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Human Tissue Kallikrein 4

Anna Frutiger '09 and Paige Roberts '09


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

Bode, W., P. Schwager, R. Huber. 1978. The transistion of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined cyrstal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution. Journal of Molecular Biology. 118: 99-112

Crawford, P. J. M., M. Aldred, A. Bloch-Zupan. 2007. Amelogenesis imperfecta. Orphanet Journal of Rare Diseases 2: 17.

Debela, M., V. Magdolen, B. V. Grimminger, C. Sommerhoff, A. Messerschmidt, R. Humber, R. Friedrich, W. Bode, P. Goettig. 2006. Crystal Structure of Human Tissue kallikrein 4: Activity Modulation by a Specific Zinc Binding Site. Journal of Molecular Biology 362: 1094-1107.

Obiezu, C. V., A. Scorilas, D. Katsaros, M. Massobrio, G. M. Yousef, S. Fracchoili, et al.. 2001. Higher human kallikrein gene 4 (KLK4) expression indicates poor prognosis of ovarian cancer patients. Clin. Cancer Res. 7: 2380-2386.

Obiezu, C. V., A. Soosaipillai, K. Jung, C. Stephan, A. Scorilas, D. H. Howarth, E. P. Kiamandis. 2002. Detection of human kallikrein 4 in healthy and cancerous prostatic tissues by immunofluorometry and immunohistochemistry. Clin. Chem. 48: 1232-1240.

Kim, J.-W., F. Seyman, B. P.-J. Lin, B. Kiziltan, K. Gencay, J. P. Simmer, J. C.-C Hu. 2005. ENAM Mutations in Autosomal-dominant Amelogenesis Imperfecta. Journal of Dental Research 84: 278-282. .

Masuya, H., K. Shimizu, H. Sezutsu, Y. Sakuraba, J. Nagano, A. Shimizu, N. Fujimoto, A. Kawai, I. Miura, H. Kaneda, K. Kobayashi, J. Ishijima, T. Maeda, Y. Gondo, T. Noda, S. Wakana, T. Shirosishi. 2005. Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI). Human Molecular Genetics 14:575-83

Silva, N. and Marcy, D. 2007. An Introduction to Jmol Scripting. /Departments/BioDev/omm/scripting/molmast.htm.

Stephanopoulos, G., M.-E. Garefalaki, K. Lyroudia. 2005. Genes and
Related Proteins Involved in Amelogenesis Imperfecta. Journal of
Dental Research 84: 1117-1126.

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