The hK4 enzyme is a subgroup of the
serine proteases that catalyzes the cutting of the peptide backbone in
proteins via hydrolysis. kallikreins been known to catalyze the break
down of bradykininogen to bradykinin and plasminogen to plasmin, in
addition to being active in initiating intracellular signaling through
protease-activated receptors. The reason this group of digestive
enzymes are named serine proteases is due to the serine residue present
in the catalytic triad within the active site. To see the catalytic
mechanism in relation to the serine protease click here.
The hK4 gene and the rest of this gene
family can be found on the 19q13.3-q13.4 chromosome locus, located near
the telomere of chromosome 19 (Stephanopoulos et al.,
2005). In particular, the hK4 protein is a calcium-independent serine
protease which is secreted as a 254 amino acid long inactive zymogen.
An unknown enzyme reduces the zymogen to the active protein form which
is 224 amino acid residues long.
(Stephanopoulous et al., 2005;
Debela et al., 2006).
The hK4 enzyme has been
found to affect numerous regions of the human body. The protein was
originally discovered in the prostate and was thus originally called
prostase. hK4 is highly expressed in the prostate and has been found to
be heavily upregulated in prostate cancer cells. The protein has also
been found in seminal fluids and urine, is a
potential prostate cancer indicator. Further, while hK4 has been found
within the ovarian tissues under healthy conditions, the enzyme has
been found to be severely upregulated in the corresponding ovarian
cancer cells, thus indicating a role in ovarian cancer (Debela et
al., 2006). hK4 has also been found
to be a key gene in the mineralization of dental enamel. Mutations in
the gene coding for the hK4 protein, are known to cause the disease
amelogenesis imperfecta, which
affects the proper formation of the enamel (Stephanopoulous et
al., 2005). While hK4 has been discovered mainly in these
three regions of the body, it is also expressed in a variety of other
tissues and organs such as the testis, the skin, and the mammary and
salivary glands (Debela et al., 2006).
The knowledge
regarding the x-ray structures of human tissue kallikrein proteins is
relatively recent, with the information known previously coming from
research performed on porcine animal models (Debela et al.,
2006). The hK4 x-ray crystal structure has also been solved, however
the crystalization has only been successful when the enzyme is in the
presence of cobalt, nickel or zinc. These metals bind at the hK4 metal
binding site and affect the conformation of the active site. These
structures represent the crystal in its inhibited form when it is bound
to a p-aminobenzamidine ligand. By understanding
these structures, it is possible to gain a
better understanding of the enzyme, and can thus lead us to look into
more of the regulatory patterns of not only hK4, but of the entire
human tissue kallikrein protein family
II.
General
Structure
hK4
has been discovered to exist as
a monomer
in
solution, but is able to be crystallized only as a
cyclic tetramer
.
The hK4 mature monomer
is
224 amino acids long and consists of a single active
site
.
The monomer, while found only in solution, is
thought to be the active form of the protein. When bound to either Ni2+,
Co2+, or Zn2+
, the metal ions occupy the cation site between Glu77 O and His25 N
.
This causes the protein to bind into cyclic
tetramers, which are further stacked into octamers in the crystal forms
of hK4-Zn and hK4-Co.
The
hK4 monomer looks like an oblate ellipsoid that has diameters of about
35 and
50 Å
.
The polypeptide chain is composed of 2 adjacent
six-stranded β-barrels
and
2 α-helices
in residues 164-172
and 234-244
.
Two 310-
helices also exist in the
monomer in segments 55-59
and 74-77
.
310-helices are rare
right-handed helices. They differ from α-helices since
the N-H group on
the peptide chain forms a hydrogen
bond
with a carbonyl group
4 residues away, while in a 310-helix, the hydrogen bond is formed with
a carbonyl group 3 residues away
.
Each
monomer also contains a catalytic triad which is located along the
junction of the β-barrels. The cleft of the active site with
recognition subsites S4 to S3 is perpendicular to this
β-barrel junction. The catalytic triad is composed of residues
His57, Ser195, and Asp102
(Debela
et al., 2006). The catalytic triad and the
oxyanion hole associated with the triad, are homologous with the active
site of trypsin. The oxyanion hole is induced when the N-terminal Ile16 forms a salt-bridge with the
carboxylate side-chain of Asp194
.
In addition to the oxyanion hole formation, this
interaction also forms an S1 pocket and a rigid activation domain (Bode
et al, 1978).
III.
Active Site
The
active site cleft of the hK4 protein has multiple unique structural
characteristics that cause the narrow substrate specificity. One of
these is the 37 loop
.
The 37 loop is slightly flexible and helps to form a
negatively charged surface patch. The other characteristics of the
active site that contribute to this negatively charged patch is the N-terminal segment
and
the 70-80 loop
.
This negative surface is composed of residues Glu20,
Asp21, Glu36, Glu38, Glu74, Asp75,
Asp109, Glu110, Glu114, Asp116 and Glu84
.
The negatively charged region is additionally
located along the same area as a positively charged anion secondary
binding site of thrombin. This binding site in thrombin has been
known to be involved in substrate and inhibitor interactions, thus
indicating that this negative region in hK4 has similar activities.
The
binding site of the inhibitor p-amino-benzamidine
(PABA) is located in the S1 site of the enzyme
.
This is the primary active site and is bordered by
residues Val213-Cys220,
Ser190-Ser195, Pro225-Tyr228 , and the Cys191-Cys220 disulfide bridge .
In this active site, the residues
Phe215 , Gly216 ,
Cys191 , and Asn192 tightly surround the inhibitor
.
The amidine group on the inhibitor forms a
salt bridge with the
Asp189 carboxylate group.
Hydrogen bonding can also be observed between the inhibitor PABA and
the Lys217 carbonyl
oxygen and to the Ser190
oxygen atom
.
The inhibitor binds in the active site by resembling
the tetrahedral intermediate present in the catalytic triad. This
binding fills up the active site and prevents the enzyme from working
properly. The inactive monomer enzyme then binds to other inactive
monomers to form an oligomer which is termed a zymogen (a large,
inactive structure with the potential to break down to the smaller,
active form).
The
S2 subsite of
the active site is a structure that is observed only in kallikreins
.
The subsite is
formed by the amino acid side chains of residues His57, Leu99,
and Phe215
.
The S3
subsite lacks specificity and is thus not clearly detectable. The S4
subsite,
however, is located on the phenyl ring of Phe215,
which is found next to
the S2 pocket. The
boundaries of this hydrophobic S4 subsite include the residues Leu99 and Leu175 . The sizes and
limitations of each of these subsites helps to determine the exact
molecules that bind into the active site, which enhances active site
specificity.
IV. Metal Substrate Binding
To
date, there have been three crystal forms discovered of the hK4
protein. Each of these three forms consists of a binding site that is
occupied by a divalent transition metal ion. For hK4-Zn and hK4-Co, the
divalent metal ion
occupies some of the cation sites between residues Glu77 O
and His25 N
,
however for hK4-Ni, the metal
ion
is
present in this cation site for all independent
hK4 molecules
.
The
formation of this metal binding site is created from the insertion of a
single
residue before Glu77
that
creates the 70-80
loop
.
This residue is not present in many of the
serine-like proteases, thus making hK4 unique. The insertion of this
residue causes the formation of one of the 310-helices,
which moves the Glu77 side chain closer to the His25 imidazoyl side
chain, thereby producing a unique metal binding site (Debela et
al., 2006).
The
hK4 molecule has been crystallized with Ni2+,
Co2+,
and Zn2+
occupying this binding site, however the best understood structure has Ni2+
bound to the metal binding site. Interestingly, when Ni2+ is
bound to hK4
,
the activity of the protein is reduced in solution,
but only when the concentrations are higher than 100μM. When Co2+
binds hK4, the protein is observed in an oligomer formed of two
octamers. Co2+
binding
was
discovered to be even less tightly bound to hK4
than Ni2+,
however Zn2+
binding
was
shown to have an extraordinary binding affinity to the cation found at
the metal binding site. Therefore, Zn2+
binding causes the most extreme inactivation of the protein hK4, and is
thus possibly involved in the regulatory pathway of the protein.
When
the metal ions are bound to the substrate binding site, many of the
protein's activities are hindered. In the
presence of zinc , the
Ile116 N terminus
becomes
more accessible for acetylating agents
.
It is probable, then that zinc
affects the hK4 active site by disrupting the salt bridge between the N
terminus and the Asp194 residue, which is necessary for a functional
hK4 protein.
Despite
the inactivity of the active site due to metal binding, all of the
different copies of the protein, both with and without occupied metal
sites, exhibit very similar structures. This suggests that both the
active metal-free monomers, and the inactive enzymes bound by the
inhibitor display a very similar conformation (Debela et al.,
2006).
V.
Medical Implications
The
hK4 protein is known to be up-regulated in prostate and ovarian
cancers. It was originally discovered in the prostate, and thus
prostate was the first area of the body this protein was studied. It is
currently thought to be a potential prognostic marker for prostate
cancer, since hK4 is significantly upregulated when prostate
cancer is present (Obiezu et al., 2002). The
upregulated hK4 expression was found to be performed by androgens in
the prostate cancer cell-derived line.
hK4
has also been found to play a role in ovarian
cancer. Because hK4 was thought to be upreglated by androgens only in
prostate cancer cell lines, it was not originally thought that this
protein had much to do with ovarian cancer. Historically, the role of
androgens in the ovary was not well known, however it has recently been
shown
that androgens are present at higher levels than estrogen at different
points in the menstrual cycle. Because of this discovery, it has been
suggested that these androgens upregulate hK4 in ovarian cancer as well
(Obiezu et al., 2001). Expression of hK4 was found
to be present in normal ovarian tissues, but only in very low levels.
Research has since suggested that hK4 is a good prognostic marker for
ovarian cancer (Obiezu et al., 2001). While the
cells were found to be upregulated in many women with ovarian cancer,
the cells were more heavily upregulated in women experiencing later,
more advanced stages of the cancer. This suggests that as the cancer
reaches the more advanced stages, hK4 upregulation will also increase.
Additionally,
hK4 has been discovered to play a pivotal role in the proper
development of tooth enamel. Mutations in this gene are involved in the
disease amelogeness imperfecta (AI). AI is a genetically inherited
group of diseases affecting the formation of dental enamel. It can be
classified as hypoplastic, hypocalcified, hypomaturation or a
combination of these forms, each causing a different phenotype in
the tooth enamel (Kim et al., 2005). Point
mutations of KLK4, the gene encoding for hK4, is
known to cause autosomal recessive hypomaturation AI. Hypomaturation
type AI is classified by a ground-glass-like rough surface of the
enamel. This disease further contributes to increased dental caries
prevalence as the proper formation of the enamel is strongly affected
(Masuya et al. 2005).
VI.
References
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